Modulators of immune escape mechanism for universal cell therapy

Abstract

To solve the following problem that: there remains a need in the art for a universal cell which is safe and effective.SOLUTION: Therapeutic agents capable of detaining bulky proteins, such as CD45, CD148, and CD43, in the middle of a cellular interface between a graft cell and a CD45 positive host effector cell (for example, a T cell, NK cell, B cell, or dendritic cell) are disclosed, as are methods for their use and products made with such therapeutic agents. The therapeutic agents prevent or inhibit the formation of functional immunologic synapses (including physiological SMAC), and they result in continuous dephosphorylation of signal transduction pathways.SELECTED DRAWING: None

Description

[Background technology]

[0001] Cell therapy, a remarkable achievement of modern science, is currently being used to replace damaged tissues and / or organs and appears promising for many diseases, including diabetes, retinitis pigmentosa, Parkinson's disease, myocardial infarction, blood cancers including lymphoma and leukemia, bone marrow failure syndromes including anemia and cytopenias, inherited immune disorders including Wiskott-Aldrich syndrome (WAS) and severe combined immunodeficiency (SCID), hemoglobinopathies including thalassemia, sickle cell anemia, and congenital dyserythropoietic anemia, inherited metabolic disorders including lysosomal storage disorders, galactosemia, phenylketonuria, and glycogen storage diseases, neurological disorders including neuromyelitis optica, cartilage replacement including knee replacement, and Crohn's disease. Like organ transplantation, cell therapy also faces the challenges of limited donor availability and immune rejection, which requires the development of mechanisms to render cells immune privileged. Immunoprivileged cells not only allow the generation of "off-the-shelf" cell products, but may also lead to the generation of "off-the-shelf" organs.

[0002] Universal cells are cells that can be administered to any patient without triggering an immune response. This has been the ultimate goal of organ transplantation and cell therapy since the creation of these fields. The lack of universal cells generally limits off-the-shelf therapies and reduces many therapies to a close tissue match between donor and recipient. In almost all cases, immunosuppressive drugs are administered with significant side effects.

[0003] An obvious side effect of administering immunosuppressants is an increased overall susceptibility to infections and cancer. Commonly used immunosuppressants include cyclosporine, azathioprine, antilymphoblastoid, antithymocyte globulin, muromonab-CD3, and porcine antilymphocyte globulin (P-ALG). Cyclosporine is known to cause nephrotoxicity, hepatotoxicity, hyperkalemia, hypertension, tremor, gingival overgrowth, and hirsutism. Azathioprine inhibits bone marrow suppression, leading to leukopenia. Antilymphoblastoid and antithymocyte globulin are foreign antibodies that can cause allergic-type reactions, such as fever, chills, and hypotension. The initial side effects of monoclonal antibodies (muromonab-CD3, OKT3) are similar to those of P-ALG, including high fever, shaking chills, headache, rigors, and hypotension. Min, DI and Monaco, AP (1991), Complications Associated with Immunosuppressive Therapy and Their Management. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 11: 119S-125S.

[0004] The art contains many examples of attempts to produce cells compatible with any recipient. The most common approach is beta-2 microglobulin (B2M) disruption, which eliminates surface expression of all class I molecules but leaves cells vulnerable to lysis by natural killer (NK) cells. Insertion of the HLA-E gene at the B2M locus in human pluripotent stem cells (PSCs) confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and peptide antigens) without surface expression of either HLA-A, B, or C. These HLA-engineered PSCs and their differentiated derivatives express CD8 + They are not recognized as allogeneic by T cells, do not bind anti-HLA antibodies, and are resistant to NK-mediated lysis. German G,Hirata, Roli K,Funk, Sarah E, Riolobos, Laura,Lopes, Vanda S, Manske, Gabriel, Plunkard, Donna, Colunga, Aric G, Hanafi, Laila-Aicha, Clegg, Dennis O, Turtle, Cameron, Russell, David W.; HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nature Biotechnology (Vol 35 p 765) 2017 / 05 / 15 / online; and Glas R, Franksson L, Ohlen C, Hoglund P, Koller B, Ljunggren HG, et al. Major histocompatibility complex class I-specific and -restricted killing of beta 2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1992;89(23):11381-5. Such approaches are genetically engineered to prevent some cells from being recognized by the immune system but do not provide truly universal cells. Furthermore, potential cis interactions between HLA-E and NKG2A and NKG2C may affect graft function, leading to suboptimal cell production. Furthermore, the cell product is generated in multiple gene editing steps consisting of simultaneous knockout of all HLA class I molecules and knock-in of the HLA-E B2M fusion protein. Summary of the Invention [Problem to be solved by the invention]

[0005] CRISPR-Cas9 and other gene editing technologies have launched a race to create "off-the-shelf" donor cells that are invisible to the immune system. A common approach to creating such cells involves manipulating genes required for immune recognition, particularly HLA class I and II proteins. Another approach leverages knowledge of immune-cloaking strategies used by certain bacterial, viral, parasitic, fetal, and cancer cells to induce tolerance to allogeneic cell-based therapies by modifying cells to express immunosuppressive molecules, such as PD-L1 and CTLA4-Ig. The same mechanisms that lead to cell and tissue rejection are also implicated in autoimmune diseases. There remains a need in the art for safe and effective universal cells. [Means for solving the problem]

[0006] Because many pathogenic and nonpathogenic microorganisms have shaped our immune system, they themselves have evolved and mastered immune evasion, particularly in chronic infections. Epstein-Barr virus (EBV) is one such immune system evader. We have discovered that it is possible to exploit immune evasion mechanisms evolved by various pathogens to render them immune-privileged. Human cytomegalovirus (HCMV) inhibits T cell activity through engagement of the UL11 protein with CD45 (Figure 27), resulting in disruption of proximal signaling required for T cell activation and / or development. Similarly, the E3 protein from adenovirus (Figure 21) engages with CD45 and inhibits NK and T cells. We have shown that graft rejection can be avoided by genetically modifying the surface of grafted cells to express binding molecules for CD45 (Figures 7-12). Following the same path, we also constructed a single-chain monoclonal antibody against CD45 (a-CD45-sc) (Figure 24). Cells engineered to express CD45 engagers are reported here for their immune evasion properties. To this end, we tested and compared the cytotoxicity of NK and T cells against target cells expressing UL11, E3.49K, a-CD45-sc, or GFP (as a control) (Figures 7-12).

[0007] CD45 is a transmembrane protein tyrosine phosphatase (PTPase) expressed on nucleated cells. It has a large, heavily glycosylated extracellular domain and tandem intracellular phosphatase domains. CD45 covers approximately 10% of the surface area of B and T cells, where it regulates cell development and activation by directing membrane-proximal signaling. After cell synapse formation, CD45 dephosphorylates inhibitory tyrosines in the tails of SRC family kinases, allowing them to assume an "open," non-inhibitory conformation. "Open" SRC family kinases achieve increased kinase activity through autophosphorylation on the activation loop of their own kinase domain. Active SRC family kinases further phosphorylate protein molecules containing immunoreceptor tyrosine-based activation motifs (ITAMs) and SYK family kinases, resulting in signal transduction, propagation, and amplification. During a successful cellular immune response, CD45 is excluded from the immune synapse and is only reintroduced into the synapse upon cessation of the response. CD45 dephosphorylates activation loop phosphorylation, reducing SRC family kinase activity and resulting in the termination of immune signaling. Furthermore, it also attenuates cytokine receptor signaling by dephosphorylating Janus kinases. CD45 can also dephosphorylate other proximal signaling molecules, including ZAP70 and CD3-zeta. CD45 is a constitutively active type I membrane phosphatase consisting of a heavily glycosylated extracellular domain and an intracellular tandem phosphatase domain, with the intrinsic catalytic activity of the membrane-proximal domain. The membrane-proximal extracellular region consists of a fibronectin type III domain, followed by a cysteine-rich domain and a heavily glycosylated distal region. The CD45 gene has multiple exons, and alternative splicing of exons 4 (A), 5 (B), and 6 (C) generates transcripts of variable length. Human CD45 can be the result of alternative exon usage, generating ABC, AB, BC, B, and O isoforms. The shortest product, in which all three exons (A, B, and C) are deleted, is called CD45RO, and the longest, which contains all these exons, is called CD45RABC.Different isoforms are used as developmental and activation markers in various lymphocytes. CD45RO is a conserved domain that is targeted among all isoforms.

[0008] CD148 is a receptor tyrosine phosphatase with a large, heavily glycosylated fibronectin extracellular domain and an intracellular catalytic domain. In addition to hematopoietic lineages, CD148 is also expressed on vascular and glandular endothelial cells, where it negatively regulates cell proliferation and transformation. Loss of CD148 has been observed in cancer cell lines, and re-expression resulted in tumor growth suppression both in vitro and in vivo. CD148 dephosphorylates multiple growth factor receptors, including VEGFR, EGFR, HGFR, and FGFR, as well as other key downstream signaling molecules such as p85, PLCγ1, and ERK1 / 2.

[0009] CD43 is a highly glycosylated mucin-type protein with a large extracellular domain and a small globular intracellular domain that is expressed on hematopoietic cells, including stem cells, T cells, monocytes, granulocytes, NK cells, and platelets. The CD43 extracellular domain promotes adhesion through interactions with E-selectin, galectin-1 and -3, Siglec-1, M-ficolin, integrins, cell surface nucleolin, and ICAM-1 (intercellular adhesion molecule type 1). The conserved intracellular domain is involved in signal transduction, mediating connection to the cytoskeleton through binding to ezrin, radixin, and moesin (ERM) proteins. CD43 contains a proline-rich sequence resembling an SH3 binding consensus and a nuclear localization signal (NLS) that accounts for the nuclear localization of CD43.

[0010] The B cell receptor (BCR) is a membrane-bound immunoglobulin with a short intracellular domain of three amino acids. BCR is made up of two identical heavy chains and two light chains. The extracellular domain has the ability to specifically recognize and bind to antigens. BCR consists of two chains, Igα (also called Igα), BCRs lack intracellular signaling, which is compensated for by two associated ITAMs containing Igβ (IgFa) and Igβ (beta). After successful binding to an antigen, the BCR transmits signals that lead to B cell activation and maturation. After class switching, the BCR switches from a membrane-bound to a shed form, called an antibody.

[0011] The immune synapse, also known as the supramolecular activation cluster (SMAC), is the interface between target cells and lymphocytes due to the accumulation of activating and regulatory molecules (Figures 1A-1E, left). Prior to immune synapse formation, molecules are stochastically distributed (Figure 1A, left). Upon ligation of the TCR with the target MHCp complex (Figure 1B, left), LCK is retained while CD45 is moved or pushed to the periphery (Figure 1C, left). This ultimately results in LCK activation. Finally, as CD45 is pushed to the periphery, coreceptors ligate, resulting in the formation of a mature synapse (Figure 1D, left). This interface, or SMAC, is composed of concentric circles of molecules involved in immune cell recognition. The innermost central SMAC (cSMAC) consists of TCR / CD3 / MHCp, CD28 / CD80, SRC family kinases, and PKCθ. Outside the cSMAC is the peripheral SMAC (pSMAC), which consists of an adhesive ring of LFA-1, ICAM-1, and talin, followed by the outermost circle termed the distal SMAC (dSMAC), which consists of glycoproteins including CD45, CD43, and CD148.

[0012] In the present system, we disrupt the formation and structural sequence of the immune synapse by placing bulky proteins, such as CD45, at the center of the cell interface between graft cells and cytotoxic cells, such as T cells or NK cells (Figures 1A-E, right; Figures 2A-2D). This not only prevents the formation of a physiological SMAC (in the case of graft cell-T cell interactions), but also results in the continuous dephosphorylation of signaling pathways, such as the TCR / CD3 / MHCp signaling pathway, so that cells may not even reach close enough to engage the TCR / CD3 / MHCp signaling pathway.

[0013] CD148 and CD43 may be used in a similar manner to CD45, albeit in a less obvious manner. In certain embodiments, CD45, CD148, and / or CD43 may be placed alone or in combination with other molecules.

[0014] In embodiments, the present invention includes a therapeutic agent comprising one or more molecules or cells configured to prevent cytotoxicity by modulating the ability of CD45, CD148, or CD43 to form a functional immunological synapse with cytotoxic cells. In embodiments, the therapeutic agent may comprise a protein, an aptamer, a peptide nucleic acid (PNA), a nanoparticle, or a cell expressing or secreting the one or more molecules. In embodiments, the therapeutic agent may comprise a protein, preferably a protein comprising an antibody, more preferably a protein comprising a single-chain antibody or a VHH nanobody. In embodiments, the therapeutic agent may comprise a nanoparticle, preferably a lipid nanoparticle (LNP), a dendrimer, or a ribonucleoprotein (RNP). In embodiments, the therapeutic agent may comprise an extracellular vesicle, preferably an exosome or a microvesicle. In embodiments, the therapeutic agent may comprise a cell, preferably a eukaryotic cell, more preferably an avian cell or a mammalian cell, e.g., a murine, porcine, bovine, canine, feline, or ovine cell, and most preferably a human cell. In embodiments, the therapeutic agent may comprise hematopoietic cells, stem cells, lymphoid cells, bone marrow cells, red blood cells, or platelets. In embodiments, the therapeutic agent may comprise one or more excipients or additives, preferably one or more of fillers, extenders, diluents, wetting agents, solvents, emulsifiers, preservatives, absorption enhancers, sustained-release matrices, salts, buffers, starches, sugars, microcrystalline cellulose, granulating agents, lubricants, binders, disintegrants, colorants, release agents, coating agents, sweeteners, flavoring agents, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, carriers, stabilizers, and combinations thereof. In embodiments, the therapeutic agent may be for oral, dermal, enteral, or parenteral administration. In embodiments, the therapeutic agent may be delivered via injection (e.g., direct injection into diseased tissue or systemic injection), patch or other transdermal delivery device, or perfusion. In embodiments, the therapeutic agent may comprise a component of viral or bacterial origin, preferably ULL or E3 / 49k, or a fragment thereof.In embodiments, the therapeutic agent may contain components of viral or bacterial origin, e.g., that do not contain ULL protein or fragments thereof or E3 / 49k protein or fragments thereof. In embodiments, the therapeutic agent may contain SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, or a protein having at least 80% identity to SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224. In embodiments, the therapeutic agent may include cells having one or more molecules expressed on their surface. In embodiments, the one or more molecules expressed on the surface of the cells include an expressed transmembrane protein, and the cells include graft cells. In embodiments, the transmembrane protein may be capable of binding to CD45, CD148, or CD43. In embodiments, the CD45, CD148, or CD43 of the therapeutic agent may be present on the surface of cytotoxic cells, preferably T cells or natural killer (NK) cells. In embodiments, the transmembrane protein may be one that retains CD45, CD148, or CD43 in the developing immunological synapse on the surface of a cytotoxic cell, thereby preventing the formation of a functional immunological synapse.

[0015] In another embodiment, the invention comprises a protein complex capable of preventing cytotoxic cell-induced lysis, the protein complex comprising an engager comprising SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, or a protein having at least 80% identity to SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224; and a CD45, CD148, or CD43 protein expressed on the surface of a T cell or NK cell.

[0016] In another embodiment, the invention includes a method of producing a composition for disrupting a functional immunological synapse, comprising expressing on the surface of a first cell one or more molecules configured to retain CD45, CD148, or CD43 on the surface of a second cell in an incomplete immunological synapse, thereby disrupting or inhibiting the formation of a functional immunological synapse between the first cell and the second cell.

[0017] In another embodiment, the present invention includes a method for promoting escape from NK-mediated lysis, comprising administering the above-described therapeutic agent to a subject in need thereof. In embodiments, the method may involve inhibiting or blocking NKG2D binding to MICA, MICB, and / or ULBP. In embodiments, the method may involve blocking an activating NK cell receptor selected from members of the human killer immunoglobulin-like receptor (KIR) family, the CD94-NKG2C / E / H heterodimeric receptor, NKG2D, natural cytotoxicity receptors such as NKp30, NKp44, and NKp46, the nectin / nectin-like binding receptors DNAM-1 / CD226 and CRTAM, receptors expressed by natural killer (NK) cells that regulate their activation, the SLAM family receptors (including 2B4 / CD244, CRACC / SLAMF7, and NTB-A / SLAMF6), as well as Fc gamma RIIIA / CD16a, CD27, CD100 / semaphorin 4D, and CD160. In embodiments, the subject may be at risk of having or suffering from one or more of the following conditions: autoimmune diseases, blood cancers including lymphoma and leukemia; bone marrow failure syndromes including anemia and cytopenias; inherited immune disorders including WAS and SCID; hemoglobinopathies including sickle cell disease (SCD) and thalassemia; neurological disorders including neuromyelitis optica; and graft-versus-host disease.

[0018] In another embodiment, the present invention includes a method for promoting escape from T cell-mediated lysis, comprising administering the above-described therapeutic agent to a subject in need thereof.In an embodiment, the method may include inhibiting or blocking T cell receptors that bind to MHC peptides.In an embodiment, the subject in need thereof may be at risk of or suffer from one or more of psoriasis and vitiligo.

[0019] In another embodiment, the invention includes a method for positionally retaining CD45 on the surface of cells expressing CD45 to disrupt the formation of a functional immunological synapse, comprising treating cells expressing CD45 with an agent that has affinity for the membrane proximal region of the extracellular domain of CD45, thereby positioning CD45 relative to other membrane proteins expressed on the surface of the cells that are necessary for the formation of a functional immunological synapse.

[0020] In another embodiment, the invention includes a non-autologous cell comprising an engager on its surface and configured to avoid synapse formation with one or more host cytotoxic cells. In an embodiment, the host cytotoxic cells are natural killer cells, T cells, or macrophages. In an embodiment, the cytotoxic cells are T cells, preferably gamma-delta T cells, CD8 + T cells, CD4 + In some embodiments, the non-autologous cells are T cells or mucosal-associated invariant T cells. In some embodiments, the non-autologous cells do not contain genetic modifications. In some embodiments, the non-autologous cells may be treated with an engager.

[0021] In another embodiment, the present invention includes a method for producing xenogeneic cells for transplantation, comprising protecting the xenogeneic cells to be transplanted with the above-described therapeutic agent. In embodiments, the therapeutic agent may be administered to a host prior to or in parallel with the xenogeneic cells for transplantation. In embodiments, the therapeutic agent may be bound to the surface of the xenogeneic cells for transplantation. In embodiments, the therapeutic agent may be a cell, and the cell may be genetically modified to express an engager on its surface or in extracellular vesicles.

[0022] In another embodiment, the present invention comprises a method for preventing the rejection of solid organ or organoid transplants, comprising transducing or transfecting a gene into the cells of solid organ or organoid to prevent or inhibit the binding of cytotoxic cells to the cells of solid organ or organoid transplants.In one embodiment, the gene can encode an engager, and the engager can be expressed in an amount or density effective to inhibit the formation of functional immunological synapses upon exposure of solid organ or organoid to cytotoxic cells.

[0023] In another embodiment, the present invention includes a method of treating cancer comprising administering hematopoietic stem cells comprising a membrane-bound engager to a subject in need thereof.

[0024] In another embodiment, the present invention includes a recombinant protein that may include (i) a signal peptide, (ii) an antibody heavy chain, (iii) a first linker, (iv) an antibody light chain, (v) optionally, a second linker, (vi) a stalk, (vii) a transmembrane region, and (viii) optionally, an intracellular region. In an embodiment, the recombinant protein may include a second linker connecting the light chain to the stalk. In an embodiment, the recombinant protein may be a single-chain antibody, preferably a single-chain antibody that specifically binds to CD45, CD148, or CD43. In an embodiment, each of (i) to (vii) may be present and may be connected in order from the amino terminus to the carboxyl terminus of the protein. In embodiments, the signal peptide may be an IL2 signal peptide; the first linker may comprise an SGGGG motif and / or may vary in length from 5 to 60, preferably from 10 to 50, more preferably from 20 to 45 amino acids; the second linker, if present, may vary in length from 5 to 60, preferably from 5 to 40, more preferably from 7 to 15 amino acids; the stalk may be at least 8 and no more than 200 amino acids in length, and the transmembrane region may be derived from CD34, CD45, CD28, and / or Cd8a.

[0025] In another embodiment, the invention includes a cell comprising an engager and an exogenous suicide gene.

[0026] In another embodiment, the invention includes a first cytotoxic cell that expresses membrane-bound CD45, CD148, and / or CD43, and that can be treated to prevent the formation of a functional immunological synapse with a second cytotoxic cell that expresses membrane-bound CD45, CD148, and / or CD43. In embodiments, the cytotoxic cell can be a natural killer cell, a T cell, or a macrophage.

[0027] In another embodiment, the invention includes a graft that has been treated to prevent binding of cytotoxic cells, the treatment comprising exposing the graft to a therapeutic agent as described above.

[0028] In another embodiment, the present invention includes a method of controlling inflammation comprising administering to a subject in need thereof mRNA or DNA encoding an engager, thereby modulating the formation of functional immunological synapses to control inflammation. In embodiments, the formation of functional immunological synapses may be inhibited, thereby reducing inflammation.

[0029] In another embodiment, the present invention includes the use of engagers to reduce cytotoxic cell responses to transplantation. In an embodiment, the use may be performed in the absence of knockout or knockdown of HLA-I and / or HLA-II. In an embodiment, the use may be performed in combination with knockout or knockdown of HLA-I and / or HLA-II.

[0030] In another embodiment, the invention comprises a cell comprising a surface-bound engager and a chimeric antigen receptor (CAR). In an embodiment, the CAR comprises a-CD38CAR (SEQ ID NO: 218) or a variant thereof having at least 80% identity thereto. In an embodiment, the CAR comprises a-CD19CAR (SEQ ID NO: 216) or a variant thereof having at least 80% identity thereto.

[0031] In another embodiment, the invention comprises an anti-CD45, anti-CD148, or anti-CD43 engager comprising a transmembrane domain configured on the surface of a cell, hi embodiments, the invention comprises an engager comprising a membrane-bound antibody, nanobody, or single chain against CD45, CD43, or CD148.

[0032] In another embodiment, the invention comprises a vector or plasmid for producing an anti-CD45, anti-CD148, or anti-CD43 engager comprising DNA encoding an anti-CD45, anti-CD148, or anti-CD43 engager operably linked to a promoter. In embodiments, the invention comprises a vector or plasmid encoding a membrane-bound antibody, nanobody, or single chain against CD45, CD148, or CD43. [Brief explanation of the drawings]

[0033] [Figure 1A] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to a mature immune synapse. [Figure 1B] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to a mature immune synapse. [Figure 1C] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to a mature immune synapse. [Figure 1D] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to a mature immune synapse. [Figure 1E] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to a mature immune synapse. [Figure 2A] Figure 2A shows the immune synapse between host T cells and graft cells. Engagement of host TCRs with donor MHC-peptide complexes leads to killing of the graft. [Figure 2B] Figure 2B shows the interaction between host T cells and graft cells expressing a novel engager that keeps CD45 at the center of the synapse, leading to non-killing of the graft and the lack of formation of a functional immunological synapse. [Figure 2C]Figure 2C shows the immune synapse between host NK cells and graft cells. Engagement of host activating receptors with recipient ligands leads to killing of the graft. [Figure 2D] Figure 2D shows the interaction between host NK cells and graft cells expressing a novel engager that keeps CD45 at the center of the synapse, leading to non-killing of the graft and the lack of formation of a functional immunological synapse. [Figure 3] Figure 3 is a map of the plasmid LeGO-iG2-UL11. [Figure 4] Figure 4 is a map of the plasmid LeGO-iG2-E3.49k. [Figure 5] Figure 5 shows the map of the plasmid LeGO-iG2-A-CD45-SC. [Figure 6] FIG. 6 shows the generation of stable cell lines. [Figure 7] Figure 7 is a bar graph showing inhibition of cell lysis in cells transfected with a-CD45-sc, E3.49K, or UL11 (control untransfected). The y-axis shows percent specific lytic 51Cr release in K562 cells incubated with PBMCs. Effector:target (E:T) ratios are shown below the bar groupings. [Figure 8] Figure 8 is a bar graph showing inhibition of cell lysis in cells transfected with a-CD45-sc, E3.49K, or UL11 (control was untransfected). The y-axis shows percent specific lytic 51Cr release in K562 cells incubated with NK92 cells; E:T ratios are shown below the bar groupings. [Figure 9] Figure 9 is a line graph showing inhibition of cytolysis in K562 cells transformed with a-CD45-sc, E3.49K, or UL11 (untransformed control) when exposed to PBMC cells. The y-axis represents the percent specific lytic 51Cr release in K562 cells incubated with PBMC; the x-axis represents the E:T ratio. [Figure 10]10 is a line graph showing inhibition of cytolysis in K562 cells transformed with a-CD45-sc, E3.49K, or UL11 (untransformed control) when exposed to NK92 cells. The y-axis represents the percent specific lytic 51Cr release in K562 cells incubated with NK92 cells; the x-axis represents the E:T ratio. [Figure 11] 11 is a line graph showing predictive data for the inhibition of cytolysis in RPMI88226 cells transfected with a-CD45-sc, E3.49K, or UL11 (untransfected control) when exposed to T cells. The y-axis indicates percent specific lytic 51Cr release; the x-axis indicates the E:T ratio. [Figure 12] Figure 12 is a line graph showing data on the inhibition of cytolysis in CD34 differentiated T-like cells transfected with a-CD45-sc, E3.49K, or UL11 (control was untransfected) when exposed to CD8+ T cells. The y-axis indicates percent specific lytic 51Cr release; the x-axis indicates the E:T ratio. [Figure 13] FIG. 13 shows the map of the plasmid LeGO-iG2-a-CD45-(M)-VHH1. [Figure 14] Figure 14 shows the map of the plasmid LeGO-iG2-a-CD45-(M)-VHH2. [Figure 15] Figure 15 is a map of plasmid LeGO-iG2-E3.49K.R1. [Figure 16] Figure 16 is a map of plasmid LeGO-iG2-E3.49K.R3. [Figure 17] FIG. 17 is a map of the plasmid LeGO-iG2-mVHH1-E3™. [Figure 18] Figure 18 is a map of the plasmid LeGO-iG2-mVHH2-E3™. [Figure 19] Figure 19 is a map of the plasmid LeGO-iG2-a-CD19CAR. [Figure 20] Figure 20 is a map of the plasmid LeGO-iG2-a-CD38CAR. [Figure 21] Figure 21 is a diagrammatic representation of E3.49K (SEQ ID NO: 3). [Figure 22] Figure 22 is a diagrammatic representation of E3.49K.R1 (SEQ ID NO: 66). [Figure 23] Figure 23 is a diagrammatic representation of E3.49K.R3 (SEQ ID NO: 68). [Figure 24] Figure 24 is a diagrammatic representation of a-CD45-sc (SEQ ID NO: 5). [Figure 25] Figure 25 is a diagrammatic representation of m-VHH1-E3-TM (SEQ ID NO: 220). [Figure 26] Figure 26 is a diagrammatic representation of m-VHH2-E3-TM (SEQ ID NO: 222). [Figure 27] FIG. 27 is a diagrammatic representation of UL11 (SEQ ID NO: 1). [Figure 28] Figure 28 is a diagrammatic representation of a-CD38CAR (sequence number 218). [Figure 29] Figure 29 is a diagrammatic representation of a-CD19CAR (SEQ ID NO: 216). [Figure 30] FIG. 30 is a line graph showing cytolysis of target cells by NK92 cells expressing a-CD45-sc. [Figure 31] FIG. 31 is a line graph showing cytolysis of target cells by TALL-104 cells expressing a-CD45-sc. [Figure 32] FIG. 32 is the experimental flow chart followed for the in vivo experiments. [Figure 33] Figure 33 is a compilation of IVIS images of PBMCs and daratumumab-treated RPMI-8226 cells transduced with luciferase and CD45 engagers. Although the same dose of RPMI-8226 cells was administered, a higher tumor burden is observed compared to that in Figure 34. [Figure 34]Figure 34 is a compilation of IVIS images of PBMCs and luciferase-transduced (but not CD45 engager-transduced) RPMI-8226 cells treated with daratumumab. Controlled and minimal residual disease is observed. [Figure 35] Figure 35 is a line graph showing the effect of a-CD45-sc on K562 cells after PBMC exposure. Figure 35 shows IVIS imaging analysis in K562 tumor-bearing mice compared to K562 with CD45 engagers. All mice depicted received PBMCs. Each line represents one mouse. [Figure 36] Figure 36 is a line graph showing the effect of a-CD45-sc on SKOV3 cells after exposure to PBMCs treated with Herceptin. Figure 36 shows IVIS imaging analysis in SKOV3 tumor-bearing mice compared to SKOV3 with CD45 engagers. All mice depicted received PBMCs and trastuzumab, except for the control group, which received only PBMCs. Each line represents one mouse. [Figure 37] Figure 37 is a schematic scheme of mRNA loading into EVs. [Figure 38] Figure 38 is a line graph showing arthritis scores after therapeutic EV injection. The higher the score, the more severe it is. Each limb was scored using a scale of 0 to 4 based on increasing levels of erythema and swelling. [Figure 39A] Figures 39A and 39B are bar graphs showing TNFa (pg / 100 μg protein) and IL1b (pg / 100 μg protein) secretion in an arthritis model after therapeutic EV injection. [Figure 39B] Figures 39A and 39B are bar graphs showing TNFa (pg / 100 μg protein) and IL1b (pg / 100 μg protein) secretion in an arthritis model after therapeutic EV injection. [Figure 40] Figure 40 is a schematic flow chart showing EV production / isolation and purification of therapeutic EVs. [Figure 41]Figure 41 is a line graph showing cytolysis of target cells (RPMI8226) by NK92 cells co-expressing a-CD45-sc and a-CD38CAR as assessed by 51Cr release assay. [Figure 42] Figure 42 is a line graph showing cytolysis of target cells (CD38KO RPMI8226) by NK92 cells co-expressing a-CD45-sc and a-CD38CAR as assessed by 51Cr release assay. [Figure 43] Figure 43 is a bar graph showing degranulation of target cells (Raji and Jurkat) by NK92 cells co-expressing a-CD45-sc and a-CD19CAR. [Figure 44] Figure 44 is a live cell imaging co-culture of target cells (K562) with NK92 co-expressing a-CD45-sc and a-CD38CAR. Dead cells appear bright. Effector cells appear dark. This is a microscopic representation of what is demonstrated in Figure 42 and Figure 43. DETAILED DESCRIPTION OF THE INVENTION

[0034] Priority Claims and Incorporation by Reference This application claims priority to U.S. Provisional Patent Application No. 62 / 943,807, filed December 5, 2019, the contents of which are incorporated herein by reference. All references cited herein are expressly incorporated by reference.

[0035] Introduction

[0036] The differentiation potential of pluripotent stem cells, such as embryonic stem cells (ESCs), allows them to provide an unlimited supply of any cell type for transplantation. ESCs were expected to offer "off-the-shelf" cell therapy for conditions such as Parkinson's disease, diabetes, and cardiovascular disease, where any damaged tissue requires repair or replacement. However, immune rejection dramatically limited the use of this opportunity. Induced pluripotent stem cells (iPSCs) offered a solution: generating pluripotent cells from patients and then differentiating them into the desired cell type. iPSC generation, gene repair, differentiation, and therapeutic and safety validation for individual patients are not affordable in terms of convenience and cost. Despite immune rejection remaining a major challenge in the field of cell therapy, notable advances have been made in the form of mesenchymal stem cells, CAR-T cells, and adult stem cells. Numerous strategies have been developed to prevent immune rejection for in vivo persistence of allogeneic grafts.

[0037] Immunosuppression with continuous cyclosporine and cyclophosphamide has been the only option in organ transplantation and autoimmune treatment. Furthermore, in the context of donor lymphocyte infusion, chimeric antigen receptor-modified T-cell therapy, and infusion of other genetically modified cells, cyclophosphamide and cyclosporine have been used to create an environment in which donor cells can be maintained for a period of approximately 2 weeks. The use of rifampin and fludarabine treatment regimens has been utilized for temporary host lymphodepletion. It is also used as an alternative drug for prolonged in vivo expression of gene therapy vectors. In cell transplantation, to prevent host T cell-mediated rejection of allogeneic grafts, donor cell HLA has been knocked out as a possible host CD8+ T cell (HLA class I knockout) and CD4+ T cell (HLA class II knockout)-mediated immune escape strategy. At the same time, non-classical HLA expression has been enforced on these cells to prevent NK cell-mediated cytotoxicity. Similarly, CTLA4Ig has been used to prevent T cell CD28 coreceptor ligation and therefore immune responses against donor cells, and CD40 mAb has been used to attenuate APC and B cell function. Some studies have utilized viral proteins redirected to intracellular signaling and antigen processing to prevent immune responses. For example, ICP4, a cytosolic protein from HSV, inhibits TAP-mediated transport of peptides into the endoplasmic reticulum (ER), whereas HCMV proteins US11 / 2 lead to degradation of MHC-I, US3 retains MHC-I in the ER, and US6 blocks TAP.

[0038] We aim to exploit the immune evasion strategy employed by viruses that targets direct extracellular, cell-to-cell trans-interactions with regulatory proteins on T cells and NK cells. UL11, a member of the RL11 protein family, is expressed on the surface of CMV-infected cells and binds to CD45 on leukocytes (Figures 3 and 27). CD45, a protein tyrosine phosphatase, is a key regulator of T cell antigen receptor (TCR) signaling. CD45 activates SRC family kinases by removing their C-terminal inhibitory phosphorylation. Activated SRC family kinases phosphorylate ITAMs in the CD3-TCR complex, propagating the signal and thereby activating T cells. CD45 inhibition blocks TCR-mediated signaling, resulting in severe combined immunodeficiency (SCID) in humans. UL11 binds to CD45 and blocks downstream signaling, thereby blocking both T cell activation and development.

[0039] The E3 transcription unit of human adenoviruses contains proteins with immunomodulatory functions that enable persistent, asymptomatic infection in immunocompetent individuals (Figures 4 and 21). E3.49K from adenovirus (Ad) species D is unique in that it acts on uninfected cells, unlike E3s from other adenovirus species, which only affect infected cells. E3.49K is a highly glycosylated type I protein that releases an extracellular 49 kDa molecule after cleavage. E3.49K has been shown to inhibit both NK cell-mediated lysis of MHC-I-deficient target cells and TCR complex-mediated activation / development of T cells. Our designs include individual proteins and chimeric proteins with the UL11 protein linked to the extracellular 49 kDa of E3.49K. A third single-chain antibody targeting CD45 was also added for the same purpose. We are also testing single-domain antibodies. We expressed these proteins on target cells and tested them for NK and T cell-mediated lysis.

[0040] Referring to Figure 1A (left side), very early in the TCR receptor activation process, CD45, along with other bulky molecules, is excluded from the immune synapse. The immune synapse consists of rings containing distinct gradients of molecules involved in immune recognition / response. The innermost central supramolecular activation cluster (cSMAC) contains TCR / CD3 / MHCp, CD28 / CD80, SRC family kinases, and PKCθ. Outside the cSMAC is the pSMAC, which contains an adhesive ring of LFA-1, ICAM-1, and talin. Glycoproteins, including CD45, CD148, and CD43, are translocated to the outside of these rings.

[0041] CD148 is a receptor tyrosine phosphatase with a large, heavily glycosylated fibronectin extracellular domain and an intracellular catalytic domain. CD148 is also expressed on vascular and glandular endothelial cells, where it negatively regulates cell proliferation and transformation. Loss of CD148 has been observed in cancer cell lines, and re-expression resulted in the suppression of tumor growth both in vitro and in vivo. CD148 dephosphorylates multiple growth factor receptors, including VEGFR, EGFR, HGFR, and FGFR, as well as other key downstream signaling molecules such as p85, PLCγ1, and ERK1 / 2.

[0042] CD43 is a highly glycosylated mucin-type protein with a large extracellular domain and a small globular intracellular domain that is expressed on hematopoietic cells, including stem cells, T lymphocytes, monocytes, granulocytes, NK cells, and platelets. The CD43 extracellular domain promotes adhesion through interactions with E-selectin, galectin-1 and -3, Siglec-1, M-ficolin, integrins, cell surface nucleolin, and ICAM-1 (intercellular adhesion molecule type 1). The conserved intracellular domain is involved in signal transduction, mediating connection to the cytoskeleton through binding to ezrin, radixin, and moesin (ERM) proteins. CD43 contains a proline-rich sequence resembling an SH3 binding consensus and a nuclear localization signal (NLS) that accounts for the nuclear localization of CD43.

[0043] The B cell receptor (BCR) is a membrane-bound immunoglobulin with a short intracellular domain of three amino acids. BCRs are made up of two identical heavy chains and two identical light chains. The extracellular domain has the ability to specifically recognize and bind to antigens. BCRs lack intracellular signaling, which is compensated for by two associated ITAMs containing Igα and Igβ chains. After successful binding to an antigen, BCRs transmit signals that lead to B cell activation and maturation. After class switching, BCRs are converted from a membrane-bound form to a released form, called an antibody.

[0044] Referring to Figure 2A, the diagram shows the formation of a synapse between a T cell and a target cell in the absence of the present invention, leading to target cell lysis. In Figure 2B, using an engager of the present invention, the physiological synapse is prevented and lysis does not occur.

[0045] Referring to Figure 2C, the diagram shows the formation of a synapse between an NK cell and a target cell in the absence of the present invention, leading to target cell lysis. In Figure 2D, using an engager of the present invention, the physiological synapse is prevented and lysis does not occur.

[0046] While the data presented herein demonstrate that enforced retention of CD45 in the immune synapse via engagers prevents cytolysis by cytotoxic cells (Figures 7-12), inhibition of cytotoxic cells through CD43 and CD148 immune synapse retention may also prevent lysis. An engager may be a molecule used to interfere with the binding of CD45, CD43, and CD148. An "engager" is a molecule or group of molecules that can bind to CD45, CD43, or CD148, thereby inhibiting or preventing the formation of a functional immunological synapse. A "functional immunological synapse" is an immune synapse that can form between CD45-, CD148-, or CD43-positive cells and graft cells, including non-autologous cells. We have used single-chain, single-domain, and antibody effectors. Using the teachings disclosed herein, one skilled in the art will be able to identify other engagers.

[0047] The engager should be present in an amount sufficient to bind to CD45, CD43, or CD148. As shown in more detail below, we have shown that they can modulate or shut down NK or T cell responses to foreign cells. In certain embodiments, the compositions and methods disclosed herein are non-agonistic.

[0048] In embodiments, variants of the amino acid sequences disclosed herein are also contemplated. For example, an amino acid sequence may have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one or more of the disclosed amino acid sequences. In certain preferred embodiments, exemplary amino acid sequences may have at least 90%, 95%, 96%, 97%, 98%, or 99% identity to one or more of the disclosed amino acid sequences. In embodiments, the variant amino acid sequence retains the function ascribed to it herein (e.g., the ability to bind to CD45, CD43, or CD148 and / or the ability to prevent or inhibit the formation of a functional immunological synapse and / or the ability to confer immune escape and / or the ability to prevent cytotoxicity).

[0049] In embodiments, variants of the nucleic acid sequences disclosed herein are also contemplated. In embodiments, the nucleic acid sequence may have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one or more of the disclosed nucleic acid sequences. In certain preferred embodiments, exemplary amino acid sequences may be nucleic acid sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to one or more of the disclosed nucleic acid sequences. In embodiments, the variant nucleic acid sequence retains the function ascribed to it herein and / or encodes the (variant) amino acid as disclosed herein.

[0050] In embodiments, engagers may comprise, for example, an amino acid sequence of SEQ ID NO: 1 (UL11), 3 (E3.49K), 5 (a-CD45-sc), 64 (a-CD148-sc), 66 (E3.49K.R1), 68 (E3.49K.R3), 71 (a-CD45(M)-VHH-1), 73 (a-CD45(M)-VHH-2), 220 (m-VHH1-E3-TM), 223 (m-VHH2-E3-TM), or 224 (a-CD43-sc), in which 1 to 50 amino acids have been deleted, substituted, inserted, and / or added, and which has activity of binding to CD45 and / or inhibiting or preventing the formation of a functional immunological synapse. In a preferred embodiment, the engager disclosed herein is selected from the group consisting of 1-49, 1-48, 1-47, 1-46, 1-45, 1-44, 1-43, 1-42, 1-41, 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-28, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-48, 1-47, 1-46, 1-45, 1-44, 1-43, 1-42, 1-41, 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-42, 1-43, 1-44, 1-45, 1-46, 1-46, 1-47, 1 and a protein sequence consisting of an amino acid sequence in which 6, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue has been deleted, substituted, inserted, and / or added, and which has the activity of inhibiting or preventing the formation of a functional immunological synapse.

[0051] We will perform antibody competition and deletion mutant assays to determine the binding site of the engager. We will use immunoprecipitation to identify interacting motifs. Top candidates will be collected for further experiments.

[0052] All three CD45 engagers (E3.49K, UL11, and a-CD45-sc) bind to all isoforms of CD45, suggesting an interaction with the membrane-proximal region containing fibronectin-III and the cysteine-rich domain. Immunoprecipitation studies revealed a physical interaction between CD45 and E3.49K, UL11, or anti-CD45-sc, and Ab competition experiments and deletion mutations revealed that E3.49K, UL11, and a-CD45 bind to all isoforms of CD45. This further supports the notion that -sc interacts primarily with the membrane-proximal region of CD45 that is common to all isoforms.

[0053] The present invention also contemplates the use of aptamers directed against CD45, CD43, and CD148. Similar to antibodies, aptamers are short chains of nucleic acid, protein, or other nature that can specifically bind to target molecules with high affinity. These aptamers have the ability to target small ions, molecules, cells, tissues, or organs. This application covers aptamers whether they are composed of nucleic acids, proteins, or other molecules that can specifically bind to target molecules CD45, CD148, and / or CD43. These aptamers may be naturally occurring or synthesized de novo. Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature. 1996;380(6574):548-50; and Zhang Y, Lai BS, Juhas M. Recent Advances in Aptamer Discovery and Applications. Molecules. 2019;24(5).

[0054] The strategies described herein can be utilized in the absence of HLA-I and HLA-II knockout or knockdown strategies, however, it is also contemplated that the combination of HLA class-I (e.g., B2M) and / or HLA class II (e.g., CIITA) along with CD45 / CD148 / CD43 engagers can lead to synergistic suppression of host cytotoxicity.

[0055] Suitable stem cells include, but are not limited to, embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, and others. [Example]

[0056] Example 1. Modulation with UL11 and E3.49K

[0057] We first set out to show that UL11 and E3.49K can be used to modulate CD45 inclusion in the immune synapse.

[0058] material and method

[0059] A vector incorporating the HCMV-M (Merlin strain; HHV5) protein UL11 sequence downloaded from uniprot was created. The UL11 sequence is shown below as SEQ ID NO: 1.

[0060] UL11 https: / / www.uniprot.org / uniprot / Q6SWB9 >sp|Q6SWB9|UL11P HCMVM protein UL11 OS=Human cytomegalovirus (Merlin strain) OX=295027 GN=UL11 PE=1 SV=1 MLFRYITFHREKVLYLTAACIFGVYISLHDACIPVVGKIGTNVTLNAVDVLPPRDQVRWSYGPGGQGYMLCIFTGTSTTTFNNTRFNFSCLSNYSLLLINVTTQYSTTYRTMTSLDHWLHQRHNHGSRWTLDTCYNLTVNENGTFPTTTTTKKPTTTTRTT TTTTQRTTTTRTTTTAKKTTISTTHHKHPSPKKSTTPNSHVEHHVGFEATAAETPLQPSPQHQHLATHALWVLAVVIVIIIIIIFYFRIPQKLWLLWQHDKHGIVLIPQTDL (SEQ ID NO: 1)

[0061] Codon optimization for human cell expression was performed using CLC Workbench 8. The gene was synthesized by GeneArt Thermofischer Scientific. The gene was cloned into the LeGO-iG2-IRES-GFP plasmid and lentiviral particles were generated. The viral particles were used to transduce K562 and RPMI82261 cells, which were grown in RPMI 1640 medium supplemented with 10% FBS. Transduced cells were expanded and selected for GFP expression. Sorted cells were expanded and subjected to killing and degranulation assays. This was performed for UL11 and E3.49K to generate the plasmids LeGO-iG2-UL11 (Figure 3), LeGO-iG2-E3.49k (Figure 4), and LeGO-iG2-a-CD45-sc (Figure 5). The sequences of the genes inserted into these plasmids are shown below.

[0062] UL11-codon optimized for human cell expression.

[0063] SEQ ID NO: 2 below is UL11 codon-optimized for human cells.

[0064] (SEQ ID NO: 2)

[0065] The human adenovirus D serotype 17 protein E3.49K was downloaded from uniprot: https: / / www.uniprot.org / uniprot / Q77N38. The E3.49K sequence is shown below as SEQ ID NO:3.

[0066] E3.49K >tr|Q77N38|Q77N38 9ADEN 48.9kDa OS=Human adenovirus D37 OX=52275 GN=E3 PE=4 SV=1 MNTVIRIVLLSLLVAFSQAGFHTINATWWANITLVGPPDTPVTWYDTQGLWFCNGSRVKNPQIRHTCNDQNLTLIHVNKTYERTYMGYNRQGTKKEDYKVVVIPPPPA TVKPQPEPEYVFVYMGENKTLEGPPGTPVTWFNQDGKKFCEGEKVLHPEFNHTCDKQNLILLFVNFTHDGAYLGYNHQGTQRTHYEVTVLDLFPDSGQMKIENHSEETE QKNDEHHNWQKQGGQKQGGQKTNQTKVNDRRKTAQKRPSKLKPATIEAMLVTVTAGSNLTLVGPKAEGKVTWFDGDLKRPCEPNYRLRHECNNQNLTLINVTKDYEGTYYGTNDKDEGKRYRVKVNTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTTVAIVVGVIAGFITLIIVFICYICCRKRPRAYNHMVDPLLSFSY (SEQ ID NO: 3)

[0067] E3.49K codon optimized for human cell expression

[0068] SEQ ID NO:4

[0069]

[0070] Example 2: Generation of a single chain that recognizes CD45 (a-CD45-sc)

[0071] Using the method described in Example 1, a single chain antibody recognizing CD45 was designed as shown below to obtain the plasmid LeGO-iG2-a-CD45-sc shown in Figure 5 and Figure 24. Lin Y, Pagel JM, Axworthy D, Pantelias A, Hedin N, Press OW. A genetically engineered anti-CD45 single-chain antibody-streptavidin fusion protein for pretargeted radioimmunotherapy of hematologic malignancies. Cancer Res. 2006;66(7):3884-92. In a preferred embodiment, the engager is actually present on the surface of the target cell, as shown in Figures 7-12.

[0072] a-CD45-sc (SEQ ID NO: 5) is a protein for an anti-CD45 antibody with the stalk and transmembrane domains joined through a linker region. SEQ ID NO: 6 is the DNA sequence of the same molecule. In the sequence below, the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane domain. myrmqllscialslalvtns qvqlvesggglvqpggslklscaasgfdfsrywmswvrqapgkglewigeinptsstinftpslkdkvfisrdnakntlylqmskvrsedtalyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGSGTKLEIK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 5)

[0073] First, a cDNA was generated using the IL-2 signal peptide, VH, linker, VL, and linker along with the single-chain (SC) stalk and CD34 transmembrane region. SEQ ID NO: 6 below encodes the plasmid LeGO-iG2-a-CD45-sc.

[0074] In the sequence below, the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane region.

[0075] atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt caggttcagctggtggaatcaggaggtggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcgatttcagtagatactggatgagttgggtccggcaggctccagggaaagggctagaatggattggagagattaatccaactagcagtac gataaactttacgccatctctaaaggataaagtcttcatctccagagacaacgccaaaaatacgctgtacctgcaaatgagcaaagtgagatccgaggacacagccctttattactgtgcaagagggaactactataggtacggagatgctatggactactggggtcaag gaacctcagtcaccgtgagcaagatc TCTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGCTCGGGTGGTGGTGGGTCGGGCGGCGGCGGCTCGAGCGACATCGTGCTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGACAGAGGGCCACCATCTCATGCAGGGCCAGCAAAGTGTCAGTACATCTGGCTATAGTTATCTGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCAT CCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG AGCTCTGGCTCTGGTTCG CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCGCAGGTGCACACGAGGGGGCTGGACTTC GCCCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTG TAA (SEQ ID NO: 6)

[0076] Generation of CD45 single chains Single chain antibodies are fusion proteins of a light and heavy chain joined by a linker. The CD45 single chain protein translation is shown below in SEQ ID NO: 7. The heavy chain is shown in lower case and the light chain in upper case. The linker is underlined and in upper case.

[0077] In the sequences below, the lower case letters are the heavy chain, the underlined upper case region is the linker, and the non-underlined upper case region is the light chain. qvqlvesggglvqpggslklscaasgfxfsrywmsxvrqapgkglewigeinptsstinxtpslkdkvfisrdnakntlylqmskvrsedtaxyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGSGGGGSGGGGSGGGGSS DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK SSGSGS (SEQ ID NO: 7)

[0078] The heavy chain is encoded by SEQ ID NO:8. QVQLVESGGGLVQPGGSLKLSCAASGFXFSRYWMSXVRQAPGKGLEWIGEINPTSSTINXTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAXYYCARGNYYRYGDAMDYWGQGTSVTVSKI (SEQ ID NO: 8)

[0079] The light chain is encoded by SEQ ID NO:9. DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK (SEQ ID NO: 9)

[0080] Stoke

[0081] The stalk is the structural domain between the single chain and the outer membrane of the cell. The stalk is sometimes referred to as a hinge or spacer. The stalk serves to position the antibody region at the desired location on the outside of the cell membrane. The stalk is preferably 8 to 200 amino acids in length. The stalk needs to protrude from the cell membrane surface, but should not be so long that it folds. This stalk is fused to a single-chain antibody, connecting it to the transmembrane domain. In this case, we used a CD8a / CD28 extracellular domain fusion construct for the stalk.

[0082] The CD8a / CD28 extracellular domain fusion construct including the stalk region is encoded by the 5'3' frame 1 SEQ ID NO:10. cccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgccc ctaggaaaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 10)

[0083] SEQ ID NO: 10 encodes the protein of SEQ ID NO: 11 below. PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 11)

[0084] The stalk contained the underlined Homo sapiens CD8a sequence, underlined in SEQ ID NO: 12 below, as part of its CD8a region. >sp|P01732|CD8A_Human T-cell surface glycoprotein CD8 alpha chain OS=Homo sapiens OX=9606 GN=CD8A PE=1 SV=1 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 12)

[0085] The underlined stalk region above is shown below as SEQ ID NO: 13: PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 13)

[0086] CD8a

[0087] The CD8a nucleotide sequence is shown below as SEQ ID NO: 14. The underlined region encodes the stalk. Nucleotide sequence (708nt): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTGCCAGGTGCTGCT GTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCCGAGGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGT TGGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAG CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC TGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCTAA (SEQ ID NO: 14)

[0088] The CD8a stalk is encoded by the polynucleotide of SEQ ID NO: 15, shown below: CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC (SEQ ID NO: 15)

[0089] CD8a translation (235aa): MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 16)

[0090] We used the underlined portion of SEQ ID NO: 16 as the stalk and the transmembrane shown in SEQ ID NO: 17. PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 17)

[0091] We used the underlined sequence from CD28 to further complete the stalk / hinge. The CD28 protein is encoded by SEQ ID NO: 18 below: >sp|P10747|CD28_Human T cell-specific surface glycoprotein CD28 OS=Homo sapiens OX=9606 GN=CD28 PE=1 SV=1 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 18)

[0092] The cDNA of CD28 nucleotide sequence is shown below as SEQ ID NO: 19: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGT TGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGC AAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 19)

[0093] The underlined portion of SEQ ID NO: 19 in the final construct is shown below as SEQ ID NO: 20, which serves as part of the stalk. KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20)

[0094] transmembrane region

[0095] The transmembrane region serves to anchor the stalk / protein to the cell. The transmembrane region is taken from CD34 FASTA and the protein sequence is shown below as SEQ ID NO: 21.

[0096] >sp|P28906|CD34_Human hematopoietic progenitor cell antigen CD34 OS=Homo sapiens OX=9606 GN=CD34 PE=1 SV=2 385AA MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT LIALVTSGALLAVLGITGYFL MNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL (SEQ ID NO: 21)

[0097] We used the following sequence taken from the underlined portion of SEQ ID NO: 20 as the transmembrane: LIALVTSGALLAVLGITGYFL (SEQ ID NO: 22).

[0098] The protein of SEQ ID NO:21 is encoded by the cDNA of SEQ ID NO:23 below. ATGCTGGTCCGCAGGGGCGCGCGCGCAGGGCCCAGGATGCCGCGGGGCTGGACCGCGCTTTGCTTGCTGAGTTTGCTGCCTTCTGGGTTCATGAGTCTTGACAACAACGGTACTGCTACCCCAGAGTTACCTACCCAGGGAACATTTTCAAATGTTTCTACAAATGTATCCTACCAAGAAACTACAACACCTAGTACCCTTGGAAGTACCAGCCTGCACCCTGTGTCTCAACATGGCAATGAGGCCACAACAAACATCACAGAAACGACAGTCAAATTCACATCTACCTCTGTGATAACCTCAGTTTATGGAAACACAAACTCTTCTGTCCAGTCACAGACCTCTGTAATCAGCACAGTG TTCACCACCCCAGCCAACGTTTCAACTCCAGAGACAACCTTGAAGCCTAGCCTGTCACCTGGAAATGTTTCAGACCTTTCAACCACTAGCACTAGCCTTGCAACATCTCCCACTAAACCCTATACATCATCTTCTCCTATCCTAAGTGACATCAAGGCAGAAATCAAATGTTCAGGCATCAGAGAAGTGAAATTGACTCAGGGCATCTGCCTGGAGCAAAATAAGACCTCCAGCTGTGCGGAGTTTAAGAAGGACAGGGGAGAGGGCCTGGCCCGAGTGCTGTGTGGGGAGGAGCAGGCTGATGCTGATGCTGGGGCCCAGGTATGCTCCCTGCTCCTTGCCCAGTCTGAGGTGAGGCCTCAGTGTCTACTGCTGGTCTTGGCCAACAGAACAGAAATTTCCAGCAAACTCCAACTTATGAAAAAGCACCAATCTGACCTGAAAAAGCTGGGGATCCTAGATTTCACTGAGCAAGATGTTGCAAGCCACCAGAGCTATTCCCAAAAG ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTGATGAATCGCCGCAGCTGGAGCCCCACAGGAGAAAGGCTGGGCGAAGACCCTTATTACACGGAAAACGGTGGAGGCCAGGGCTATAGCTCAGGACCTGGGACCTCCCCTGAGGCTCAGGGAAAGGCCAGTGTGAACCGAGGGGCTCAGGAAAACGGGACCGGCCAGGCCACCTCCAGAAACGGCCATTCAGCAAGACAACACGTGGTGGCTGATACCGAATTGTGA (SEQ ID NO: 23)

[0099] The cDNA for the transmembrane protein of SEQ ID NO:22 is taken from the underlined region of SEQ ID NO:23 and is shown below as SEQ ID NO:24.

[0100] ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTG (SEQ ID NO: 24)

[0101] Transmembrane regions from other proteins (membrane-bound) can also be used, such as the transmembrane domain of CD34. There appears to be no limitation on the use of transmembrane domains. Commonly used examples of other proteins with transmembrane domains include, but are not limited to, CD45, CD28, and CD8a, which are provided below.

[0102] CD45

[0103] The transmembrane region of CD45 is underlined in the protein of SEQ ID NO: 25 below. >sp|P08575|PTPRC_Human receptor tyrosine-protein phosphatase C OS=Homo sapiens OX=9606 GN=PTPRC PE=1 SV=3MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPA STFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSA GTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTATFPTDPVSPLTTTLSLAHHSAAL PARTSNTTITANTSDAYLNASETTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYL YNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVP PGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLE PEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFH NFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTT KSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFR VKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK ALIAFLAFLIIVTSIALLVVL YK IYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSI PRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPR KYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNNRNKCAEYWPSMEEGTRAFGDVV VKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLLKLRRRVNA FSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEA QYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQ HIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINA SFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGK QTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISM IQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQV VKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDAN CVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS (SEQ ID NO: 25)

[0104] CD45 transmembrane domain

[0105] >sp|P08575|578~598

[0106] ALIAFLAFLIIVTSIALLVVL (SEQ ID NO: 26)

[0107] CD45 DNA sequence ATGACCATGTATTTGTGGCTTAAACTCTTGGCATTTGGCTTTGCCTTTCTGGACACAGAAGTATTTGTGACAGGGCAAAGCCCAACACCTTCCCCCACTGGATTGACTACAGCAAAGATGCCCAGTGTTCCACTTTCAAGTGACCCCTTACCTACTCACACCACTGCATTCTCACCCGCAAGCACCTTTGAAAGAGAAAATGACTTCTCAGAGACCACAACTTCTCTTAGTCCAGACAATACTTCCACCCAAGTATCCCCGGACTCTTTGGATAATGCTAGTGCTTTTAATACCACAGGTGTTTCATCAGTACAGACGCCTCACCTTCCCACGCACGCAGACTCGCAGACGCCCTCTGCTGGAACTGACACGCAGACATTCAGCGGCTCCGCCGCCAATGCAAAACTCAACCCTACCCCAGGCAGCAATG GCACTGATAGCATTTCTGGCATTTCTGATTATTGTGACATCAATAGCCCTGCTTGTTGTTCTC TACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTTAGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGATTGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTATAAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATTATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCACAAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGCCACAGTTATTGTCATGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAATACTGGCCGTCAATGGAAGAGGGCACTCGGGCTTTTGGAGATGTTGTTGTAAAGATCAACCAGCACAAAAGATGTCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAAAAAGAAAAAGCAACTGGAAGAGAG

[0108] CD28

[0109] The transmembrane region of CD28 is underlined in the protein of SEQ ID NO: 28 below. >sp|P10747|CD28_Human T-cell specific surface glycoprotein CD28 OS=Homo sapiens OX=9606 GN=CD28 PE=1 SV=1 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 28)

[0110] CD28 transmembrane domain >sp|P10747|153~179 FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29)

[0111] CD28 DNA sequence ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTT CAGGTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTCTGGGTG AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 30)

[0112] The underlined region of SEQ ID NO:30 is the transmembrane domain that encodes SEQ ID NO:29.

[0113] CD8a

[0114] The protein sequence of CD8a is shown in SEQ ID NO: 31. The transmembrane region of CD8a is underlined. >sp|P01732|CD8A_Human T-cell surface glycoprotein CD8 alpha chain OS=Homo sapiens OX=9606 GN=CD8A PE=1 SV=1 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVIT LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 31)

[0115] CD8a transmembrane domain

[0116] >sp|P01732|183~203

[0117] IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 32)

[0118] CD8a DNA sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTGCCAGGTGCTGCTGTCCAACCCG ACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCGGCCGAGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCG GCCCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC CTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCTAA (SEQ ID NO: 33)

[0119] The underlined region of SEQ ID NO: 33 encodes the transmembrane domain of the protein.

[0120] signal peptide

[0121] We used the underlined signal peptide encoding sequence of IL-2 human in SEQ ID NO:34.

[0122] Nucleotide sequence (462nt): AGTTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAG TGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA (SEQ ID NO: 34)

[0123] The protein sequence of IL-2 is shown in SEQ ID NO: 35. The signal peptide is underlined. https: / / www.uniprot.org / uniprot / P60568 MYRMQLLSCIALSLALVTNS APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 35)

[0124] IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 36)

[0125] Similar to the IL-2 signal peptide, signal peptides of other proteins (secreted or membrane-bound) can also be used. Such proteins with signal peptides Examples include, but are not limited to, IFNg, as given below, and IL2Ra / CD25.

[0126] The protein sequence of IFNg is shown in SEQ ID NO: 37. The signal peptide is underlined. IFNg>sp|P01579|IFNG_Human interferon gamma OS=Homo sapiens OX=9606 GN=IFNG PE=1 SV=1 MKYTSYILAFQLCIVLGSLGCYC QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ (SEQ ID NO: 37)

[0127] IFN gamma signal peptide >sp|P01579|1~23 MKYTSYILAFQLCIVLGSLGCYC (SEQ ID NO: 38)

[0128] IFNg DNA sequence. The signal peptide nucleotide sequence is underlined in SEQ ID NO:39. ATGAAATATACAAGTTATATCTTGGCTTTTCAGCTCTGCATCGTTTTGGGTTCTCTTGGCTGTTACTGC CAGGACCCATATGTAAAAGAAGCAGAAAACCTTAAGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAATGGAACTCTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAGAAAAATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTTAAAAACTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCATCA AGGAAGACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAAACGAGATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTTGAATGTCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAACTGTCGCCAGCAGCTAAAACAGGGAAGCGAAAAAAGGAGTCAGATGCTGTTTCGAGGTCGAAGAGCATCCCAGTAA (SEQ ID NO: 39)

[0129] The protein sequence of IL2Ra / CD25 is shown in SEQ ID NO: 40. The signal peptide is underlined.

[0130] >sp|P01589|IL2RA_Human interleukin-2 receptor subunit alpha OS=Homo sapiens OX=9606 GN=IL2RA PE=1 SV=1 MDSYLLMWGLLTFIMVPGCQA ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 40)

[0131] CD25 signal peptide

[0132] >sp|P01589|1~21

[0133] MDSYLLMWGLLTFIMVPGCQA (SEQ ID NO: 41)

[0134] IL2Ra DNA sequence. The DNA encoding the IL2Ra signal peptide is underlined in SEQ ID NO:42. ATGGATTCATACCTGCTGATGTGGGGACTGCTCACGTTCATCATGGTGCCTGGCTCGCCAGGCA(SEQ ID NO: 42)

[0135] The a-CD45-sc translation is shown as SEQ ID NO:43.

[0136] In the sequences below, the bold lower case letters are the heavy chain, the underlined upper case region is the linker, and the non-underlined bold upper case region is the light chain.

[0137] CD45 V H V L qvqlvesggglvqpggslklscaasgfxfsrywmsxvrqapgkglewigeinptsstinxtpslkdkvfisrdnakntlylqmskvrsedtaxyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK SSGSGS (SEQ ID NO: 43)

[0138] Homo sapiens CD8a molecule (CD8A), transcript variant 4, non-coding RNA Sequence ID: NR_027353.1 Length: 2621 Matches: 1 Related Information Gene - Details of related genes UniGene-Clustered Expressed Sequence Tags GEO Profile - Microarray Expression Data PubChem Bioassays - Bioactivity Screening Genome Data Viewer - Context of Aligned Genomes Range 1:885~1015 GenBankGraphics

[0139] [Table 1]

[0140] Homo sapiens CD28 molecule (CD28), transcript variant 1, mRNA Sequence ID: NM_006139.4 Length: 4721 Matches: 1 Related Information Gene - Details of related genes PubChem Bioassays - Bioactivity Screening Genome Data Viewer - Context of Aligned Genomes Range 1:395-514 GenBankGraphics

[0141] [Table 2]

[0142] a-CD45-sc; additional engagers

[0143] We have generated an additional engager: anti-CD45 (9.4) single chain, which is derived from human HIB-10508 = 9.4 = IgG2a = mouse anti-human CD45.

[0144] The protein sequence is shown below in SEQ ID NO: 54. The underlined lowercase region is the IL2 signal peptide, the lowercase is the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is CD34.

[0145] myrmqllscialslalvtnsq vqlqqlgaelarpgasvkmsckasgytftsysiqwvkqrpgqglewigyinpssgyikynqhfrdratltadrssstaymqlssltsedsavyycargnsgsfdywgqgttltvssa SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVLTQAAPSVPVTPGESLSISCRSSKSLLHSSGITYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 54)

[0146] This results in the anti-CD45(9.4) single codon-optimized cDNA shown below as SEQ ID NO:55. ATGTACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCCAGGTGCAGCTGCAGCAGCTGGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAGCA

[0147] We generated a single-chain anti-CD45 (GAP8.3) antibody from the light and heavy chains of the GAP8.3 hybridoma (ATCC® HB-12™) (IgG2a, kappa = immunoglobulin; monoclonal antibody; directed against human leukocytes (monocytes, lymphocytes, granulocytes); directed against CD45).

[0148] In the sequence below (SEQ ID NO: 56), the underlined lowercase section is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane region. myrmqllscialslalvtns evqlqlqqsgpelvktgasvkisckasgysftgyfihwvkqshgkslewigyiscyngatsynqkfkgkatftvdtssstaymqfnsvtsedsavyycvrnyygnldamdywgqgtsvtvssa SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKILIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPRTFGGGTKLEIKRADAAQTCI SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 56)

[0149] The anti-CD45 (GAP8.3) single codon-optimized cDNA for the protein of SEQ ID NO:56 is shown below as SEQ ID NO:57.

[0150] Anti-CD45m (M1) single chain was generated from mouse M1 / 89.18.7.HK (ATCC® TIB-124™) = IgG2b. = rat anti-mouse CD45.

[0151] In the sequence below (SEQ ID NO: 58), the underlined lowercase section is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is CD34. myrmqllscialslalvtns qvqlkesgpglvkpsltlsltctvsgfslnsygviwvrqppgkglewlgvkwgygntnynsalksrlninrdtsksqvflkmdnvqtedtamyfcarsrfnyggpldywgqgvmvtvssa SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVLTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRADAAPTVS SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 58)

[0152] Anti-CD45(M1) codon-optimized for human cell expression.

[0153] Anti-CD45 (4B2) single chain 4B2 (ATCC® HB-196™) = mouse anti-human CD45.

[0154] In the sequence below (SEQ ID NO: 60), the underlined lowercase section is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is CD34. myrmqllscialslalvtns qvqlkesgaelarpgasvkmsckasgytftsytmqwvkqrpgqglewigyinpssgyikynqkfkdkvtltadkssttaymqlsrltsedsavyycarrgsyffdfwgqgtsvtvssa SGGGGSGGGGSGGGGSGGGGSGGGGSS DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPFTFGGGTKLEVKRADAAPTVS SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 60)

[0155] Anti-CD45 (4B2) codon-optimized for human cell expression

[0156] Arakawa F, Kuroki M, Kuwahara M, Senba T, Ozaki H, Matsuoka Y, Misumi Y, Kanda H, Watanabe T. Cloning and sequencing of the VH and V kappa genes of an anti-CD3 Anti-CD3 (OKT3) single chain antibody taken from J Biochem. 1996 Sep;120(3):657-62. doi: 10.1093 / oxfordjournals.jbchem.a021462. PMID: 8902633. In the sequence below (SEQ ID NO: 62), the underlined lowercase section is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is CD34.

[0157] myrmqllscialslalvtns qvqlqqsgaelarpgasvkm skkasgytftrytmhwvkqrpgqglewigyinpsrgytnynqkfkdkatlttdkssstaymqlssltsedsavyycaryyddhycldywgqgttltvssak SGGGGSGGGGSGGGGSGGGGSGGGGSS QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRS SGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 62)

[0158] The anti-CD3 (OKT3) single chain codon-optimized for human cell expression is shown below as SEQ ID NO:63.

[0159] The anti-CD148 single chain was taken from sequence AB1 of US Patent Application Publication No. 2005 / 0287,140(A1). In the sequence below (SEQ ID NO: 64), the underlined lowercase section is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the transmembrane region of CD34.

[0160] myrmqllscialslalvtns evqllesggglvqpggslrl scaasgftfssyamswvrqapgkglewvsaisgsggstyyadsvkgrftisrdnskntlylqmnslraedtavyycargrtevatpgaywgqgtmvtvssa SGGGGSGGGGSGGGGSGGGGSGGGGSS QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAVTGLQAEDEADYYCQSYDSSLSDVFGGGTKLTVLSGSGSGSTGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 64)

[0161] The anti-CD148 single chain codon-optimized for human cell expression is shown as a cDNA in SEQ ID NO:65 below.

[0162] E3.49K R1 mutant

[0163] The E3.49K R1 mutant was created through deletion of a portion of the extracellular region of E3.49k taken from Uniprot (Figures 15 and 22). In the sequence below (SEQ ID NO: 66), the underlined lowercase section is the E3.49K signal peptide, the lowercase letters are the R1 domain, the underlined uppercase region is the linker, and the non-underlined uppercase letters are the The region is the E3.49K extracellular membrane proximal region, the bold underlined region is the transmembrane region of E3.49K, followed by the bold capital letters the intracellular region of E3.49K.

[0164] mntvirivllsllvafsqagfhtinatwwanitlvg ppdtpvtwydtqglwfcngsrvknpqirhtcndqnltlihvnktyertymgynrqgtkkedykvvvi GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (SEQ ID NO: 66)

[0165] E3.49K R1

[0166] The E3.49K R1 codon-optimized for human cell expression is shown in the cDNA sequence SEQ ID NO:67 below.

[0167] In the sequence below (SEQ ID NO: 67), the underlined lowercase section is the E3.49K signal peptide, the lowercase letters are the R1 domain, the underlined uppercase region is the linker, the non-underlined uppercase region is the E3.49K extracellular membrane-proximal region, the bold underlined region is the transmembrane region of E3.49K, followed by the uppercase bold text is the intracellular region of E3.49K.

[0168] atgaacacggtgatccgcatagtccttctgtctctgctggtggctttctcccaggccggcttccacacaatt aatgccacctggtgggctaacattactctcgtaggccccccggatacccccgtgacttggtacgacactcagggtctgtggttctgtaacgggagtcgagtgaaaaatcctcaaattc gccatacctgtaacgaccaaaatctgaccttgatccacgtgaacaagacatacgagcgtacatatatgggctacaataggcagggtacaaagaaagaggactataaagtggtagtgatt GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA (SEQ ID NO: 67)

[0169] E3.49K-Ig-R3 mutant from E3.49k taken from Uniprot (Figure 16 and Figure 23).

[0170] In the sequence below (SEQ ID NO: 68), the underlined lowercase section is the E3.49K signal peptide, the underlined uppercase region is the linker, the lowercase letters are the R3 domain, the non-underlined uppercase region is the E3.49K extracellular membrane-proximal region, the bold underlined region is the E3.49K transmembrane region, followed by the bold uppercase letters is the E3.49K intracellular region. mntvirivllsllvafsqagfhtinatwwanitlvGGGGS vtvtagsnltlvgpkaegkvtwfdgdlkrpcepnyrlrhecnnqnltlinvtkdyegtyygtndkdegkryrvkvNTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VA IVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (SEQ ID NO: 68)

[0171] The cDNA of E3.49K-Ig-R3, codon-optimized for human cell expression, is set forth below in SEQ ID NO: 69. atgaacacggtgatccgcatagtccttctgtctctgctggtggctttctcccaggccggcttccacacaattaatgccacctggtgggctaacattactctcgtaGGCGGCGGCGGCAGC gtgacagtaactgctggaagtaacctgaccctcgtggggcccaaggcggaggggaaagtaacctggttcgacggcgatctaaaacgcccctgtgaaccaaactacagacttagacacgaatgcaacaaccagaacctgactctgattaacgtgaccaaggactacgaaggaacatac tacgggacgaatgataaggatgagggaaaacggtaccgggttaaggttAACACCACAAACTCCCAGAGTGTCAAAATTCAGCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA (SEQ ID NO: 69)

[0172] All three of the CD45 engagers developed above, E3.49K, UL11, and anti-CD45 single-chain (a-CD45-sc), bind to all isoforms of CD45, suggesting an interaction with the membrane-proximal region of CD45, including the fibronectin-III and cysteine-rich domains. Immunoprecipitation studies revealed a physical interaction between CD45 and E3.49K, UL11, or a-CD45-sc, and antibody competition experiments and deletion mutations further support the notion that E3.49K, UL11, and a-CD45-sc interact primarily with the membrane-proximal region of CD45 common to all isoforms. We will generate additional antibodies specific for different isoforms and epitopes of CD45, which will also be evaluated for CD43 and CD148.

[0173] Example 3: Generation of VHH-nanobodies

[0174] Nanobodies are single monomeric variable antibody domains that selectively bind to specific antigens, like antibodies. Nanobodies are much smaller (12-15 kDa) compared to typical antibodies (150-160 kDa). Nanobodies are commonly engineered from heavy chain antibodies found in camelids, also known as VHH fragments or single domains. The VHH fragment provided below is specific for mouse CD45. Codon optimization was performed using the CLC Main Workbench, as described above.

[0175] VHH generation method

[0176] Female camelids receive intramuscular and / or intradermal injections of purified (human) antigen every three weeks. Purified protein antigen in phosphate-buffered saline (PBS) / HEPES-buffered saline (HBS) is prepared and concentrated to ≥1 mg / mL. Approximately 3 mg of protein is used for the complete protocol, including immunization, panning, and confirmation of clones. Small test bleeds are performed in each animal 3-4 days after the third and fifth injections to obtain test serum. The presence of antigen-specific antibodies is confirmed by ELISA using serum obtained from test bleeds at pre-immunization, 3 weeks, and 5 weeks. A final bleed is taken while antibody titers are still increasing.

[0177] After immunization, peripheral blood lymphocytes are isolated by centrifugation on a discontinuous Ficoll gradient. Total RNA is extracted from peripheral blood lymphocytes, and first-strand cDNA is synthesized from total or polyA+ RNA using a cDNA synthesis kit. A bacteriophage library is generated from this cDNA. Single-domain antibodies are panned by adding the phage solution to antigen-coated plate wells. Specific phages (eluted) are added to TG1 phage display competent cells and grown at 37°C for 30 minutes. Serial dilutions of bacteria are plated and grown overnight at 37°C. Colonies from the plates are inoculated into 96-well plates and incubated overnight at 37°C without shaking. The next day, the plates are shaken at 170 rpm at 37°C for 1 hour. 2 μL of medium is used for PCR amplification to screen positive clones. Positive clones are grown in 10 ml of Luria Bertani broth (LB) and grown overnight at 37°C with shaking. Minipreps are performed and clones are sequenced. Repeatedly identified sequences are likely to be high-affinity binding sequences. These sequences can be used to generate engagers, and their affinity and avidity can be confirmed using pull-down assays and ELISA.

[0178] We generated a VHH-nanobody a-CD45-1 (mouse) cDNA codon-optimized for human cell expression (xenografted into mice and transduced into human cells; directed against mouse CD45). Rossotti M, Tabares S, Alfaya L, Leizagoyen C, Moron G, Gonzalez-Sapienza G. Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells. Biochim Biophys Acta. 2015;1850(7):1397-404. The cDNA is shown in Figure 13 as part of LeGO-iG2-a-CD45(M)-VHH-1 and is set forth below in SEQ ID NO: 70. ATGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCACCCCGGCGACAGCCTGAGACTGAGCTGCGCCGCCAGCGGCAGCGTGTTCAACAGCGCCACCATGGGCTGGTACAGACAGAGCCCCGGCAGCCAGAGAGAGCTGGTGGCCACCATCGTGGTGGGCACCCCCACCTACGCCG ACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACATCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCAACTACAGAGCCACCTACACCAGCGGCTACAGCAGAGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGC (SEQ ID NO: 70)

[0179] VHH-nanobody a-CD45-1 (mouse) protein sequence MAQVQLVESGGGLVHPGDSLRLSCAASGSVFNSATMGWYRQSPGSQRELVATIVVGTPTYADSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNYRATYTSGYSRDYWGQGTQVTVS (SEQ ID NO: 71)

[0180] Currently, we have two different VHH engagers against mouse CD45 that bind to different epitopes to be tested (5).

[0181] VHH-nanobody a-CD45-2 (mouse) codon-optimized for human cell expression (DNA sequence), shown in Figure 14 as LeGO-iG2-a-CD45(M)-VHH-2. ATGGCCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCAGAGCCTTCAACAGCGCCGCCATGGGCTGGTACAGACAGGCCCCCGGAGCCAGAGAGAGCTGGTGGCCAGCATCAGCGCCGGCACCGCCAGCTACGCCG ACGCCGTGAAGGGCAGATTCACCATCAGCAGAGACTACGCCAAGAACATCATCTACCTGCAGATGAACAGCCTGAAGCCCGACGACACCGCCGTGTACTTCTGCAACTACAGAACCACCTACACCAGCGGCTACAGCGAGGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGC (SEQ ID NO: 72)

[0182] VHH-nanobody a-CD45-2 (mouse) (amino acid sequence) MAQVQLVQSGGGLVQPGGSLRLSCAASGRAFNSAAMGWYRQAPGSQRELVASISAGTASYADAVKGRFTISRDYAKNIIYLQMNSLKPDDTAVYFCNYRTTYTSGYSEDYWGQGTQVTVS (SEQ ID NO: 73).

[0183] Example 4: Single-domain human nanobody sequences

[0184] We have generated engagers comprising human single domain / nanobody sequences using the methods disclosed above for Example 3. The generated protein and cDNA sequences are shown in SEQ ID NOs: 74-215. These were generated for us by Nanotag.

[0185] a-CD45-h-VHH-01

[0186] EVQLVESGGGLVQPGGSLRLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVLSAHHSEDPIS (SEQ ID NO: 74)

[0187] This protein sequence is encoded by the cDNA shown in SEQ ID NO:75 below.

[0188] GAGGTGCAGCTGGTGGAGTCTGGCGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGTTCCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCATTGATAGCAGCACAAATTATGCAGACTCCGTGAGGGGCC GATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 75)

[0189] a-CD45-h-VHH-02

[0190] EVQLLESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 76)

[0191] This protein sequence is encoded by the cDNA shown in SEQ ID NO:77 below.

[0192] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCTTCTGGACTGACTAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGCGACTCCGTGAAGGGCCGATT CACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 77)

[0193] a-CD45-h-VHH-03

[0194] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVAVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCAARVRGSTGDFGSWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 78)

[0195] This protein sequence is encoded by the cDNA shown in SEQ ID NO:79 below.

[0196] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGA GGGGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAGCGAGGGTACGCGGCAGCACAGGGGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 79).

[0197] a-CD45-h-VHH-04

[0198] EVQLVESGGGLVETGGSLRLSCAGSGRTFSSRHVGWFRQTPGKEREFVASIRWSGGHTYYADSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 80)

[0199] This protein sequence is encoded by the cDNA shown in SEQ ID NO:81 below.

[0200] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTCGAAACTGGGGGTTCTCTGAGACTCTCCTGTGCAGGTTCTGGACGCACCTTCAGTAGCCGGCACGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGAGTTTGTAGCATCCATTAGGTGGAGTGGCGGTCACACATACTATGCAGACTCCGTGAAGGGCCG ATTCACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 81)

[0201] a-CD45-h-VHH-05

[0202] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVAVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 82)

[0203] This protein sequence is encoded by the cDNA shown in SEQ ID NO:83 below.

[0204] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAG GGGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 83)

[0205] a-CD45-h-VHH-06

[0206] EVQLQESGGGLVQPGGSLRLSCVASGFTFSIYAMSWVRQAPGKGPERVAVIGSVGGATGVTSYADSVKDRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 84)

[0207] This protein sequence is encoded by the cDNA shown in SEQ ID NO:85 below.

[0208] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTATCTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGTCACAAGTTATGCAGACTCCGTGAA GGACCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 85)

[0209] a-CD45-h-VHH-07

[0210] EVQLVESGGGLVQAGGSLKLSCAASGRTLTYYTAWFRQAPGKEREFVASLGWSGDVTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCNVMQAWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 86)

[0211] This protein sequence is encoded by the cDNA shown in SEQ ID NO:87 below.

[0212] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAAACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTATTATACTGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCGCTAGGGTGGAGTGGCGATGTCACATACTATGCAGACT CCGTGAAGGGCCGATTCACCATCTCCGGCGACAACGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGTAATGTCATGCAGGCTTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 87)

[0213] a-CD45-h-VHH-08

[0214] EVQLLESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 88)

[0215] This protein sequence is encoded by the cDNA shown in SEQ ID NO:89 below.

[0216] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCTTCTGGACTGACCAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGGGACTCCGTGAAGGGCCGATT CACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 89)

[0217] a-CD45-h-VHH-09

[0218] EVQLLESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGHTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREDLYDVWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 90)

[0219] This protein sequence is encoded by the cDNA shown in SEQ ID NO:91 below.

[0220] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGG CGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCCATTAGGTGGAGTGGCGGTCACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCA AGAACACGGTGTATCTACAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGACCTCTATGATGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 91)

[0221] a-CD45-h-VHH-10

[0222] EVQLQESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPIS (SEQ ID NO: 92)

[0223] This protein sequence is encoded by the cDNA shown in SEQ ID NO:93 below.

[0224] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCAC CATCACCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 93)

[0225] a-CD45-h-VHH-11

[0226] EVQLEESGGGLVQAGDSLRLSCACSERAYRNRLLGWFRQAPGKEREFVANIRPIDSASDYAGSVKGRFTISRDIAKRTVYLQMNSLKPEDTAVYYCASTYMFDSVREDEYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 94)

[0227] This protein sequence is encoded by the cDNA shown in SEQ ID NO:95 below.

[0228] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCTTGCTCTGAACGCGCCTATAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAATTTGTAGCAAATATCAGACCCATTGATAGCGCTCCGATTAT GCAGGCTCCGTGAAGGGCCGATTCACCATCTCTAGAGACATCGCCAAGAGAACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGTCCGGGAGGATGAATATGACTACTGGGGCCAGGGAACCCAGGTCACC GTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 95)

[0229] a-CD45-h-VHH-12

[0230] EVQLVESGGGLVQAGGSLRLSCVVSGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 96)

[0231] This protein sequence is encoded by the cDNA shown in SEQ ID NO:97 below.

[0232] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCGTGTGTAGTCTCTGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCG ATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTTCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGGCCAGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 97)

[0233] a-CD45-h-VHH-13

[0234] EVQLLESGGGLVQAGGSLRLSCVASGRGFSRYDMGWFRQASGKEREFVAAISWSNSTTAYADSVKGRFAISRDNNKNMVYLQMNSLKPEDTAVYYCAARVRGSTGDFGSWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 98)

[0235] This protein sequence is encoded by the cDNA shown in SEQ ID NO:99 below.

[0236] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGGGGCTTCAGTAGGTATGACATGGGCTGGTTCCGCCAGGCTTCAGGGAAGGAGCGTGAGTTTGTAGCAGCAATTAGCTGGAGTAATAGTACCACGGCCTATGCAGACTCCGTGAAGGG CCGATTCGCCATCTCAAGAGACAACAACAAGAATATGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCGTGTATTACTGTGCAGCGAGGGTACGCGGCAGCACAGGGGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 99)

[0237] a-CD45-h-VHH-14

[0238] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYNYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 100)

[0239] This protein sequence is encoded by the cDNA shown in SEQ ID NO:101 below.

[0240] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCGGTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTGGCACGAATTACTCTGATTGGCCACGGCACATACTATGCAGATGCCTTGAAGGGCCGATTCACCATT TCCAGAGACCACGCTAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTATATTACTGTGTAGCGCGAGACAGCCCGTGCGTGGTAATTGTTGGTACGAGAATGCGGGCGACTATAATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 101)

[0241] a-CD45-h-VHH-15

[0242] EVQLLESGGGLVQAGGSLRLSCVSSGDSISGVVVRWYRQVPGKQREWIGGIGTSDNPEYADSVWGRFVLSRDNAGSRVNLQMNNLKLEDTATYYCNAVHKWGPGTQVTVSSEPKTPKPQ (SEQ ID NO: 102)

[0243] This protein sequence is encoded by the cDNA shown in SEQ ID NO:103 below.

[0244] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCCTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAAGTTCTGGAGACAGTATCAGTGGAGTGGTCGTCCGTTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTGGATCGGAGGTATTGGTACTAGTGATAACCCAGAATATGCGGACT CCGTCTGGGGCCGATTCGTCCTCTCCAGAGACAATGCCGGGAGCCGCGTAAATCTGCAAATGAACAACCTGAAACTTGAGGACACGGCCACCTATTACTGCAATGCAGTGCACAAATGGGGCCCGGGTACCCAGGTCACCGTCTCTTCTGAACCCAAGACACCAAAACCACAAAC (SEQ ID NO: 103)

[0245] a-CD45-h-VHH-16

[0246] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAVMSWVRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSEPKTPKPQT (SEQ ID NO: 104)

[0247] This protein sequence is encoded by the cDNA shown in SEQ ID NO:105 below.

[0248] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCGTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGA GTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCACCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 105)

[0249] a-CD45-h-VHH-17

[0250] EVQLEESGGGLVQAGGSLRLSCAASGRTFSSYRLGWFRQAPGKEREFVAGWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 106)

[0251] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:107.

[0252] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATCGACTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGCTGGAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTCAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 107)

[0253] a-CD45-h-VHH-18

[0254] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 108)

[0255] This protein sequence is encoded by the cDNA shown in SEQ ID NO:109 below.

[0256] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCTTCTGGACTGACCAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 109)

[0257] a-CD45-h-VHH-19

[0258] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 110)

[0259] This protein sequence is encoded by the cDNA shown in SEQ ID NO:111 below.

[0260] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACAGCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 111)

[0261] a-CD45-h-VHH-20

[0262] EVQLEESGGGLVQAGDSLRLSCVVSGSISSIYAMGWVREDPGKERVVVAGINSGAIRWYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 112)

[0263] This protein sequence is encoded by the cDNA shown in SEQ ID NO:113 below.

[0264] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTAGTCTCTGGAAGCATCTCCAGTATCTATGCCATGGGATGGGTCCGCGAGGATCCAGGGAAGGAGCGCGTAGTGGTTGCAGGTATTAATAGCGGAGCTATCAGATGGTACGCAGACTCTGTGAAGGGCCGATTCACC ATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 113)

[0265] a-CD45-h-VHH-21

[0266] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQV TVSAEPKTPKPQT (SEQ ID NO: 114)

[0267] This protein sequence is encoded by the cDNA shown in SEQ ID NO:115 below.

[0268] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCGCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 115)

[0269] a-CD45-h-VHH-22

[0270] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNAVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 116)

[0271] This protein sequence is encoded by the cDNA shown in SEQ ID NO:117 below.

[0272] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 117)

[0273] a-CD45-h-VHH-23

[0274] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 118)

[0275] This protein sequence is encoded by the cDNA shown in SEQ ID NO:119 below.

[0276] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGC CTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGGGCCGATTCACCATCTCCAG AGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTCAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 119)

[0277] a-CD45-h-VHH-24

[0278] EVQLEESGGGLVETGDSLRLSCSASGGGFSFNAIGWYRQGPGKGRELVAAGTSGSTTYYAPSVKGRFIFSRDSAKNTVYLQMNNLNPEDTAIYYCATPALGQMEYDVVSGDGLAHWGKGTLVIVSSAHHSEDPNS (SEQ ID NO: 120)

[0279] This protein sequence is encoded by the cDNA shown in SEQ ID NO:121 below.

[0280] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCCTGGTGGAGACTGGGGATTCTCTGAGACTCTCCTGCTCTGCCTCTGGGGGCGGTTTTAGTTTCAATGCCATAGGCTGGTACCGGCAGGGGCCGGGAAAGGGGCGCGAATTGGTCGCAGCAGGTACTAGTGGAAGTACCACATATTACGCGCCCTCTGTGAAGGGCCGATTCATCTTC TCCAGAGACAGTGCCAAAAACACCGTCTATCTGCAAATGAACAACCTGAACCCTGAAGACACGGCCATCTATTACTGTGCCACACCGGCACTTGGACAAATGGAGTATGACGTAGTGAGCGGCGACGGCTTGGCCCACTGGGGCAAAGGGACCCTGGTCATCGTCTCTTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 121)

[0281] a-CD45-h-VHH-25

[0282] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 122)

[0283] This protein sequence is encoded by the cDNA shown in SEQ ID NO:123 below.

[0284] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCG GGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGGGGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACA CGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGGAGTGACTACTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 123)

[0285] a-CD45-h-VHH-26

[0286] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNHVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 124)

[0287] This protein sequence is encoded by the cDNA shown in SEQ ID NO:125 below.

[0288] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCACGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 125)

[0289] a-CD45-h-VHH-27

[0290] EVQLVESGGGLVQPGGSLRLSCATSGLTNPERRLAWFRQEPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYYCAARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 126)

[0291] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:127.

[0292] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAACTTCTGGACTGACCAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGAACCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGGGACTCCGTGAAGGGCCGATTCACCA TCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCGCGAGACAGCCCGTGCGTGGTAATTGTTGGTACGAGAATGCGGGCGACTATGAGTACTGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 127)

[0293] a-CD45-h-VHH-28

[0294] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYNYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 128)

[0295] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:129.

[0296] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCGGTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTGGCACGAATTACTCTGATTGGCCACGGCACATACTATGCAGATGCCTTGAAGGGCCGATTCACCATT TCCAGAGACCACGCTAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTATATTACTGTGTAGCGCGAGACAGCCCGTGCGTGGTAATTGTTGGTACGAGAATGCGGGCGACTATAATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 129)

[0297] a-CD45-h-VHH-29

[0298] EVQLVESGGGLVQAGDSLTLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 130)

[0299] This protein sequence is encoded by the cDNA shown in SEQ ID NO:131 below.

[0300] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCGCTGACACTCTCCTGTGCAGCCTCTGAACGCGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGTTCCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCATTGATAGCAGCACAAATTATGCAGACTCCGTGAAGGGCC GATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 131)

[0301] a-CD45-h-VHH-30

[0302] EVQLEESGGGSVQAGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 132)

[0303] This protein sequence is encoded by the cDNA shown in SEQ ID NO: 133 below. .

[0304] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATCGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTCCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTACAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 133)

[0305] a-CD45-h-VHH-31

[0306] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 134)

[0307] This protein sequence is encoded by the cDNA shown in SEQ ID NO:135 below.

[0308] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACAGCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 135)

[0309] a-CD45-h-VHH-32

[0310] EVQLLESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS (SEQ ID NO: 136)

[0311] This protein sequence is encoded by the cDNA shown in SEQ ID NO:137 below.

[0312] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCTTCTATTAGGTGGAGTG GCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGACTTAGATCTTGGACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 137)

[0313] a-CD45-h-VHH-33

[0314] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNAKNMVYLQMDNIKPEDTARYFCASSYTFSSVREDDYDYWGQGTQVTVLSAHHSEDPIS (SEQ ID NO: 138)

[0315] This protein sequence is encoded by the cDNA shown in SEQ ID NO:139 below.

[0316] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCTTCTGGACTGACCAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGAGACTCCGTGAAGGGCCGA TTTACCATCTCTCGAGATAACGCCAAGAACATGGTGTACCTGCAAATGGACAACATAAAACCTGAAGACACGGCCCGTTATTTCTGTGCGTCCTCATACACCTTCAGCAGTGTCCGGGAGGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 139)

[0317] a-CD45-h-VHH-34

[0318] EVQLVESGGGLVQAGGSLRLSCAASGRTVSRYDMGWFRQAPGAERVVVAISWSGGSTYYVDSVKGRFTMSRDNSKNTVYLQMNSLKPEDTAVYYCAVRTERSSLDFHSWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 140)

[0319] This protein sequence is encoded by the cDNA shown in SEQ ID NO:141 below.

[0320] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGTCAGTAGATATGACATGGGCTGGTTCCGCCAGGCTCCAGGGGCGGAGCGTGTCGTTGTAGCTATTAGCTGGAGCGGTGGTAGTACATACTATGTAGACTCCGTGAAGGGCC GATTCACCATGTCCAGAGACAACAGCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCAGAACCGAACGCTCCAGTCTTGACTTTCATTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 141)

[0321] a-CD45-h-VHH-35

[0322] EVQLEESGGGLVQAGDSLRLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVKGRFTISRDNDKNTVYLQMDNMKPEDTALYYCASTYYYSSIREDDYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 142)

[0323] This protein sequence is encoded by the cDNA shown in SEQ ID NO:143 below.

[0324] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGTTCCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCATTGATAGCAGCACAAATTATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCTAGAGATAACGACAAGAACACGGTGTATTTGCAAATGGACAATATGAAACCTGAGGACACGGCCCTCTATTATTGTGCGTCCACATACTACTACAGTAGTATCCGGGAGGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 143)

[0325] a-CD45-h-VHH-36

[0326] EVQLVESGGGLVQAGGSLRLSCAASGRAFSNRALGWFRQAPGKEREFVAWIRGIGSSTNYAGSVQGRFTISRDNAKNTLYLQMDKLKPEDTAVYYCASTYMFDSVREDEYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 144)

[0327] This protein sequence is encoded by the cDNA shown in SEQ ID NO:145 below.

[0328] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCGCCTTCAGTAACCGTGCACTTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTGGATTAGAGGCATCGGTAGCAGCACAAATTATGCAGGCTCCGTACAGGGCCGAT TCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAGATGGACAAGCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGCGGGAGGATGAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 145)

[0329] a-CD45-h-VHH-37

[0330] EVQLQESGGGLLQTGDSLRLACEASEIVVENYVMAWFRQAPGKEREWLARIIWNTGGTHLQEFVKGRLTISRDIAKKTVYLQMNSLKPEDTAVYYCAGGSFDAIADPFSARRYGFWGQGT QVTVSSEPKTPKPQT (SEQ ID NO: 146)

[0331] This protein sequence is encoded by the cDNA shown in SEQ ID NO:147 below.

[0332] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGCTGCAGACTGGGGACTCACTGAGACTCGCTGTGAAGCCTCTGAAATCGTCGTCGAAAATTATGTCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTGGCTAGCGCGTATTATTTGGAATACCGGTGGCACACATCTTCAAGAATTTGTGAAGGGCCGACTCACC ATCTCTAGAGACATCGCCAAGAAAACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCCGGTGGAAGTTTTGACGCTATAGCCGATCCCTTCGGCCCGCCGGTATGGGTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 147)

[0333] a-CD45-h-VHH-38

[0334] EVQLQESGGGLVQAGGSLRLSCVSSGDSISGVVVRWYRQVPGKQREWIGGIGTSDNPEYADSVWGRFVLSRDNAGSRVNLQMNNLKLEDTATYYCNAVHKWGPGTQVTVSSEPKTPKPQT (SEQ ID NO: 148)

[0335] This protein sequence is encoded by the cDNA shown in SEQ ID NO:149 below.

[0336] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCCTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAAGTTCTGGAGACAGTATCAGTGGAGTGGTCGTCCGTTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTGGATCGGAGGTATTGGTACTAGTGATAACCCAGAATATGCGGACT CCGTCTGGGGCCGATTCGTCCTCTCCAGAGACAATGCCGGGAGCCGCGTAAATCTGCAAATGAACAACCTGAAACTTGAGGACACGGCCACCTATTACTGCAATGCAGTGCACAAATGGGGCCCGGGTACCCAGGTCACCGTCTCTTCTGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 149)

[0337] a-CD45-h-VHH-39

[0338] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKEREFVAWIRGIGGSTHYAGSVEGRFTISRDSAKNTLYLQMDNVKPEDTAVYYCASTYMFDSVREDEYDYWGQGTEVTVSSAHHSEDPNS (SEQ ID NO: 150)

[0339] This protein sequence is encoded by the cDNA shown in SEQ ID NO:151 below.

[0340] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTGGATTAGAGGCATCGGTGGCAGCACACATTATGCAGGCTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGTTGTATCT ACAGATGGACAACGTGAAACCGGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGTCCGGGAGGATGAATATGACTACTGGGGCCAGGGGACCGAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 151)

[0341] a-CD45-h-VHH-40

[0342] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDSAKNTLYLQMDNVKPEDTAVYYCASTYMFDSVREDEYDYWGQGTEVTVSSEPKTPKPQT (SEQ ID NO: 152)

[0343] This protein sequence is encoded by the cDNA shown in SEQ ID NO:153 below.

[0344] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCACGTCATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGGGGC CGATTCACCATCTCCAGAGACAGCGCCAAGAACACGTTGTATCTACAGATGGACAACGTGAAACCCGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGTCCGGGAGGATGAATATGACTACTGGGGCCAGGGGACCGAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 153)

[0345] a-CD45-h-VHH-41

[0346] EVQLEESGGGLVQTGGSLRLSCAASGGTFSSYVMGWFRQAPGKEREFVAWIRPIDSSTNYADSVKGRFTISRDDAKNSLYLQMDNMKPEDTALYYCASTYYYSSIREDDYDYWGRGTQVTVLSAHHSEDPNS (SEQ ID NO: 154)

[0347] This protein sequence is encoded by the cDNA shown in SEQ ID NO:155 below.

[0348] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGACCGGGGGATCTTTGAGACTCTCCTGTGCAGCCTCTGGCGGCACCTTCAGTAGCTATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCA TTGATAGCAGCACAAATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGGGATGACGCCAAGAACTCGCTGTATCTGCAAATGGACAATATGAAACCTGAGGACACGGCCCTCTATTATTGTGCGTCCACATACTACTACAGTAGTATCCGGGA GGATGACTATGACTACTGGGCCGGGGGGACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 155)

[0349] a-CD45-h-VHH-42

[0350] EVQLVESGGGLVQPGGSLRLSCATSGFTFSNNVMSWVRQAPGKGPERVAVIGSVGGTTGATSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 156)

[0351] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:157.

[0352] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTCAGTAACAACGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTACCACGGGTGCCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 157)

[0353] a-CD45-h-VHH-43

[0354] EVQLVESGGGLVQARGSLRLSCVASGRTLTYYTGWFRQAPGKEREFVASFAWSGGNTYYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 158)

[0355] This protein sequence is encoded by the cDNA shown in SEQ ID NO:159 below.

[0356] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGAGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCCGGCCGCACCCTCACTTACTATACTGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCATCTTTTGCGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 159)

[0357] a-CD45-h-VHH-44

[0358] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQA PGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 160)

[0359] This protein sequence is encoded by the cDNA shown in SEQ ID NO:161 below.

[0360] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTCAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 161)

[0361] a-CD45-h-VHH-45

[0362] EVQLEESGGGLVQAGDSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 162)

[0363] This protein sequence is encoded by the cDNA shown in SEQ ID NO:163 below.

[0364] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTACCACAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGTCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 163)

[0365] a-CD45-h-VHH-46

[0366] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 164)

[0367] This protein sequence is encoded by the cDNA shown in SEQ ID NO:165 below.

[0368] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACAGCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 165)

[0369] a-CD45-h-VHH-47

[0370] EVQLLESGGGLVQAGDSLRLSCTQSGRTFSRYAIGWFRQAPGKEREFVASIRWSGGHTYYADSVKGRFTISKDNAKDTVYLQMNSLKPEDTAVYYCAGGSFDAIADPFSARRYGFWGQGTQVTVSSAHHSEDPIS (SEQ ID NO: 166)

[0371] This protein sequence is encoded by the cDNA shown in SEQ ID NO:167 below.

[0372] GAGGTGCAGCTGCTGGAGTCTGGGGGGGGATTGGTGCAGGCAGGGGACTCTCTGAGACTCTCCTGTACACAATCTGGACGCACCTTCAGCAGATATGCCATAGGCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCCATTAGGTGGAGTGGCGGTCACACATACTATGCAGACTCCGTGAAGGGTCGCTTCACC ATTTCCAAGGACAACGCCAAAGACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGGTGGAAGTTTTGACGCTATAGCCGATCCCTTCGGCCCGCCGGTATGGATTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 167)

[0373] a-CD45-h-VHH-48

[0374] EVQLEESGGGLVQAGGSLRLSCAASGRTLTYYTGWFRQAPGKEREFVASFAWMGDNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCAALRFWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS (SEQ ID NO: 168)

[0375] This protein sequence is encoded by the cDNA shown in SEQ ID NO:169 below.

[0376] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTATTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTTTTGCGTGGATGGGTGATAACACATACTACGCTGACTCCGTGAAGGGCCGGTT CACCATCTCCGGCGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCATTAAGATTTTGGACTACTACACCGCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 169)

[0377] a-CD45-h-VHH-49

[0378] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT (SEQ ID NO: 170)

[0379] This protein sequence is encoded by the cDNA shown in SEQ ID NO:171 below.

[0380] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCTTCTGGACTGACCAACCCTGAAAGACGCTTGGCCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACACACTATGGAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 171)

[0381] a-CD45-h-VHH-50

[0382] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 172)

[0383] This protein sequence is encoded by the cDNA shown in SEQ ID NO:173 below.

[0384] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 173)

[0385] a-CD45-h-VHH-51

[0386] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 174)

[0387] This protein sequence is encoded by the cDNA shown in SEQ ID NO:175 below.

[0388] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 175)

[0389] a-CD45-h-VHH-52

[0390] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 176)

[0391] The protein sequence is encoded by the cDNA shown below in SEQ ID NO:177.

[0392] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTACCACAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 177)

[0393] a-CD45-h-VHH-53

[0394] EVQLVESGGGLVQAGGSLRLACTASGSDFKRAALGWYRQAPGQERELVAAFNSGGKTYYTDSVKDRFTISRDNAKSTLYLQMNSLKPDDTAMYYCALSRFDYYLPPTQFDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 178)

[0395] This protein sequence is encoded by the cDNA shown in SEQ ID NO:179 below.

[0396] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCGCCTGTACAGCCTCTGGAAGCGACTTCAAGCGCGCCGCCCTGGGCTGGTACCGCCAGGCTCCAGGACAGGAGCGCGAGTTGGTCGCAGCTTTTAATAGTGGAGGTAAAACATACTACACAGATTCTGTGAAGGACCGATTC ACCATCTCCAGAGACAATGCCAAGAGTACGCTGTATCTCCAAATGAACAGCCTGAAACCTGACGACACGGCCATGTATTACTGTGCGTTATCACGGTTCGATTACTATCTTCCACCCACCCAATTTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 179)

[0397] a-CD45-h-VHH-54

[0398] EVQLVESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTDYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 180)

[0399] This protein sequence is encoded by the cDNA shown in SEQ ID NO:181 below.

[0400] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGAGTGGCGGTAACACAGACTATGCAGACTCCGTGAAGGGCCGATTCACC ATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCGGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACG (SEQ ID NO: 181)

[0401] a-CD45-h-VHH-55

[0402] EVQLVESGGGLVQAGGSLKLSCAASGRTLTYYTAWFRQAPGKEREFVASLGWSGDVTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPNS (SEQ ID NO: 182)

[0403] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:183.

[0404] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAAAACTCTCCTGTGCAGCCTCCGGACG CACCCTCACTTATTATACTGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCGCTAGGGTGGAGTGGCGATGTCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGCGACAACGCCAAGAACACGGTATATCTGCAAA TGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACCGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 183)

[0405] a-CD45-h-VHH-56

[0406] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 184)

[0407] This protein sequence is encoded by the cDNA shown in SEQ ID NO:185 below.

[0408] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 185)

[0409] a-CD45-h-VHH-57

[0410] EVQLVESGGGLVQAGDSLKLSCVGSGRTFSSYGLGWFRQAPGKEREFLAHITWTAGGTYHADNVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAARSSGDWRVERYYDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 186)

[0411] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:187.

[0412] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAAACTCTCCTGTGTAGGCTCTGGACGCACCTTCAGCAGCTATGGTTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTAGCACATATTACCTGGA CTGCTGGTGGAACATACCATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAATACGGTGTATCTACAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGCACGTTCCTCTGGGGATTGGCGTGTCGA GAGATATTATGACTACTGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAACCACAAACT (SEQ ID NO: 187)

[0413] a-CD45-h-VHH-58

[0414] EVQLEESGGGLVQPGGSLRLSCATSGFTFSNNVMSWVRQAPGKGPERVAVIGSVGGATGATSYADSVKGRFTITRDNARSTLHLQMNGLKPEDTAMYYCAAETSSGLYYSYDDLQTIDFDSWGQGTQVTVSSAHHSEDPNS (SEQ ID NO: 188)

[0415] This protein sequence is encoded by the cDNA shown in SEQ ID NO:189 below.

[0416] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTCAGTAACAACGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAAGGGCCGATTCACC ATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACGGCCTGAAACCGGAGGACACGGCAATGTATTACTGTGCGGCGGAGACCAGTAGCGGTCTTTACTACAGTTACGATGACCTTCAAACAATTGACTTTGATTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 189)

[0417] a-CD45-h-VHH-59

[0418] EVQLVESGGGLVQAGGSLRLSCAASERAFKNRALGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 190)

[0419] This protein sequence is encoded by the cDNA shown in SEQ ID NO:191 below.

[0420] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTTCAAGAACCGTGCACTTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCA CCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 191)

[0421] a-CD45-h-VHH-60

[0422] EVQLVESGGGLVQAGGSLRLSCAASEFTFSGYWMHWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVRGRFTVSRDDAKNTVYLHMDSLKAEDTAVYYCNVMQAWGQGTQVTVLSAHHSEDPIS (SEQ ID NO: 192)

[0423] This protein sequence is encoded by the cDNA shown in SEQ ID NO:193 below.

[0424] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAATTCACCTTCAGTGGCTACTGGATGCACTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATG CAGACTCCGTGAGGGGCCGATTCACTGTCTCCAGAGACGACGCCAAGAACACGGTGTATCTGCATATGGATAGTTTGAAAGCTGAGGACACGGCCGTGTATTACTGTAATGTCATGCAGGCTTGGGGCCAGGGCACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 193)

[0425] a-CD45-h-VHH-61

[0426] EVQLVESGGGLVETGGSLRLSCAGSGRTFSSRHVGWFRQTPGKEREWVGSVAWNTGSEYYADSVKGRFTISKDNAKDTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS (SEQ ID NO: 194)

[0427] This protein sequence is encoded by the cDNA shown in SEQ ID NO:195 below.

[0428] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTCGAAACTGGGGGTTCTCTGAGACTCTCCTGTGCAGGTTCTGGACGCACCTTCAGTAGCCGGCACGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGAGTGGGTTGGAAGTGTTGCCTGGAACACTGGTAGTGAATATTATGCAGACTCCGTGAAGGGTCGCTTCA CCATTTCCAAGGACAACGCCAAAGACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCGGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 195)

[0429] a-CD45-h-VHH-62

[0430] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGVGGVTSYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 196)

[0431] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:197.

[0432] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATTGGCAGTGTGGGAGGTGTCGGAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 197)

[0433] a-CD45-h-VHH-63

[0434] EVQLQESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVLSAHHSEDPNS (SEQ ID NO: 198)

[0435] This protein sequence is encoded by the cDNA shown in SEQ ID NO:199 below.

[0436] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 199)

[0437] a-CD45-h-VHH-64

[0438] EVQLVESGGGLVQAGGSLRLSCVASGEEDFQPYAMGWFRQAPGKEREYVAATTWNGGRIRYGDSVKGRFTISRDHPKNTITLQMTSLKPDDTAVYYCAARYGTVLLTREDYQHWGRGTQVTVSAAHHSEDPIS (SEQ ID NO: 200)

[0439] This protein sequence is encoded by the cDNA shown in SEQ ID NO:201 below.

[0440] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGCGTAGCCTCTGGAGAGGAGGATTTTCAGCCGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATACGTGGCCGCGACTACATGGAATGGTGGTAGAATAAGATATGGAGACTCCGTGAAGGG CCGATTCACCATCTCCAGAGACCACCCCAAGAACACGATCACTTTACAAATGACCAGTTTGAAACCTGACGACACGGCCGTTTATTACTGTGCAGCACGGTACGGTACAGTCCTACTTACACGCGAAGACTATCAACACTGGGGCCGTGGGACCCAGGTCACCGTTTCCGCGGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 201)

[0441] a-CD45-h-VHH-65

[0442] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 202)

[0443] This protein sequence is encoded by the cDNA shown in SEQ ID NO:203 below.

[0444] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGTCTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACCGGTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTGGCACGAATTACTCTGATTGGCCACGGCACATACTATGCAGATGCCTTGAAGGGCCGATTCACCATT TCCAGAGACCACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTATATTACTGTGTAGCGCGAGACAGCCCGTGCGTGGTAATTGTTGGTACGAGAATGCGGGCGACTATGAGTACTGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 203)

[0445] a-CD45-h-VHH-66

[0446] EVQLLESGGGLVQAGGSLRLSCAASGFTFSNYAMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSAHHSEDPIS (SEQ ID NO: 204)

[0447] This protein sequence is encoded by the cDNA shown in SEQ ID NO:205 below.

[0448] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAG GGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTTCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 205)

[0449] a-CD45-h-VHH-67

[0450] EVQLVESGGGLVQAGGSLRLSCAASERTVSVYTMGWFRQAPGKEREFVASIRWSGGPNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 206)

[0451] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:207.

[0452] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCACCGTCAGTGTCTATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTCCATTCGCTGGAGTGGTGGTCCCAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCA TCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTATATTACTGTGTAGCGCGAGACAGCCCGTGCGTGGTAATTGTTGGTACGAGAATGCGGGCGACTATGAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 207)

[0453] a-CD45-h-VHH-68

[0454] EVQLVESGGGLVQPGDSLRLSCAASGFTFSSYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 208)

[0455] This protein sequence is encoded by the cDNA shown in SEQ ID NO:209 below.

[0456] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTACCACAGGTGTCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 209)

[0457] a-CD45-h-VHH-69

[0458] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQV TVSSAHHSEDPIS (SEQ ID NO: 210)

[0459] This protein sequence is encoded by the cDNA shown in SEQ ID NO:211 below.

[0460] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTCCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 211)

[0461] a-CD45-h-VHH-70

[0462] EVQLLESGGGLVQAGDSLRLSCAASERAYRNRLLGWFRQAPGAERVVVAISWSGGSTYYVDSVKGRFTMSRDNSKNTVYLQMNSLKPEDTATYYCAALRFWTTTPQKEGLYDTWGQGTQVTVSSEPKTPKPQT (SEQ ID NO: 212)

[0463] This protein sequence is encoded by the cDNA shown in SEQ ID NO:213 below.

[0464] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGCTCCAGGGGCGGAGCGTGTCGTTGTAGCTATTAGCTGGAGCGGTGGTAGTACATACTATGTAGACTCCGTGAAGGGCCGATTCACC ATGTCCAGAGACAACAGCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCACTTAGATTTTGGACTACAACACCTCAGAAAGAGGGCCTCTATGACACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCCGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 213)

[0465] a-CD45-h-VHH-71

[0466] EVQLQESGGGSLQTGDSLRLACEASEIVVENYVMAWFRQAPGKEREWLARIIWNTGGTHLQEFVKGREGIGYSVKTSTRTVMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPIS (SEQ ID NO: 214)

[0467] This protein sequence is encoded by the cDNA shown in SEQ ID NO:215 below. GAGGTGCAGCTGCAGGAGTCTGGGGGAGGATCGCTGCAGACTGGGGACTCACTGAGACTCGCCTGTGAAGCCTCTGAAAT CGTCGTCGAAAATTATGTCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTGGCTAGCGCGTATTATCTGGAATACCGGTGGCACACATCTTCAAGAATTTGTGAAGGGCCGAGAAGGGATCGGCTATAGCGTCAAAACTTCCACCCGCACAGTAA TGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACCGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 215)

[0468] Example 5: Generation of Chimeric Antigen Receptors (CARs)

[0469] In the sequences below, the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, the bold underlined region is the CD28 transmembrane region, the uppercase italics are the CD28 intracellular region, and the underlined uppercase italics are the CD3Z intracellular region. a-CD19CAR mlllvtslllcelphpaflli pdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleit GGGGSGGGGSGGGGS EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTV SSSGSGSG KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 216)

[0470] This protein sequence is encoded by the cDNA shown below in SEQ ID NO:217.

[0471] Codon-optimized

[0472] a-CD38CAR

[0473] In the sequences below, the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, the bold underlined region is the CD28 transmembrane region, the uppercase italics are the CD28 intracellular region, and the uppercase italics are the CD3Z intracellular region.

[0474] myrmqllscialslalvtns qvqlvqsgaevkkpgssvkvsckafggtfssyaiswvrqapgqglewmgriirflgianyaqkfqgrvtliadkstntaymelsslrsedtavyycagepgredpdavdiwgqgtmvtvss SGGGGSGGGGSGGGGSGGGGSGGGGSS DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 218)

[0475] This protein sequence is encoded by the cDNA shown in SEQ ID NO:219 below.

[0476] Codon-optimized a-CD38CAR

[0477]

[0478] m-VHH1-E3-TM

[0479] In the protein sequence below (SEQ ID NO: 220), the region in lower case is anti-mouse CD45 The VHH is represented by the underlined capital letters, which are the linkers, the non-underlined capital letters, which are the extracellular membrane-proximal region of E3.49K, the bold capital letters, which are the underlined region of E3.49K transmembrane region, and the bold capital letters, which are the intracellular region of E3.49K. maqvqlvesggglvhpgdslrlscaasgsvfnsatmgwyrqspgsqrelvativvgtptyadsvkgrftisrdnaknivylqmnslkpedtavyycnyratytsgysrdywgqgtqvtvs GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (SEQ ID NO: 220)

[0480] In the DNA of the following sequence (SEQ ID NO:221), the region in lowercase is the anti-mouse CD45 VHH, the region in underlined capital letters is the linker, the region in non-underlined capital letters is the extracellular membrane-proximal region of E3.49K, the region in bold capital letters underlined is the E3.49K transmembrane region, and the region in bold capital letters is the intracellular region of E3.49K. This sequence encodes the protein of SEQ ID NO:220.

[0481] atggcccaggtgcagctggtggagagcggcggcggcctggtgcaccccggcgacagcctgagactgagctgcgccgccagcggcagcgtgttcaacagcgccaccatgggctggtacagacagagccccggcagccgagagagctggtggccaccatcgtggtgggcacccccacctac gccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacatcgtgtacctgcagatgaacagcctgaagcccgaggacaccgccgtgtactactgcaactacagagccacctacaccagcggctacagcagagactactggggccagggcacccaggtgaccgtgagc GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA (SEQ ID NO: 221)

[0482] mVHH2-E3-TM

[0483] In the protein sequence below (SEQ ID NO: 222), the region in lower case is anti-mouse CD45 The VHH is represented by the underlined capital letters, which are the linkers, the non-underlined capital letters, which are the extracellular membrane-proximal region of E3.49K, the bold capital letters, which are the underlined region of E3.49K transmembrane region, and the bold capital letters, which are the intracellular region of E3.49K. maqvqlvqsggglvqpggslrlscaasgrafnsaamgwyrqapgsqrelvasisagtasyadavkgrftisrdyakniiylqmnslkpddtavyfcnyrttytsgysedywgqgtqvtvs GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (SEQ ID NO: 222)

[0484] In the DNA of the following sequence (SEQ ID NO:223), the region in lowercase is the anti-mouse CD45 VHH, the region in underlined capital letters is the linker, the region in non-underlined capital letters is the extracellular membrane-proximal region of E3.49K, the region in bold capital letters underlined is the E3.49K transmembrane region, and the region in bold capital letters is the intracellular region of E3.49K. This sequence encodes the protein of SEQ ID NO:222.

[0485] atggcccaggtgcagctggtgcagagcggcggcggcctggtgcagcccggcggcagcctgagactgagctgcgccgccagcggcagagccttcaacagcgccgccatgggctggtacagacaggcccccggcagccgagagagctggtggccagcatcagcgccggcaccgccagctac gccgacgccgtgaagggcagattcaccatcagcagagactacgccaagaacatcatctacctgcagatgaacagcctgaagcccgacgacaccgccgtgtacttctgcaactacagaaccacctacaccagcggctacagcgaggactactggggccagggcacccaggtgaccgtgagc GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA (SEQ ID NO: 223)

[0486] a-CD43-sc a-CD43-sc (SEQ ID NO: 224) is a protein for an anti-CD43 antibody with the stalk and transmembrane domains joined through a linker region. SEQ ID NO: 225 is the DNA sequence of the same molecule. In the sequence below, the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane domain. myrmqllscialslalvtns evqlqqsgpelvkpgasvrmsctasgytftsyvmhwikqkpgqgldwigyinpynggtqynekfkgkatltsdkssstaymelssltsedsavyycarrtfpyyfdywgqgttltvss SGGGGSGGGGSGGGGSGGGGSGGGGSS DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHAPLTFGAGTKLELK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (SEQ ID NO: 224) In the DNA of the sequence below (SEQ ID NO: 225), the underlined lowercase region is the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase region is the linker, the non-underlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane region. atgtacagaatgcagctgctgagctgcatcgccctgagcctggccctggtgaccaacagc gaggtgcagctgcagcagagcggccccgagctggtgaagcccggcgccagcgtgagaatgagctgcaccgccagcggctacaccttcaccagctacgtga tgcactggatcaagcagaagcccggccagggcctggactggatcggctacatcaaccccctacaacggcggcacccagtacaacgagaagttcaagggcaaggccaccctgaccagcgacaagagcag cagcaccgcctacatggagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgccagaagaaccttcccctactacttcgactactggggccagggcaccaccctgaccgtgagcagc AGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGC GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGAAGCAGCCAGAGCATCCTGCACAGCAACGGCAACACCTACCTGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG AGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTTCCAGGGCAGCCACGCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG AGCAGCGGCAGCGGCAGC CCCACCACCACCCCCGCCCCCAGACCCCCCACCCCGCCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCCGCCGGCGGCGCCGTGCACACCAGAGGCCTGGACTTC GCCCCCAGAAAGATCGAGGTGATGTACCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCAGCAAGCCC ACCCTGATCGCCCTGGTGACCAGCGGCGCCCTGCTGGCCGTGCTGGGCATCACCGGCTACTTCCTGTAA (SEQ ID NO: 225)

[0487] Example 6. Creation of lentiviral vectors

[0488] The transfer vector containing the gene of interest is transfected into 293T cells along with packaging vectors (pMDLg / pRRE and pRSV-Rev) and envelope vector (phCMV-VSV-G), and viral supernatant is harvested.

[0489] Small-scale production of VSV-G pseudotyped lentiviral vectors

[0490] This method describes the production of VSV-G pseudotyped lentiviral vectors in 6-well plates using a calcium phosphate transfection kit. The volume of viral supernatant produced per well is 4 ml, and the viral concentration depends on the vector used.

[0491] [Table 3]

[0492] Common materials:

[0493] T75 flask, cell culture pipette, micropipette and tips, 1.5ml microcentrifuge tube, 5ml syringe, Trypan Blue, Virkon, M-ytdes.

[0494] Plasmid Vector:Depends on choice Gag-pol plasmid: pMDLg / pRRE Rev plasmid: pRSV-Rev Envelope plasmid: phCMV-VSV-G

[0495] procedure

[0496] Maintenance of 293FT cells

[0497] Maintain cells by splitting 1:5 to 1:4 every other day and keep them in T75 or T150 flasks. After thawing, culture them for at least three passages before using them for virus production to recover and initiate exponential growth. The use of high-passage cells is not recommended as it negatively impacts virus production.

[0498] Day 1: Plating & Transfection

[0499] Plate 500,000 cells / well in complete growth medium. - Remove the medium from the flask with a pipette - Wash the cells with 10 ml of room temperature PBS. - Add 1 ml (T75) or 2 ml (T150) of TrypLE Express and incubate at 37°C for 5 minutes. - After incubation, add 10-20 ml of complete medium to the flask and resuspend thoroughly, dissociating any clumps by pipetting up and down several times. - Count cells using Trypan Blue and make a cell suspension of 250,000 cells / ml - Place 2 ml of this suspension into wells (poly-L-lysine coated 6-well plates), one well for each vector. - Place the plate in an incubator for at least 7-8 hours

[0500] After incubation, the cells are transfected. Ensure that the cells are attached, optimally at 80% confluency. If the cells are viable and at the appropriate density, return the plate to the incubator and proceed to prepare the transfection mix. If the confluency is less than 60%, stop the transfection.

[0501] - Prepare 1 ml of complete growth medium with the addition of chloroquine to a final concentration of 25 μM. Place it in an incubator for pre-warming. It is important to bring the calcium phosphate precipitation kit components to room temperature before starting the transfection.

[0502] - Prepare a plasmid mixture in a microcentrifuge tube as follows (4 μg total DNA): - 2 μg of LeGO-iG2 vector (containing the transgene) - 1 μg of pMDLg / pRRE(Gag / Pol) - 0.75 μg pRSV-REV(Rev) - 0.25 μg phCMV-VSV-G (envelope) - Mix the plasmids and adjust the volume to 54 μl with ddH2O - Add 6 μl of 2.5 M CaCl2 solution to the DNA mixture - In a separate microtube, add 60 μl of 2X HeBS buffer. Add the CaCl2 / DNA mixture and vortex. - Let the mixture stand at room temperature for 15 minutes, but do not let it stand for longer than 30 minutes, as this may decrease transfection efficiency. During these 15 minutes, remove the dish, discard the medium and add 1 ml of pre-warmed complete growth medium containing 25 μM chloroquine. After 15 minutes of incubation, 120 μl of the mixture is added dropwise to the wells while gently swirling the dishes in a circular motion. - Close the lid of the dish and return it to the incubator for 10-12 hours of incubation.

[0503] Day 2: Medium change

[0504] 10-12 hours after transfection: - Aspirate the medium containing the transfection mix and chloroquine from the wells and discard. - Add 2 ml of complete growth medium per well. Ensure the medium is pre-warmed to at least room temperature, preferably 37°C. Place the cells in the incubator.

[0505] Day 3: Supernatant collection 1 24 hours after medium change: - Check the cells for GFP expression under a UV microscope. Transfection efficiency should be greater than 90%. - Prepare a 0.45 μm filter, a 5 ml syringe, a 5 ml microtube and a 1.5 tube per well. - Harvest the medium from the dish using a 5ml syringe. Apply to the filter and filter the supernatant into a 5ml microfuge tube. Be gentle during the filtration, avoid creating bubbles, and do not use excessive force. Once complete, drop the syringe and filter into the Virkon solution. - Take a 100 μl aliquot from the filtered supernatant into a 1.5 ml microcentrifuge tube (virus titration). If desired, aliquot the remainder of the supernatant and freeze at -80°C for long term storage. - Add 2 ml of complete growth medium per dish. Ensure the medium is pre-warmed to at least room temperature, preferably 37°C. Place the cells in the incubator.

[0506] Day 4: Supernatant collection 2 48 hours after medium change: - Collect the viral supernatant in the same manner as the previous day. - Discard the plate.

[0507] Example 7: Generation of cell lines

[0508] Lentiviral particles harvested in the previous examples were used to transduce, select, and expand K562 and RPMI8226 (FIG. 6) cells, and the resulting cells were tested in the following manner.

[0509] Preparation of target cells 1. Take a sample from the target cells for cell counting. 2. Put 1-2 x 10 6 Taking cells 3. Centrifuge the cells 4. Discard the supernatant 5. Wash with PBS 6. Discard the supernatant 7. Centrifuge again and remove the liquid with a pipette to obtain a "dry" pellet 8. 0.1 ml 51 Add Cr to the cell pellet and mix thoroughly. 9. Incubate for 1 hour, shaking the vial every 15 minutes.

[0510] Preparation of effector cells 1. Take a sample from the cultured cells for cell counting. 2. Centrifuge the cells and collect 0.3 x 10 6 Resuspend the pellet in warm RPMI+10% FCS at a concentration of cells / ml. 3. Add 150 μl / well of diluted sample in triplicate to column 1. 4. In triplicate, add 100 μl / well of RPMI + 10% FCS to columns 2-4 and Add to three wells of the same plate. 5. Add 100 μl / well of dH2O + 1% Triton X100 to the three wells of the plate for maximum release. Add to wells. 6. Prepare serial dilutions at a ratio of 1:3 across the wells. 7. Place the plate in the incubator.

[0511] target cell 1. Wash target cells twice in PBS 2. Resuspend cells in 1 ml of RPMI + 10% FBS. 3. Take a sample for cell counting 4. Add 100 μl / well of target cells and mix. 5. Incubate for 4 hours 6. Spin the plate at 300g for 3 minutes. 7. Add 20ul of cell suspension to each well of a 96-well LumaPlate. Add with a pipette. 8. Place the plate in the chrome hood overnight to dry. Calculate percent specific lysis: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100

[0512] Differentiation of functionally mature NK cells from induced pluripotent stem cells (iPSCs)

[0513] Using a feeder-independent differentiation protocol, induced pluripotent stem cells (iPSCs) were differentiated into functionally mature NK cells, which exhibited both functional maturation and the typical phenotypic signature of blood-derived NK cells and possessed potent antitumor effector functions.

[0514] Human iPSC generation culture and differentiation into hematopoietic cells

[0515] Healthy male skin fibroblasts were reprogrammed using the StemRNA 3rd Generation Reprogramming kit.

[0516] Thawed iPSC lines are cultured on hESC-Qualified Matrigel (Corning, 354277) coated 6-well plates in mTesRTM 1 (StemCell Technologies, 85850) feeder-free maintenance medium for 5 days to reach 80% confluency.

[0517] iPSC lines were passaged using 0.5 mM EDTA (01-862-1B) in PBS.

[0518] Prior to passaging, hematopoietic differentiation medium (HPDM) consisting of StemDiff™ APEL™2 (StemCell Technologies, 05270), 40 ng / mL SCF (PeproTech, 300-07), 20 ng / mL BMP4 (PeproTech, 120-05), and 20 ng / mL VEGF (PeproTech, 100-20B), supplemented with 10 μM Rock inhibitor (Y-27632, Tocris, 1254), was prepared for the first 3 days.

[0519] iPSCs were passaged and seeded at a density of 3,000 cells / well in 100 uL of HPDM supplemented with Rock inhibitor in each well of an ultra-low attachment round-bottom 96-well plate (Corning, CLS3474).

[0520] The cells were centrifuged at 220 g for 5 min to encourage the formation of embryoid body (EB) structures and incubated gently for 3 days at 37° C. and 5% CO 2 .

[0521] Media changes were performed on days 3, 6, and 9 by removing 70 uL of media from each well and adding 100 uL of freshly prepared HPDM without Rock inhibitors.

[0522] Hematopoietic progenitor cells were collected on day 11 using a wide-bore p200 pipette (Fisher Scientific, 14-222-730) for flow cytometry analysis or transfer to NK cell differentiation cultures.

[0523] Hematopoietic cell differentiation into NK cells

[0524] In the second phase of NK cell differentiation from iPSCs, hematopoietic progenitor cells were seeded at a concentration of 32 EBs / well in standard cell culture-treated 6-well plates in 4 mL of NK cell differentiation medium (NKDM) consisting of StemDiff APEL 2 (StemCell Technologies, 05270), 20 ng / mL SCF (PeproTech, 300-07), 20 ng / mL IL-7 (PeproTech, 200-07), 10 ng / mL IL-15 (PeproTech, 1110-15), and 10 ng / mL Flt3L (PeproTech, 300-19), supplemented with 5 ng / mL IL-3 (PeproTech, 200-03).

[0525] Half of the medium was replaced twice a week for 4 weeks with freshly prepared NKDM containing IL-3 for the first week only and without IL-3 for the following 3 weeks.

[0526] After 4 weeks of NK cell differentiation culture, cells were harvested and analyzed for phenotype via flow cytometry, or for cytotoxicity and functional assays after 3–4 weeks of expansion in CTS OpTmizer™ T Cell Expansion medium (ThermoFisher, A1048501) supplemented with 5% human anti-body serum (Corning, 35-060-CI), 1% penicillin / streptomycin (Gibco, 15140122), 0.2 mM L-glutamine (Gibco, 25030081), 10 ng / mL rhIL-15 (Gold Biotechnology, 1110-15), 500 IU / mL rhIL-2 (Akron Biotech, AK8223), and 25 ng / mL rhIL-21 (Gold Biotechnology, 1110-21).

[0527] Example 8: Test Assay

[0528] Khan FA, Almohazey D, Alomari M, Almofty SA. Isolation, Culture, and Functional Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) were cultured according to the procedure described in "Characterization of Human Embryonic Stem Cells: Current Trends and Challenges." Stem Cells Int. 2018;2018:1429351. All cell lines were tested for mycoplasma contamination, and only mycoplasma-free cells were used in the study. Karyotype analysis of cell lines was performed in the cytogenetics facility of the present laboratory using standard protocols.

[0529] ESC / iPSC cultures were dissociated with dispase and plated at 5 x 10 per well of an ultra-low attachment 6-well plate containing X-VIVO medium with supplements. 6 Embryoid bodies are generated by plating 1000 cells. The medium is changed every 3 days and the cultures are maintained for 15-20 days. do.

[0530] Hematopoietic differentiation and genetic modification of ESCs / iPSCs are achieved by electroporation of standard mammalian expression vectors and / or excisable lentiviral vectors. They were differentiated into NK cells by co-culture with OP9 and OP9-DLL1 cells as described in Zeng J, Tang SY, Toh LL, Wang S. Generation of "Off-the-Shelf" Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells. Stem Cell Reports. 2017;9(6):1796-812.

[0531] Differentiation of ESCs / iPSCs into RPE (retinal pigment epithelium)

[0532] ESC / iPSC colonies were passaged using EDTA and differentiated into RPE using a protocol developed by Buchholz (Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coffey PJ, Clegg DO. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med. 2013;2(5):384-93), which was used in the treatment of macular degeneration. Briefly, the hESC line HS980 was established and cultured on rhLN-521 under xeno-free, defined conditions and passaged using standard protocols. For differentiation, cells were plated onto dishes coated with 20 mg / mL hrLN-111 at a density of 2.4 × 104 cells / cm2 using NutriStem hESC XF medium and Rho kinase inhibitor for the first 24 h. The medium was then replaced with NutriStem hESC XF lacking basic fibroblast growth factor and transforming growth factor b, and 100 ng / mL activin A was added to the medium starting on day 6 after plating for a total of 5 weeks.

[0533] Normal human CD56+ NK cells and CD8+ T cells were isolated by positive / negative enrichment (Miltenyi CliniMACS system) of blood cells collected from healthy human donors. NK-92 cells (CRL-2407) were obtained from the American Type Culture Collection (Manassas, VA) and cultured as described on the product sheet.

[0534] Cytotoxicity testing by chromium release assay

[0535] Target cells were analyzed for their susceptibility to NK cell-mediated lysis for 4 h. 51NK cells were cultured in NK medium containing IL2 for 48 h prior to the assay. Target cells were treated with 50 μCi of IL2. 51 Label with Cr at 37°C for 2 hours. 51 Cr-labeled cells are plated per well of a 96-well U-bottom plate. NK cells are added to target cells at different ratios and incubated at 37°C for 4 h. Controls include labeled cells without NK cells (spontaneous release) and labeled cells lysed with 1% Triton X-100 (complete lysis). 20 μl of each reaction supernatant is added to a Luma scintillation plate and allowed to dry overnight in a hood. After reading the radioactivity, the percentage of specific lysis is calculated. A more detailed protocol is provided below. a. Take a sample from K-562 for cell count. b. Place 1-2 x 106 cells in a tube. c. Centrifuge the cells d. Discard the supernatant e. Add 0.1 ml of 51Cr to the cell pellet. f. Incubate for 1 hour g. Take a sample from the cultured cells for cell count. h. Dilute sample to 0.3 x 106 cells / ml in RPMI + 10% FCS. Total volume 1 ml. i. Label a 96-well plate. j. Add 150 μl / well of diluted sample in triplicate to column 1. k. 100 μl / well of RPMI + 10% FCS in 3 steps and minimum release Add to three wells of the same plate. l. Add 100 μl / well of dH2O + 2M HCl to three wells of maximum release. can. m. Take 50 μl from column 1 and add to column 2, mix, take 50 μl from column 2, Place in column 3. Continue in this manner, discarding 50 μl from the last column. n. Place the plate in the incubator. o. Wash K-562 twice in PBS p. Resuspend cells in 1 ml of 10% RPMI. Q. Take a sample for cell count r. Calculate the number of cells required. s. Add 100 μl / well of target cells. t. Incubate for at least 4 hours. u. Label the tubes for gamma counter. v. Take 70 μl sample / tube. Work carefully and avoid cells. w. Analyze in a gamma counter. x. Incucyte-based cytotoxicity assay.

[0536] Incucyte-based cytotoxicity assay We used Incucyte to measure immune cell-mediated cytotoxicity and invasion of single tumor spheroids. Spheroids mimic in vivo conditions more accurately than cell monolayers and exhibit several characteristics that determine solid tumor killing and invasion, such as cell-cell adhesion within tumors, increased cell survival, and diffusion gradients of oxygen, nutrients, and waste products from the outer cell ring to the inner core. Incucyte-based measurements of immune cell cytotoxicity allow for real-time observation.

[0537] procedure: 1. Add 21.5ul of DMSO to a new vial to prepare Cytolight Resuspend the Green vial to prepare a 5 mM stock solution. 2. Add 2.8 ul of stock solution to 360 ul of PBS to make a 100x dilution. 3. Place the effector cells in a 15 ml tube and spin at 400 x g for 5 minutes. 4. Wash the pellet with 5 ml of PBS and carefully remove the wash solution. 5. Resuspend the pellet in 6 ml of PBS and add 60 μl of 100x Cytolight Green solution to each tube. 6. Incubate the cells at 37°C for 20 minutes, mixing every 5 minutes. 7. Place the clean plate with the lid in the incubator to pre-warm the lid. 8. Add 3.6 ml of 100% FBS to bind excess Cytolight reagent. Mix the cells and centrifuge at 400 x g for 5 minutes. Aspirate the supernatant and resuspend the cells in 500 ul of medium. 9. Count the cells and adjust the concentration through the addition of medium. 10. Bring one vial of CytotoxRed (5 μL) to RT, centrifuge briefly, CytotoxRed staining was performed by adding 45 μL of PBS to the CytotoxRed. Prepare the tick solution 11. Add 32.5 ul of C to a total volume of 6.5 ml of SCGM containing 10% FBS. Prepare CytotoxRed working concentration by adding CytotoxRed. 12. Assemble plates and add 100ul CytotoxRed. Add 50ul target cells, 50ul effector cells or media. 13. Place plates in Incucyte and count red cells for 4 hours.

[0538] Cytotoxicity test using CD8+ T cells

[0539] Normal human CD8+ T cells were cultured in a 2×10 -well culture of IFN-γ-treated target cells (2×10 per 2 ml). 6 CD8+ cells and 5 × 10 5 The cells were primed by co-culturing with 1.75 × 10 target cells in culture medium supplemented with 50 U / ml IL-2 and 25 ng / ml IFN-γ. On day 7, the co-cultures were primed with 1.75 × 10 target cells. 6 On day 14, these primed CD8+ T cells are collected by centrifugation and used in a chromium release assay as described above for NK cells.

[0540] Generation of cell lines

[0541] K562 and RPMI cells were transduced and expanded using the lentiviral particles harvested in Example 10. The resulting cells were tested for expression of the target gene using GFP as a marker or by labeling the cells with the corresponding antibody or fluorescently labeled protein. 1. Prepare medium: DMEM / RPMI 10% FBS = 400 ul / well 2. Remove 50 ul of supernatant from each well. 3. Add lentiviral vector and tx media. 4. Label the 24-well plates with the date, name, cell type and virus used for transduction. 5. Set the centrifuge temperature to 32°C. 6. Detach the cells using a cell scraper and resuspend all the cells by pipetting up and down several times using a serological pipette. 7. Count cells using trypan blue and culture for 10 min in medium + 10% FBS. 6 Make a cell suspension of 100 cells / ml. 8. Distribute 250ul of cells into a 24 well plate. 9. Take the required amount of protamine sulfate stock at a final concentration of 8ug / ml. 10. Avoid repeated freezing / thawing of stocks. 11. Add medium according to the calculation. 12. Keep virus on dry ice until use. Thaw required amount of virus quickly. 13. Mix the virus carefully to minimize contact with air. 14. Add the calculated amount of virus to each well. 15. For NK cells, protamine sulfate (8 μg / ml) and IL-2 (1000 1.1U / ml) is pipetted into each well. 16. Carefully mix the cells by pipetting up and down. 17. Centrifuge the plate at 1000 x g for 1 hour at 32°C without interruption. 18. Remove plates and incubate in incubator for 4 hours to overnight (depending on construct and viral titer; to be tested). 19. At the end of the incubation, the plates are centrifuged again at 1000 x g for 1 hour at 32°C. 20. Carefully remove 80% of the medium from all wells and fill with 500 ul of fresh pre-warmed medium containing serum. 21. Place the plate back into the incubator. Days 1 & 2: Check cells under a microscope for colonies. Day 3: Analyze cells by flow cytometry.

[0542] In vivo reactivity with allogeneic CD8+ T cells and NK cells

[0543] All mouse care, breeding, and surgical procedures were approved by the Animal Ethics Committee of Stockholm, Sweden. Mice were purchased from Charles River Laboratories. NSG mice have been previously described (Shultz LD, Lyons BL, Burzenski LM, Gott B., Chen X., Chaleff S., Kotb M., Gillies SD, King M., Mangada J., Greiner DL, Handgretinger R. (2005) Human lymphoid and myeloid cell development in NOD / LtSz-scid IL2Rγ null mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174, 6477-6489 [PubMed: 15879151]) and were bred and maintained in the AKM5 animal facility at the Karolinska Institute, Huddinge, Sweden.

[0544] Mice were obtained from Jackson laboratories (NOD.Cg-Prkdcscid Il2rgtm1WjI / SzJ - JAX stock number 005557, originating from The Jackson Laboratory and bred under license by Charles River in Europe).

[0545] Male mice (8-10 weeks old) were subcutaneously injected with UC or K562 cell lines (1 x 10). All cells were tested and found to be free of mycoplasma prior to injection. Mice were intravenously injected with human PBMCs (10 x 10). Subcutaneous tumor size measurements were initiated when mice had measurable tumors. Tumor size was measured with a slide caliper at least twice weekly for 4 weeks, and tumor volume was calculated. When tumor volume reached 1 cm, mice were euthanized, and tumors and organs were removed.

[0546] In a parallel study, male NSG mice (6-8 weeks old) were subcutaneously injected with K562, RPMI8226, and SKOV3 cell lines (1x106) modified with a CD45 engager and luciferase reporter gene, or modified with a luciferase reporter gene alone (without CD45 engager modification). All cells were tested and found to be free of mycoplasma prior to injection. One day after tumor challenge, mice were intravenously injected with human PBMCs (10x106), split over two consecutive days (5x106 PBMCs / day). Mice were then subcutaneously injected with daratumumab (an ADCC-competent anti-CD38 antibody) in the RPMI8226-injected group and trastuzumab (an anti-Her2 antibody) in the SKOV3-injected group, both at 8 mg / kg, 3 days after tumor cell injection. Mice under isoflurane were fluorescently imaged using an In Vivo Imaging System (IVIS) Spectrum (Perkin Elmer, Santa Clara, CA, USA) and analyzed using IVIS imaging software (Perkin Elmer). Imaging was performed on all animals on day 0 and twice weekly until the mice were euthanized and tumors and organs were removed (Figure 32).

[0547] IVIS imaging demonstrated that control mice in the RPMI-8226 group given PBMC and daratumumab controlled tumor development (Figure 34). Injection of RPMI-8226 cells expressing a CD45 engager together with PBMCs and daratumumab leads to tumor development (Figure 33: Photograph from IVIS imaging depicting RPMI-8226 expressing luciferase and a CD45 engager. Mice treated with PBMCs and daratumumab.) In a similar manner, CD45 engager-modified K562 cells, even with PBMC administration, led to greater immune escape compared to administration of K562 cells with sequential PBMC administration (Figure 35). Finally, IVIS imaging of control mice in the SKOV3 group given PBMCs and trastuzumab controlled tumor development, whereas injection of SKOV3 cells expressing a CD45 engager together with PBMCs and trastuzumab led to tumor development (Figure 36).

[0548] Flow cytometry

[0549] Staining and washing are performed in flow cytometry acquisition buffer. Single-cell suspensions of cells are incubated with blocking reagent for 10 minutes on ice, then stained with antibodies and viability stains for 30-60 minutes on ice. Samples are analyzed on a Fortessa / Symphony flow cytometer (BD Biosciences), and data are analyzed using FlowJo software (TreeStar, Ashland, OR). For sorting, an AriaFusion (BD Biosciences) instrument is used. Sorted cells are cultured in medium containing antibiotics for 2 weeks. Thereafter, cells are cultured without antibiotics.

[0550] Extracellular vesicle (EV)-mediated a-CD45-sc mRNA delivery ameliorates collagen-induced arthritis

[0551] Extracellular vesicles (EVs) from target cells are isolated / purified using ultracentrifugation, tangential flow filtration, or size exclusion chromatography. The number and size of EVs are analyzed using a Nanosight tracking analysis system (NTA). EVs are used for mRNA delivery of transgenes used to generate antibodies or nanobodies in vivo. We also tested engagers expressed on EVs at different densities.

[0552] EV isolation and purification

[0553] Conditioned medium (CM) was harvested and pre-clarified by low-speed centrifugation at 700 × g for 5 minutes. To remove large cell debris and apoptotic bodies, the CM was centrifuged at 2,000 × g for 10 minutes. Finally, to eliminate any remaining undesirable large vesicles, the CM was then filtered using a bottle-top filter (Corning, low protein binding) with a 0.22 μm pore-size cellulose acetate membrane. The CM medium was then diafiltered by ultrafiltration using a tangential flow filtration (TFF, MicroKross, 20 cm², SpectrumLabs) with a 300 kDa cutoff. Finally, the CM was concentrated using an Amicon Ultra-15 10 kDa weight cutoff spin filter (Millipore) at 4,000 × g for a specific time based on the sample concentration. EV quality and concentration were then analyzed using ZetaView (Figure 40).

[0554] Endogenous passive loading of a-CD45-sc mRNA into EVs. EV producer cells are engineered to overexpress a-CD45-sc mRNA, which is then overloaded into vesicles during EV biogenesis along with their original cargo and the protein translated from the overexpressed mRNA transcript. EV-mediated cargo delivery loaded with a-CD45-sc mRNA. Bioengineered EVs are internalized by autoimmune cells. Endosomal degradation leads to delivery of a-CD45-sc mRNA into the cytoplasm. Translation of the delivered a-CD45-sc mRNA into protein results in inhibition of autoimmune events (Figure 37).

[0555] The collagen-induced arthritis (CIA) mouse model is a well-established and frequently used model that mimics the clinical symptoms and immunopathogenesis of human RA. Mice immunized with collagen II (CII) exhibited increased arthritis scores, whereas the control group showed no significant changes. Interestingly, a-CD45-sc mRNA-loaded MSC EVs exhibited an inhibitory effect on arthritis severity (Figure 38). In contrast, mRNA mock MSC EVs had no effect. Additionally, RA pathogenesis involves activated immune cells, which promote macrophages to release pro-inflammatory cytokines. Therefore, serum TNF-α and IL-1β levels were measured by sandwich ELISA. Notably, a-CD45-sc mRNA-loaded MSC EVs reduced serum TNF-α and IL-1β levels in CIA mice (Figures 39A and B). These results indicated that a-CD45-sc effectively attenuated inflammation in CIA mice (Figure 38). a-CD45-sc EVs ameliorate the severity of collagen-induced arthritis (CIA). CIA was induced in DBA / 1J mice by active immunization with chicken collagen II (CII). a-CD45-sc mRNA or mock mRNA-loaded MSC EVs were injected on days 0, 7, 14, and 21 after arthritis induction. 2.5E11 EVs were injected into the tail vein. Arthritis scores were assessed every 5 days. Data are presented as mean ± SD (n=5).

[0556] Referring to Figures 39A and 39B, a-CD45-sc EVs inhibit the production of pro-inflammatory cytokines in CIA mice. CIA was induced in DBA / 1J mice by active immunization with chicken collagen II (CII). a-CD45-sc mRNA or mock mRNA-loaded MSC EVs were injected on days 0 and 10 after arthritis induction. Cytokine (TNF-α and IL-1β) levels were measured on day 40 after intravenous injection of 5E11 EVs from a-sc-CD45 or mock mRNA. Data are presented as mean ± SD.

[0557] Transgene Expression System

[0558] Lentivirus and retrovirus systems are used for transgene expression in target and / or effector cells.For transient expression, electroporation or chemical-based methods are used.Transferred gene is delivered by vector-based or as mRNA with or without nanoparticles through chemical or electrochemical delivery systems.For gene delivery, biocompatible materials such as lipids, naked DNA, chromosomes, plasmids, cationic polymers and conjugate complex systems can be used.

[0559] Suicide gene

[0560] Depending on the clinical application and the cells, a suicide gene may be incorporated into the cells. This allows for the destruction of the cells, usually using a non-toxic agent, such as ganciclovir. Representative suicide genes are listed in Table 3 below.

[0561] [Table 4-1] [Table 4-2] [Table 4-3]

[0562] Example 9: Results

[0563] According to the chromium cytotoxicity assay described above, and with reference to FIG. 51 Percent specific lysis of K562 cells with peripheral blood mononuclear cells (PBMCs) using a chromium assay. K562 control cells, and K562 expressing E3.49K, UL11 ai-CD45-sc were incubated with PBMCs for 4 hours at effector:target (E:T) ratios of 10:01, 3:01, 1:01, and 0.3:1. Cells were centrifuged, and 20 μL of supernatant was added to Luma plates. Plates were dried overnight and read the next day in a gamma counter. Figure 7 shows reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing a-CD45-sc.

[0564] Referring to Figure 8, the experiment was repeated using NK92 cells instead of PBMCs. K562 control cells, K562 expressing E3.49K, K562 expressing engager UL11, and K562 and a-CD45-sc were incubated with PBMCs for 4 hours at E:T ratios of 10:01, 3:01, 1:01, and 0.3:1. Cells were centrifuged, and 20 μL of supernatant was added to Luma plates. Plates were allowed to dry overnight and read the next day in a gamma counter. As in Figure 7, the results in Figure 8 clearly demonstrate reduced cell lysis for cells expressing UL11 and E3.49K, as well as complete inhibition of lysis for cells expressing a-CD45-sc.

[0565] Figure 9 shows 51Figure 9 shows the percent specific lysis in K562 cells using a Cr release assay. K562 control cells, K562 expressing E3.49K, UL11, and a-CD45-sc were incubated with NK92 for 4 hours at the E:T indicated in Figure 9. Cells were centrifuged, and 20 uL of supernatant was added to Luma plates. Plates were allowed to dry overnight and read in a gamma counter. As in Figures 7 and 8, the results in Figure 9 clearly show reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing a-CD45-sc.

[0566] Figure 10 shows 51 Figure 10 shows the percent specific lysis of K562 cells using a Cr release assay. K562 control cells, K562 expressing E3.E3.49K, UL11, and a-CD45-sc were incubated with PBMC and the indicated E:T for 4 hours. Cells were centrifuged, and 20 μL of supernatant was added to a Luma plate. Plates were allowed to dry overnight and read in a gamma counter. As in Figures 7, 8, and 9, the results in Figure 10 clearly show reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing a-CD45-sc.

[0567] Figure 11 shows 51 Figure 1 shows the percent specific lysis of RPMI88226 using a Cr release assay. RPMI88226 control cells, RPMI88226 expressing E3.49K, UL11, or a-CD45-sc were incubated with E:T as indicated for T cells for 4 hours. Cells were centrifuged, and 20 μL of supernatant was added to Luma plates. Plates were allowed to dry overnight and read in a gamma counter. As in Figures 7-12, the results clearly show reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing a-CD45-sc.

[0568] In an attempt to assess whether expression of the CD45 engager affects graft function when the grafted cells are effector cells (NK cells or T cells), the NK-92 and TALL-104 cell lines were transduced with a-CD45-sc. NK92 cells were maintained as described above. TALL-104 cells were maintained at 37°C, 10% CO2, in IMDM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, Ga.) and 100 units / ml of recombinant human IL-2. Cell lines were repeatedly tested for mycoplasma contamination using a commercial polymerase chain reaction kit. Both NK-92 and TALL-104 cells were cultured at concentrations of 4–6 and at a fixed number (10 4 / well) 51 4 h for Cr-labeled K562 cells 51 The specificity of the NK-92 and TALL-104 cells was examined in a Cr release assay. Unmodified NK-92 and TALL-104 cells were used as control effector cells. 51 The percentage of Cr release was calculated from the average of three replicates. Figure 30 depicts a comparative evaluation of NK-92 cells with a-CD45-sc gene modification. Figure 31 depicts a comparative evaluation of TALL-104 cells with a-CD45-sc gene modification.

[0569] Example 10: Chimeric Antigen Receptor (CAR) Modified Cells

[0570] The inventors evaluated the present invention in a-CD38CAR and a-CD19CAR cells through the following experiments.

[0571] A.(CD38 + ) Evaluation of the influence of a-CD45-sc on the CD38-directed chimeric antigen receptor (a-CD38CAR)-mediated cytotoxicity of NK / T cells against target cells.

[0572] To analyze the impact of a-CD45-sc on the function of a-CD38CAR (SEQ ID NO: 218 / 219), a-CD45-sc (SEQ ID NO: 5) was expressed on cells expressing CAR, and their cytotoxicity against target cells was evaluated (Figure 24, Figure 41). This experiment can be easily adapted using single chains and single domains against CD45, with the expectation of similar results. The following SEQ ID NOs can be used: NK92 cells were transduced with viral particles carrying a-CD38CAR and sorted. The sorted and expanded NK92 cells were again transduced with either a-CD45-sc, UL11, E3.49K, or a control. CD38 knockout RPMI8226 or wild-type cells were used as targets. Effector cells were labeled with CytoLight Green, and target cells were labeled with Cytotox Red. Both effector and target cells were incubated 1:1 in a 96-well flat-bottom plate in Incucyte. Referring to Figure 44, red cells indicate target cell death and were counted over a 4 hour period. Data was analyzed in GraphPad Prism.

[0573] Referring to Figure 41, RPMI8226 cells were incubated with NK92 control cells, NK92 cells expressing a-CD38CAR, a-CD38CAR+a-CD45-sc, or a-CD45-sc. After 4 hours of incubation, the cells were centrifuged and 20 uL of the supernatant was added to the Luma plate.

[0574] B.(CD19 + ) To assess whether the chimeric antigen receptor for CD19 (CARCD19)-mediated cytotoxicity of NK / T cells against target cells is affected by a-CD45-sc.

[0575] To analyze the impact of a-CD45-sc on the function of a-CD19CAR (SEQ ID NO: 216 / 217), a-CD45-sc (SEQ ID NO: 5) was expressed on cells expressing CAR, and their cytotoxicity against target cells was evaluated. PBMCs were transduced with viral particles carrying a-CD19CAR (Figure 29). Expanded PBMCs expressing a-CD19CAR were again transduced with either a-CD45-sc or a control. Jurkat and Raji cells were used as targets. Degranulation assays were performed for 4 hours, and cells were labeled with CD107a along with CD3, CD56, Live / Dead-APC-H7, and CD19h-biotin. After degranulation, the cells were run on a flow cytometer. Data were analyzed using Flowjo (Figure 43).

[0576] CRISPR-Cas9 technology was used to generate the RPMI8226 CD38.KO cell line. More specifically, the cells were transduced with a lentiviral vector encoding the Cas9 gene, a gRNA targeting exon 1 of the CD38 gene, and a puromycin selection gene. After evaluating the transduction efficacy by flow cytometry, the cells were treated with puromycin for 2 weeks to allow selective survival of the transduced cells. Further flow cytometry analysis confirmed the knockout of CD38 in selected populations.

[0577] Referring to Figure 42, RPMI8226 CD38 KO cells were incubated with NK92 control cells, NK92 cells expressing a-CD38 CAR, a-CD38 CAR+a-CD45-sc, or a-CD45-sc. After 4 hours of incubation, the cells were centrifuged and 20 uL of the supernatant was added to the Luma plate.

[0578] Example 11: Clinical Applications

[0579] The present invention can be used to treat any cell or tissue before being introduced into the body.The present invention can also be used to treat autoimmune diseases; blood cancers including lymphoma and leukemia; bone marrow failure syndromes including anemia and cytopenia; inherited immune disorders including WAS and SCID; hemoglobinopathies including sickle cell disease (SCD) and thalassemia; neurological disorders including neuromyelitis optica; cartilage replacement, for example, joint replacement, for example, knee and hip replacement; preventive management of cytotoxicity.

[0580] Currently, tissue transplants require immunosuppression with drugs. Immunosuppression may be required for cell transplants. Immunosuppressants leave patients severely immunocompromised and at high risk for opportunistic infections. Using the constructs and methods taught in Examples 1-13 above, tissue and cell therapies can be designed that do not require additional immunosuppression or may require only low doses of immunosuppressant drugs.

[0581] The cells and tissues to be treated can be any mammalian cell or hybrid between human and other mammalian cells. Cyranoski D. Japan approves first human-animal embryo experiments. Nature. 2019.

[0582] Those skilled in the art will recognize that the scope of the present invention is not limited to these examples, and the present invention can be applied to any It is understood that the present invention is potentially applicable to any cell or tissue therapy that involves the introduction of desired non-autologous cells or tissues or modified autologous cells or tissues into a living mammal where the body may recognize and reject the introduced cells or tissues.

[0583] A. Graft

[0584] 1. Solid organ grafts

[0585] It is envisioned that this strategy could be utilized by temporary or permanent genetic modification of solid organs with vectors encoding CD43, CD45, and / or CD148 engagers, thus rendering the graft safe from T- and NK-cell-based immune responses due to the lack of synapse formation. This could hypothetically be utilized in any organ or organ portion or organoid graft, including, but not limited to, the muscular system (including joints, ligaments, muscles, tendons), digestive system (including mouth, teeth, tongue, salivary glands, parotid glands, submandibular glands, sublingual glands, pharynx, esophagus, stomach, small intestine, duodenum, jejunum, ileum, large intestine, liver, gallbladder, mesentery, pancreas, anal canal), respiratory system (including nasal cavity, pharynx, larynx, trachea, bronchi, lungs, diaphragm), urinary system (including kidneys, ureters, bladder, urethra); female reproductive system (ovaries, fallopian tubes, uterus, vagina, vulva, clitoris, placenta); male reproductive system (testes, epididymis, vas deferens, seminal vesicles, prostate, bulbourethral glands, penis, scrotum); endocrine system (including pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands, pancreas); circulatory system (including heart, patent foramen ovale, arteries, veins, capillaries); lymphatic system (including lymphatic vessels, lymph nodes, bone marrow, thymus, spleen, gut-associated lymphoid tissue, tonsils, interstitium); nervous system (brain, cerebrum, cerebral hemispheres, diencephalon, brainstem, midbrain, pons, medulla oblongata, cerebellum, spinal cord, ventricular system, choroid plexus, peripheral nervous system, cranial nerves, spinal nerves, ganglia, enteric nervous system, sensory organs, eye, cornea, iris, ciliary body, lens, retina, ear, outer ear, earlobe, tympanic membrane, middle ear, ossicles, inner ear, cochlea, ear vestibule, semicircular canals, olfactory epithelium, tongue, taste buds, integumentary system, main article Article): Integumentary system, mammary gland, skin, and subcutaneous tissue. Organs in these areas can be genetically modified using previously defined genetic modification strategies. Aravalli RN, Belcher JD, Steer CJ. Liver-targeted gene therapy: Approaches and challenges. Liver Transpl. 2015;21(6):718-37. Essentially, a batch of vectors can be utilized for in vivo or ex vivo gene delivery via hydrodynamic or similar strategies. This potentially allows for the use of tissues across species.

[0586] 2. Tissue transplantation

[0587] Similar to solid organ transplants, the use of these engagers may allow for the use of tissue or portions of the organ to be transplanted. Composite transplants (hand, limb, face) may be made possible by utilizing CD45 engagers. The first face transplant was performed in 2005. The ethical issues surrounding face transplants are even more pronounced than those surrounding limb transplants because the surgical procedure is extremely demanding and the required immunosuppression places the recipient at considerable risk of opportunistic infections.

[0588] Immunosuppression usually consists of induction therapy (antithymocyte globulin [ATG] and / or IL-2 receptor blockers), followed by triple maintenance immunosuppression with corticosteroids, antiproliferative drugs (e.g., basiliximab), and calcineurin inhibitors (see table). Occasionally, topical creams containing calcineurin inhibitors or corticosteroids are used. Access to engagers for these tissues through genetic modification would reduce or even abolish the need for lifelong immunosuppression.

[0589] Skin allografts use donor skin (typically from a cadaver). Skin allografts are used for patients with extensive burns or other conditions that cause significant skin loss such that the patient does not have enough intact skin to provide a graft. Allografts can be used to cover large denuded areas, thereby reducing fluid and protein loss and preventing invasive infections. Unlike solid organ transplants, skin allografts are ultimately rejected due to immune rejection. The use of engagers for these tissues through genetic modification would prolong engraftment without the need for immunosuppression and risk of infection. When valves become damaged or diseased and do not function as they should, they may require repair or replacement. Conditions that can cause heart valve dysfunction are valvular stenosis (stiffness) and valvular regurgitation (leaky valves). Diseased valves can be repaired using rings to support the damaged valve, or the entire valve can be removed and replaced with a prosthetic valve. Prosthetic valves may be made from carbon-coated plastic or tissue (made from animal valves or human valves taken from donors). Allogeneic and xenogeneic valves pose the challenge of immune rejection. Patients may therefore need to undergo lifelong immunosuppression. Modification of valve grafts with CD45 engagers may abrogate this need for immunosuppression and prolong the time to rejection.

[0590] Nerve transplants and nerve transfers offer new hope for patients whose limbs have been paralyzed or severely injured in accidents. In most cases, replacement nerves come from cadavers or occasionally from living donors. In either case, patients must be given immunosuppressant drugs until their nerves regenerate, which can take up to two years. Modification of nerve grafts with CD45 engagers, as envisioned herein, may abrogate this need for immunosuppression and prolong the time to rejection.

[0591] Cartilage transplants are used for children with congenital nasal or ear defects and adults with severe injuries or joint destruction (e.g., severe osteoarthritis). Chondrocytes are more resistant to rejection, likely because the sparse population of cells in hyaline cartilage is protected from cellular attack by the surrounding cartilage matrix. However, grafts still carry a risk of rejection, especially in elderly populations. Inclusion of CD45 engagers for these tissues through genetic modification would increase engraftment.

[0592] Bone grafts are used for the reconstruction of large bone defects (e.g., after extensive resection of bone cancer). Viable donor bone cells do not survive in the recipient; the dead matrix from the allograft can stimulate recipient osteoblasts to regenerate the matrix and lay new bone. This matrix acts as a scaffold to bridge and stabilize the defect until new bone is formed. Cadaveric allografts are preserved by freezing to reduce bone immunogenicity and by adding glycerol to maintain chondrocyte viability. The use of CD45 engagers for soft bone tissue through genetic modification would reduce the need for this processing, thereby reducing perioperative handling and postoperative morbidity due to more rapid engraftment of bone tissue.

[0593] A similar strategy can be utilized for the creation of adrenal tissue allografts for fetal thymus transplant patients with Parkinson's disease or DiGeorge syndrome.

[0594] In the United States, the most commonly transplanted tissues are bone, tendons, ligaments, skin, and heart valves. Of the approximately 2 million tissue grafts performed each year, only about 1 million grafts are thought to be transplanted.

[0595] 3.Cell graft

[0596] The engineered cells in the engager express suicide genes, chemokine receptors, and activating or inhibiting receptors. It is envisioned that the cells may be co-modified with essentially any transgene, including, but not limited to, CD43, CD45, and / or CD148 engager-modified cells, using endonucleases or CRISPR / Cas9 or other techniques to remove immunological checkpoint receptors, chemokine receptors, hypoxia-responsive receptors, central differentiation regulators, among other genes. The use of non-human cells and tissues is envisioned for use in humans.

[0597] a. Stem cell transplantation for cancer treatment and gene corrective stem cells for single gene disorders or complex genetic disorders. Allogeneic stem cell transplantation (from umbilical cord blood, peripheral blood, bone marrow, or other sources) is an accepted therapeutic approach for a variety of diseases, including, but not limited to, acute myeloid leukemia, myelodysplasia, and multiple myeloma, and has been tested in various solid organ tumors / cancers, including liver, breast, and kidney cancers, including metastases. Similarly, this approach has shown success in treating genetic disorders affecting the hematopoietic system, such as severe combined immunodeficiency syndrome (SCID-X), Wiskott-Aldrich syndrome, and anemia. This approach has several drawbacks, including failure to engraft and the need for high chimerism levels. Donor-derived hematopoietic stem cells can be genetically modified ex vivo with vectors encoding the CD43, CD45, and / or CD148 engager constructs or their derivatives described herein, potentially achieving partial or complete chimerism without lymphodepleting regimens. In this way, host immune cells can be rendered ineffective against the graft, promoting engraftment.

[0598] b. Platelet transfusion

[0599] It is also envisioned that engagers may be utilized in platelets by direct modification of platelets or platelet-producing cells to avoid rapid platelet rejection potentially mediated by NK cells and other effector cells, which could be utilized prior to the emergence of platelet refractory states for patients receiving multiple platelet infusions over their lifetime.

[0600] c. Red blood cell / red blood cell transfusion

[0601] This strategy could also potentially be utilized to modify red blood cell progenitor cells and RBCs to avoid cellular rejection of RBC infusion products for patients requiring multiple RBC transfusions if the patient has not developed antibodies to RBC antigens prior to the time of administration.

[0602] d. Multipotent and pluripotent cell therapy or their cell derivative therapy

[0603] The above methodology can also be used to produce or generate iPSC or hES cell lines and cells derived therefrom. Thus, in one embodiment of the present invention, compositions and methods are provided for producing target cells bearing CD43, CD45, and / or CD148 engagers, thereby creating hypoimmunogenic cells. Such hypoimmunogenic cells are expected to be less prone to immune rejection by the subject into whom they are transplanted. When transplanted, the hypoimmunogenic cells should engraft (not be rejected). In one embodiment, such target cells can engraft and survive with little or no immunosuppression required from the recipient.

[0604] This methodology has been applied to treat conditions such as cartilage degeneration, age-related macular degeneration (ESC / iPSC-derived RPE administration), Stargardt disease, osteogenesis imperfecta (embryonic or postpartum MSC administration), and other diseases. It can be used to generate a variety of tissues / cells differentiated from pluripotent / multipotent cells for patients undergoing cell replacement therapy.

[0605] e. Donor leukocyte infusion

[0606] It is envisioned that donor leukocyte infusion (DLI), including NK cell, T cell, and macrophage infusion with or without additional genetic modifications encoding transgenes, such as T cell receptors, chimeric antigen receptors, dimeric antigen receptors, or any other genes, may utilize co-transduction of CD43, CD45, and / or CD148 engagers to make the graft safe for infusion by avoiding the formation of functional immunological synapses that, without the engager, could lead to recipient cell-mediated rejection of the grafted cells. T cells can include any T cell, including, but not limited to, suppressor T cells, regulatory T cells, gamma delta T cells, and mucosal-associated invariant T cells (MAIT), as well as all subtypes of innate lymphoid cells.

[0607] f. Genetically modified T cell therapy

[0608] This strategy can also be utilized in T cells from allogeneic sources, such as bone marrow CD34, donor-derived T cells, iPSC-derived T cells, and hESC-derived T cells, with or without additional genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activation receptors, and cell adhesion receptors. This can be achieved by additional transduction or cotransduction of CD43, CD45, and / or CD148 engagers to make the graft safe for infusion by avoiding the formation of a functional immunological synapse that, without the engager, could lead to recipient cell-mediated rejection of the grafted cells, thereby avoiding cell-mediated graft rejection. Currently, CAR-modified T cells are commonly used to treat cancer.

[0609] Representative CAR T cells include CARs currently being investigated for hematological cancers, including, but not limited to, the following targets and genes: BCMA (TNFRSF17), CD123 (IL3RA), CD138 (SDCl), CD19 (CD19) (commercially available CD19 CARs include axicabtadine-ciloreucel (Yescarta™) and tisagenlecleucel (Kymriah™)), CD20 (MS4A1), CD22 (CD22), CD38 (CD38), CD5 (CDS), Ig K chain (IgK), LeY (FUT3), NKG2D ligand (NKG2D), ROR1 (ROR1), and WT1 (WT1).

[0610] CARs for solid tumors include, but are not limited to, the following targets and genes: target (gene), C-Met (MET), CAIX (CA9), com (PROMl), CD171 (LlCAM), CD70 (CD70), CEA (CEACAMS), EGFR (EGFR), EGFR viii(EGFRVIII), Ep-CAM(EPCAM), EphA2(EPHA2), FAP(FAP), GD2), GPC3(GPC3), HER2(ERBB2), HPV16-E6(HPVE6), IL13Ra2(IL13RA2), LeY(FUT3), MAGEA3(MAGEA3), MA GEA4 (MAGEA4), MARTl (MLANA), mesothelin (MSLN), MUCl (MUCl), MUC16 (MUC16), NY-ES0-1 (CTAGlB), PD-Ll (CD274), PSCA (PSCA), PSMA (FOLHl), RORI (RORI) and VEGFR2 (KOR).

[0611] g. Genetically modified NK cell therapy

[0612] This strategy can also be utilized in NK cells from allogeneic sources, such as bone marrow CD34, donor-derived T cells, iPSC-derived T cells, hESC-derived NK cells, with or without further genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activating receptors, cell adhesion receptors. This can be done by additional transduction or co-transduction of CD43, CD45, and / or CD148 engagers to make the graft safe for infusion by avoiding the formation of a functional immunological synapse that could lead to recipient cell-mediated rejection of the grafted cells without the engager, thereby avoiding cell-mediated graft rejection.

[0613] h. Genetically modified macrophage therapy

[0614] This strategy can also be utilized in macrophages from allogeneic sources, such as bone marrow CD34, donor-derived T cells, iPSC-derived T cells, hESC-derived macrophages, with or without further genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activation receptors, cell adhesion receptors. This can be done by additional transduction or co-transduction of CD43, CD45, and / or CD148 engagers to make the graft safe for infusion by avoiding the formation of a functional immunological synapse that, without the engager, could lead to recipient cell-mediated rejection of the grafted cells, thereby avoiding cell-mediated graft rejection.

[0615] i. Gene correction cells for other genetic disorders

[0616] In a similar manner, it can be envisioned that transplanted cells for metabolic disorders could be modified with transgenes encoding engagers for CD43, CD45, and / or CD148 for optimal engraftment, potentially without the need for lymphocyte ablation.

[0617] j. Other cells envisioned for use / transplantation

[0618] Other cells contemplated for use / implantation herein include: endodermally derived cells, such as: exocrine epithelial cells (Brunner's gland cells in the duodenum (enzymes and alkaline mucus), shielded goblet cells of the respiratory and digestive tract (mucus secretion), stomach, pit cells (mucus secretion), chief cells (pepsinogen secretion), parietal cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), type II alveolar epithelial cells of the lung (surfactant secretion), club cells of the lung); barrier cells (type I alveolar epithelial cells (lung), gallbladder epithelial cells, central acinar cells (pancreas), intercalated duct cells (pancreas), intestinal brush border cells (with microvilli); hormone-secreting cells: enteroendocrine cells, K cells (secreting gastric inhibitory peptide), L cells (secreting glucagon-like peptide-1, peptide YY3-36, oxyntomodulin, and glucagon-like peptide-2), I cells These include thyroid cells (which secrete cholecystokinin (CCK)), G cells (which secrete gastrin), enterochromaffin cells (which secrete serotonin), enterochromaffin-like cells (which secrete histamine), N cells (which secrete neurotensin), S cells (which secrete secretin), D cells (which secrete somatostatin), Mo cells (or M cells) (which secrete motilin), and other hormones secreted: vasoactive intestinal peptide, substance P, alpha- and gamma-endorphin, and bombesin; thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, and eosinophilic cells; pancreatic islets (islets of Langerhans), alpha cells (which secrete glucagon), beta cells (which secrete insulin and amylin), delta cells (which secrete somatostatin), epsilon cells (which secrete ghrelin), and PP cells (gamma cells) (which secrete pancreatic polypeptide).

[0619] Ectoderm-derived cells, such as exocrine epithelial cells, salivary gland mucous cells, salivary gland serous cells, and tongue phosphatase Neubner's gland cells (cleansing taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), earwax gland cells (secreting earwax), eccrine sweat gland dark cells (secreting glycoproteins), eccrine sweat gland clear cells (secreting small molecules), apocrine sweat gland cells (secreting odor-producing substances, sex hormone sensitive), Molar gland cells (specialized sweat glands) in the eyelid, sebocytes (secreting lipid-rich sebum), Bowman's gland cells in the nose (cleansing the olfactory epithelium); hormone-secreting cells, hormone-secreting cells, anterior / intermediate pituitary cells, adrenocorticotropic hormone-producing cells, gonadotropic hormone-producing cells, mammary gland cells, melanocorticotropic hormone-producing cells, neurocytes, somatotrophs, thyrotrophs, magnocellular neurosecretory cells (which secrete oxytocin and vasopressin), parvocellular neurosecretory cells (which secrete thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), vasopressin, oxytocin, neurotensin, and prolactin), chromaffin cells (adrenal glands); epithelial cells, e.g., keratinocytes (differentiating epithelial cells); epithelial basal cells (stem cells); melanocytes; trichocytic cells (which give rise to hair and nail cells), e.g., medulla hair Stem cells, cortical hair stem cells, cuticle hair stem cells, Huxley's layer root sheath cells, Henle's layer root sheath cells, outer root sheath hair follicle cells; surface epithelial cells of the cornea, tongue, mouth, nasal cavity, distal anal canal, distal urethra, and distal vagina; basal cells (stem cells) of the cornea, tongue, mouth, nasal cavity, distal anal canal, distal urethra, and distal vagina; intercalated duct cells (salivary glands); striate duct cells (salivary glands); mammary duct cells (mammary glands); ameloblasts (deposit tooth enamel), oral cells, e.g., odontoblasts (dentin formation), cementoblasts (dental cement formation); sensory transduction cells, e.g., auditory inner hair cells of the organ of Corti, auditory outer hair cells of the olfactory epithelium, basal cells of the olfactory epithelium (stem cells for olfactory neurons), cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of the epithelium, olfactory receptor neurons, pain-sensitive primary sensory neurons; photoreceptor cells of the retina of the eye: photoreceptor rod cells, photoreceptor blue-sensitive cone cells of the eye, photoreceptor green-sensitive cone cells of the eye, photoreceptor red-sensitive cone cells of the eye; proprioceptive primary sensory neurons; touch-sensitive primary sensory neurons; chemoreceptor glomus cells of the carotid body cells (blood pH sensors); outer hair cells of the vestibular system of the ear (acceleration and gravity);Inner hair cells of the vestibular system of the ear (acceleration and gravity); taste receptor cells of the taste buds; autonomic neuronal cells, e.g., cholinergic neurons (various types); adrenergic neurons (various types); peptidergic neurons (various types); sensory organ and peripheral neuronal supporting cells, e.g., inner column cells of the organ of Corti, outer column cells of the organ of Corti, inner phalangeal cells of the organ of Corti, outer phalangeal cells of the organ of Corti, marginal cells of the organ of Corti, Hensen's cells of the organ of Corti, vestibular supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, olfactory ensheathing cells, Schwann cells, satellite glial cells, enteric glial cells, central nervous system neurons Neuronal and glial cells, such as neuronal cells (interneurons, basket cells, cartwheel cells, astrocytes, Golgi cells, granule cells, Lugaro cells, unipolar brush cells, Martinozzi cells, chandelier cells, Cajal-Retzius cells, double bouquet cells, neurogliaform cells, retinal horizontal cells, amacrine cells, starburst amacrine cells, spinal interneurons, Renshaw cells); principal cells (spindle neurons, fork neurons, pyramidal cells, place cells, grid cells, speed cells, head direction cells, Betz cells, stellate cells, border cells, tufted cells (Bushy cells), Purkinje cells, medium spiny neurons); astrocytes; oligodendrocytes; ependymal cells, tanycytes; pituitary cells; nervous system cells, such as sensory transduction cells, autonomic neuronal cells, sensory organ and peripheral neuron supporting cells, central nervous system neurons and glial cells; lens cells (anterior lens epithelial cells, crystallin-containing lens fiber cells);

[0620] Cells derived primarily from the mesoderm, such as metabolic and storage cells (adipocytes: (white and brown adipocytes, hepatic adipocytes); secretory cells (cells of the adrenal cortex, including cells of the zona glomerulosa, produce mineralocorticoids, cells of the zona fasciculata, produce glucocorticoids, and cells of the zona reticularis, produce androgens); theca cells of the ovarian follicles, which secrete estrogen; luteal cells of the ruptured follicles, which secrete progesterone (granulosa lutein cells, theca lutein cells); Leydig cells of the testes, which secrete testosterone; seminal vesicle cells, which secrete seminal fluid components, including fructose for sperm migration; prostate cells, which secrete seminal fluid components); urethral bulbGlandular cells (mucus-secreting); Bartholin's gland cells (vaginal lubricant-secreting); Littley's gland cells (mucus-secreting); endometrial cells (carbohydrate-secreting); juxtaglomerular cells (renin-secreting); macula densa cells of the kidney; peripolar cells of the kidney; mesangial cells of the kidney; urinary system barrier cells (parietal epithelial cells; podocytes, proximal tubule brush border cells, cells of the thin segment of Henle's loop) cell), kidney distal tubule cells, kidney collecting duct cells, chief cells, interstitial cells, transitional epithelium (lining the bladder); reproductive system: ductal cells (seminal vesicles, prostate, etc.), efferent duct cells, epididymal principal cells, epididymal basal cells; circulatory system: endothelial cells; extracellular matrix cells: semilunar planar epithelial cells of the vestibular system of the ear (secreting proteoglycans), interdental epithelial cells of the organ of Corti (secreting the tectorial membrane covering the hair cells), loose connective tissue fibroblasts, corneal fibroblasts (keratocytes), tendon fibroblasts Cells, bone marrow reticular tissue fibroblasts, other non-epithelial fibroblasts, pericytes (liver stellate cells (Ito cells)), nucleus pulposus cells of the intervertebral disc, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts / osteocytes, osteoprogenitor cells (stem cells of osteoblasts), vitreous cells of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, pancreatic stellate cells; contractile cells, e.g.: skeletal muscle cells (red skeletal muscle cells (slow contraction), white skeletal muscle cells (fast contraction), intermediate skeletal muscle cells, nuclear sacs of muscle spindles) cells, muscle spindle nuclear chain cells, muscle satellite cells (stem cells), cardiomyocytes (cardiomyocytes, SA node cells, Purkinje fiber cells); smooth muscle cells (various types); myoepithelial cells of the iris; myoepithelial cells of the exocrine glands; blood and immune system cells, such as erythrocytes (red blood cells) and precursor erythroblasts, megakaryocytes (platelet precursors), platelets, which are currently a subject of debate but are considered as separate cells, monocytes (white blood cells), connective tissue macrophages (various types), epithelial Langerhans cells Bone cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophilic granulocytes and precursors (myeloblasts, promyelocytes, myelocytes, metamyelocytes), eosinophilic granulocytes and precursors, basophilic granulocytes and precursors, mast cells, helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, plasma cells, natural killer cells, hematopoietic stem cells and precursor cells (various types) committed to the blood and immune system;Germ cells, such as oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells for spermatocytes), spermatozoa; nurse cells, granulosa cells (in the ovaries), Sertoli cells (in the testes), epithelial reticular cells (in the thymus); and interstitial cells: interstitial kidney cells.

[0621] 2. Therapeutic particles

[0622] A extracellular vesicles

[0623] The use of CD43, CD45 and / or CD148 engagers and / or introduction of CD43, CD45 and / or CD148 engagers directly into extracellular vesicles or into parent cells utilized for the production of extracellular vesicles for targeted or systemic delivery of therapeutic transgenes or proteins can also be envisioned.

[0624] B. Adenoviral gene delivery

[0625] One of the challenges of in vivo viral gene delivery is cellular rejection of the genetically modified cells due to the immunogenicity of the vector proteins. Co-introduction of engagers against CD43, CD45, and / or CD148 proteins may lead to better genetic modification by avoiding cellular responses to the modified cells in vivo.

[0626] C. oncolytic viruses

[0627] Despite active research in virotherapy, this seemingly safe modality has not achieved widespread success. The immune response to viral infection appears to be an essential factor determining the efficacy of oncolytic virotherapy. The challenge is to determine whether the virus-induced immune response will be an obstacle or a tool for viral treatment. NK cells NK cell responses are a key component of innate immunity, mediating antiviral immunity while synchronizing tumor clearance. Various reports suggest that NK cell responses to oncolytic virotherapy are an essential factor in premature viral clearance while also mediating downstream antitumor immunity. Consequently, particular attention should be paid to how NK cell responses to various oncolytic viral vectors and their antiviral properties can be suppressed while maintaining tumor clearance. In this context, it is possible to envision that oncolytic viruses could be engineered to rescue OV-infected cells from NK cell-mediated killing by including genes encoding engagers for CD43, CD45, and / or CD148.

[0628] This strategy can also be utilized for cellular drug delivery, where the grafted cells carry the drug to the target tissue, and these grafted cells are co-modified with CD43, CD45, and / or CD148 engagers.

[0629] C. Specific conditions

[0630] 1. Control of chronic inflammatory diseases by repeated transient gene delivery

[0631] It is also envisioned that mRNA or DNA encoding CD43, CD45, and / or CD148 engagers can be delivered in a local or systemic manner to patients with chronic inflammatory diseases where cytotoxicity is part of the disease physiology. This can be done locally or systemically in patients with autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, and Crohn's disease.

[0632] 2. Wound healing and skin grafting

[0633] The present invention can be used in conjunction with conventional stem cell therapies to produce cells and tissues for wound treatment without the risk of rejection. (3) Kosaric N, Kiwanuka H, Gurtner GC. Stem cell therapies for wound healing. Expert Opin Biol Ther. 2019;19(6):575-85.

[0634] 3. Inherited metabolic disorders

[0635] The present invention may be used to treat inherited metabolic disorders such as: 17-alpha-hydroxylase deficiency, 17-beta hydroxysteroid dehydrogenase 3 deficiency, 18 hydroxylase deficiency, 2-hydroxyglutaric aciduria, 2-methyl-3-hydroxybutyric aciduria, 2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase 1 deficiency, 3-alpha hydroxyacyl-CoA dehydrogenase deficiency, 3-hydroxyisobutyric aciduria, 3-methylcrotonyl-CoA carboxylase deficiency, 3-methylglutaconyl-CoA hydratase deficiency (AUH deficiency), 5-oxoprolinase deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, abdominal obesity metabolic syndrome, abetalipoproteinemia, acatalasemia, aceruloplasminemia, acetyl-CoA acetyltransferase 2 deficiency, acetyl-carnitine deficiency, acrodermatitis enteropathica, acromegaly, acute intermittent porphyria, adenine phosphoribosyltransferase deficiency, adenosine deaminase deficiency, adenosine monophosphate deaminase Ze1 deficiency, adenylosuccinase deficiency, adrenomyeloneuropathy, adult polyglucosan body disease, adult-onset type II citrullinemia, albinism deafness syndrome, ocular albinism late-onset sensorineural hearing loss, ALG1-CDG (CDG-Ik), ALG11-CDG (CDG-Ip), ALG12-CDG (CDG-Ig), ALG13-CDG, ALG2-CDG (CDG-Ii), ALG3-CDG (CDG-Id), ALG6-CDG (CDG-Ic), ALG8-CDG (CDG-Ih), ALG9-CDG (CDG-IL), alkaptonuria, Alpers syndrome, Alpha-1 antitrypsin deficiency, alpha-ketoglutarate dehydrogenase deficiency, alpha-mannosidosis, aminoacylase 1 deficiency, anemia due to adenosine triphosphatase deficiency, sideroblastic anemia and spinocerebellar ataxia, apparent mineralocorticoid excess, arginase deficiency, argininosuccinic aciduria, aromatic L-amino acid decarboxylase deficiency, arthrogryposis, renal dysfunction, cholestatic syndrome, Arts syndrome, aspartylglycosaminuria, ataxia with oculomotor apraxia type 1, ataxia with vitamin E deficiency,Atransferrinemia, atypical Gaucher disease due to saposin C deficiency (Gaucher disease), autoimmune polyglandular syndrome type 2, autosomal dominant neuronal ceroid lipofuscinosis 4B, autosomal dominant optic atrophy and cataract, autosomal dominant optic atrophy plus syndrome, autosomal recessive neuronal ceroid lipofuscinosis 4A (adult neuronal ceroid lipofuscinosis), autosomal recessive spastic ataxia 4, autosomal recessive spinocerebellar ataxia 9, B4GALT1-CDG (CDG-IId), Bantu siderosis, Barth syndrome, Bartter syndrome, prenatal Bartter syndrome type 1, prenatal Bartter syndrome Type 2, Bartter syndrome type 3, Bartter syndrome type 4, beta-ketothiolase deficiency, biotin-thiamine-responsive basal ganglia disease, biotinidase deficiency, Bjornstad syndrome, Blue Diaper syndrome, carbamoylphosphate synthetase 1 deficiency, carnitine palmitoyltransferase 1A deficiency, carnitine-acylcarnitine translocase deficiency, carnosinemia, central diabetes insipidus, cerebral folate deficiency, cerebrotendinous xanthomatosis, neuronal ceroid lipofuscinosis 1, Shanarin-Dorfman syndrome, Chediak-Higashi syndrome, CHILD syndrome, childhood hypothyroidism Phosphatasia, childhood-onset cerebral X-linked adrenoleukodystrophy, cholesteryl ester storage disease, chondrocalcinosis 1, chondrocalcinosis 2, chondrocalcinosis due to apatite crystal deposition, chondrodysplasia punctata 1, X-linked recessive, chronic progressive external ophthalmoplegia, chylomicron storage disease, citrulline transport defect, COG1-CDG (CDG-IIg), COG4-CDG (CDG-IIj), COG5-CDG (CDG-IIi), COG7-CDG (CDG-IIe), COG8-CDG (CDG-IIh), combined oxidative phosphorylation deficiency 16, congenital bile acid synthesis deficiency type 1, Congenital bile acid synthesis deficiency type 2, congenital glycosylation disorder type I / IIX, congenital dyserythropoietic anemia type 2, congenital erythropoietic porphyria, congenital lactase deficiency, congenital muscular dystrophy-dystroglycanopathy (type B) with or without intellectual disability, copper deficiency, benign familial, CoQ-responsive OXPHOS deficiency, Crigler-Najjar syndrome type 1, Crigler-Najjar syndrome type 2, cystinosis, cystinosis, ocular non-nephropathy, cytochrome c oxidase deficiency, D-2-hydroxyglutaric aciduria, D-bifunctional protein deficiency,D-glycerinemia, Danon disease, DCMA syndrome, DDOST-CDG (CDG-Ir), hearing loss, dystonia, and cerebral hypomyelination, dentatorubral-pallidoluysian atrophy, desmosterolism, Diamond-Blackfan anemia, dicarboxylic acid amino aciduria, dihydrolipoamide dehydrogenase deficiency, dihydropteridine reductase deficiency, dihydropyrimidinase deficiency, dihydropyrimidine dehydrogenase deficiency, thirst-induced diabetes insipidus, DOLK-CDG (CDG-Im), Dopa-responsive dystonia, dopamine beta-hydroxybenzoate enzyme deficiency, Dowling-Degos disease, DPAGT1-CDG (CDG-Ij), DPM1-CDG (CDG-Ie), DPM2-CDG, DPM3-CDG (CDG-Io), Dubin-Johnson syndrome, encephalopathy (sphingolipidosis) due to prosaposin deficiency, erythropoietic protoporphyria, erythropoietic uroporphyria associated with myeloid malignancies, ethylmalonic acid encephalopathy, Fabry disease, familial HDL deficiency, familial hypocalciuric hypercalcemia type 1, familial hypocalciuric hypercalcemia type 2, familial hypocalciuric hypercalcemia Mucouric hypercalcemia type 3, familial LCAT deficiency, familial partial lipodystrophy type 2, Fanconi-Bickel syndrome, Farber disease, fatal infantile encephalomyopathies, fatty acid hydroxylase-associated neurodegeneration, fish eye disease, fructose-1,6-bisphosphatase deficiency, fucosidosis, Fukuyama muscular dystrophy, fumarase deficiency, galactokinase deficiency, galactosialidosis, gamma-aminobutyric acid transaminase deficiency, gamma-cystathionase deficiency, Gaucher disease, Gaucher disease-ophthalmoplegia-cardiovascular calcification (Gaucher disease), perinatal fatal Gaucher disease Gaucher disease, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, diabetes insipidus of pregnancy, Gilbert syndrome, Gitelman syndrome, glucose transporter type 1 deficiency syndrome, glucose-galactose malabsorption, glutamate formiiminotransferase deficiency, glutamine deficiency, congenital, glutaric acidemia type I, glutaric acidemia type II, glutaric acidemia type III, glutathione synthetase deficiency, glutathioniuria, glycine N-methyltransferase deficiency, glycogen storage disease type 8, glycogen storage disease type 0, liver, glycogen storage disease type 12,Glycogen storage disease type 13, glycogen storage disease type 1A, glycogen storage disease type 1B, glycogen storage disease type 3, glycogen storage disease type 5, glycogen storage disease type 6, glycogen storage disease type 7, glycoprotein storage diseases, GM1 gangliosidosis type 1, GM1 gangliosidosis type 2, GM1 gangliosidosis type 3, GM3 synthase deficiency, GRACILE syndrome, Greenberg dysplasia, GTP cyclohydrolase I deficiency, guanidinoacetate methyltransferase deficiency, choroidal and retinal Gyrate atrophy, Heim-Munk syndrome, Hartnup disease, Hawkinskin syndrome, hemochromatosis type 2, hemochromatosis type 3, hemochromatosis type 4, hepatic lipase deficiency, myelohepatic porphyria, hereditary amyloidosis, hereditary coproporphyria, hereditary folate malabsorption, hereditary fructose intolerance, hereditary hyperalgesia, hereditary multiple osteochondromas, hereditary sensory and autonomic neuropathy type 1E, hereditary sensory neuropathy type 1, Hermansky-Pudlak syndrome 2, histidinemia, HMG CoA lyase deficiency, homocarnosinemia, homocysteinemia, homocystinuria due to CBS deficiency, homocystinuria due to MTHFR deficiency, Hurler syndrome, Hurler-Scheie syndrome, hydroxykynureninuria, hyper-IgD syndrome, hyperbeta-alaninemia, hypercoagulability syndrome due to glycosylphosphatidylinositol deficiency, hyperglycemia, hyperinsulinism due to glucokinase deficiency, hyperinsulinemia-hyperammonemia syndrome, hyperlipidemia type 3, hyperlipoproteinemia type 5, hyperlysinemia, hyperphenylalaninemia due to dehydratase deficiency, hyper Prolinemia, hyperprolinemia type 2, hypertryptophanemia, hypolipoproteinemia, hypophosphatasia, I-cell disease, Immerslund-Grassbeck syndrome, iminoglycinuria, inclusion body myopathy 2, inclusion body myopathy 3, infantile free sialic acid storage disease (free sialic acid storage disease), infantile neuroaxonal dystrophy, infantile-onset spinocerebellar ataxia, insulin-like growth factor I deficiency, intrinsic factor deficiency, isobutyryl-CoA dehydrogenase deficiency, isovaleric acidemia, Kanzaki disease, Kearns-Sayre syndrome, atypical Krabbe disease due to saposin A deficiency, L-2-hydroxyglutaric aciduria,L-arginine:glycine amidinotransferase deficiency, lactate dehydrogenase A deficiency, lactate dehydrogenase deficiency, thrombocytopenia, LCHAD deficiency, Leber's hereditary optic neuropathy, Leigh syndrome, French Canadian type, Lesch-Nyhan syndrome, leucine-sensitive hypoglycemia of infancy, leukoencephalopathy-dystonia-motor neuropathy, leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2K, limb-girdle muscular dystrophy type 2M, limb-girdle muscular dystrophy type 2N, limb-girdle muscular dystrophy type 2O, limb-girdle muscular dystrophy type 2T (limb-girdle muscular dystrophy), type 2C, lipase deficiency combined, lipoic acid synthetase deficiency, Urbach syndrome Habetes lipoid proteinosis, Rowe oculocerebrorenal syndrome, lysinuric protein intolerance, malonyl-CoA decarboxylase deficiency, MAN1B1-CDG, mannose-binding lectin protein deficiency, mannosidosis, beta A, lysosomal, maple syrup urine disease type 1A, maple syrup urine disease type 1B, maple syrup urine disease type 2, maternal hyperphenylalaninemia, maternally inherited diabetes mellitus and hearing loss, medium-chain acyl-coenzyme A dehydrogenase deficiency, megaloblastic anemia due to dihydrofolate reductase deficiency, Menkes disease, metachromatic leukodystrophy, metachromatic leukodystrophy due to saposin B deficiency, methionine adenosyltransferase deficiency, methylcobalamin deficiency cbl G, methylmalonic acidemia cbl C with homocystinuria, homocystinuria, Methylmalonic acidemia with homocystinuria cblD, Methylmalonic acidemia with homocystinuria cblF, Methylmalonic acidemia with homocystinuria cblJ, Methylmalonic aciduria, cblA, Methylmalonic aciduria, cblB, Mevalonic aciduria, MGAT2-CDG (CDG-IIa), Mild phenylketonuria, Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial DNA depletion syndrome, Encephalomyopathic forms with methylmalonic aciduria, Mitochondrial DNA Associated Leigh syndrome, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, mitochondrial myopathy with diabetes, mitochondrial myopathy with lactic acidosis, mitochondrial neurogastrointestinal encephalopathy syndrome, mitochondrial trifunctional protein deficiency, MOGS-CDG (CDG-IIb), Mohr-Tranebjaerg syndrome, molybdenum cofactor deficiency, monogenic diabetes, Morquio syndrome B, MPDU1-CDG (CDG-If), MPI-CDG (CDG-Ib), MPV17 Associated liver-cerebral mitochondrial DNA depletion syndrome, mucolipidosis III alpha / beta, mucolipidosis type 4, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, multiple congenital anomaly-hypotension-seizure syndrome, multiple congenital anomaly-hypotension-seizure syndrome type 2, multiple endocrine neoplasia type 2B, multiple sulfatase deficiency, multiple symmetric lipomatosis, myo-oculo-encephalopathy, muscular dystrophy, congenital , giant conus, muscle phosphorylase kinase deficiency, muscle contracture Ehlers-Danlos syndrome, myoclonic epilepsy with ragged-red fibers, recurrent myoglobinuria, N-acetyltransferase deficiency, N-acetyl-alpha-D-galactosaminidase deficiency type III, N-acetylglutamate synthase deficiency, NBIA / DYT / PARK-PLA2G6, neonatal adrenoleukodystrophy, neonatal hemochromatosis, neonatal intrahepatic cholestasis caused by citrin deficiency, nephrogenic diabetes insipidus, Neu Laxova syndrome, neuroferritinopathy, neuronal ceroid lipofuscinosis 10,Neuronal ceroid lipofuscinosis 2, Neuronal ceroid lipofuscinosis 3, Neuronal ceroid lipofuscinosis 5, Neuronal ceroid lipofuscinosis 6, Neuronal ceroid lipofuscinosis 7, Neuronal ceroid lipofuscinosis 9, Neuropathy-Ataxia, Retinitis Pigmentosa Syndrome, Neutral Lipid Storage Disease with Myopathy, Niemann-Pick Disease Type A, Niemann-Pick Disease Type B, Niemann-Pick Disease Type C1, Niemann-Pick Disease Type C2, Northern Epilepsy, 3-MGAuria Type Not Otherwise Specified, Occupational-Horn Syndrome, Ocular Albinism Type 1, Oculocutaneous Albinism Type 1B, Oculocutaneous Albinism Type 2, Oculocutaneous Albinism Type 3, OPA3 Defect, Optic Atrophy 1, Ornithine Transcarbamylase Deficiency, Ornithine Translocase Deficiency Syndrome, Orotic Aciduria Type 1, Papillon-Lefèvre syndrome, Parkinson's disease type 9, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, pentosuria, permanent neonatal diabetes mellitus, peroxisome biogenesis disorder, peroxisomal disorders, Perot syndrome, Peters-Plus syndrome, PGM1-CDG, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphoribosyl pyrophosphate synthetase deficiency, PMM2-CDG (CDG-Ia), pontocerebellar hypoplasia type 6, porphyria cutanea tarda, primary calcification Nitin deficiency, primary hyperoxaluria type 1, primary hyperoxaluria type 2, primary hyperoxaluria type 3, primary hypomagnesemia with secondary hypocalcemia, progressive external ophthalmoplegia, autosomal recessive 1, progressive familial intrahepatic cholestasis type 1, progressive familial intrahepatic cholestasis type 2, progressive familial intrahepatic cholestasis type 3, prolidase deficiency, propionic acidemia, pseudocholinesterase deficiency, pseudoneonatal adrenoleukodystrophy, purine nucleoside phosphorylase deficiency, pyknodysostosis, pyridoxal 5'-phosphate-dependent epilepsy, pyridoxine-dependent epilepsy, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, pyruvate dehydrogenase phosphatase deficiency, pyruvate kinase deficiency, Refsum disease, Refsum disease with increased pipecolic acidemia, Refsum disease, infantile type, renal glucosuria, renal hypomagnesemia-2, renal hypomagnesemia-6, renal tubulopathy, diabetes mellitus, and cerebellar ataxia due to mitochondrial DNA replication, RFT1-CDG (CDG-In),Radicular chondrodysplasia punctata type 3 (radicular chondrodysplasia punctata), Rotor syndrome, saccharopinuria, Salla disease (free sialic acid storage disease), sarcosinemia, Scheie syndrome, Schimke immune dysplasia, Schindler disease type 1, cochlear pelvic dysplasia, SCOT deficiency, See-Blue histiocytosis, Sengels syndrome, sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, sepiapterin reductase deficiency, severe combined immunodeficiency, short-chain acyl-CoA dehydrogenase deficiency, sialidosis type I, sialidosis type II, sialic aciduria, furan SLC35A1-CDG (CDG-IIf), SLC35A2-CDG, SLC35C1-CDG (CDG-IIc), Smith-Lemli-Opitz syndrome, spastic paraplegia 7, spinocerebellar ataxia 28, autosomal recessive spinocerebellar ataxia 3, spondylocostal dysostosis 1, spondylocostal dysostosis 2, spondylocostal dysostosis 3, spondylocostal dysostosis 4, spondylocostal dysostosis 5, spondylocostal dysostosis 6, spondylodysplastic Ehlers-Danlos syndrome, spondyloepiphyseal dysplasia-related Ganglion chalazioni, SRD5A3-CDG (CDG-Iq), SSR4-CDG, succinic semialdehyde dehydrogenase deficiency, Tangier disease, Tay-Sachs disease, thiamine-responsive megaloblastic anemia syndrome, thiopurine S-methyltransferase deficiency, tiglic acidemia, TMEM165-CDG (CDG-IIk), transaldolase deficiency, transcobalamin 1 deficiency, transient neonatal diabetes mellitus, trehalase deficiency, trimethylaminuria, triosephosphate isomerase deficiency, tyrosine hydroxylase deficiency, transient thiglic acidemia Leucine oxidase deficiency, tyrosinemia type 1, tyrosinemia type 2, tyrosinemia type 3, urea cycle disorders, valineemia, variegate porphyria, VLCAD deficiency, Walker-Warburg syndrome, Wilson disease, Wolfram syndrome, Wolman disease, wrinkled skin syndrome, X-linked adrenoleukodystrophy, X-linked Charcot-Marie-Tooth disease type 5, X-linked creatine deficiency, X-linked dominant chondrodysplasia punctata 2, X-linked sideroblastic anemia, xanthinuria type 1, xanthinuria type 2, and Zellweger syndrome.

Claims

1. comprising one or more molecules or cells configured to modulate the ability of CD45, CD148, or CD43 to prevent cytotoxicity by forming a functional immunological synapse with cytotoxic cells; Therapeutic agents that do not contain ULL proteins or fragments thereof.

2. The therapeutic agent of claim 1, comprising a surface-bound engager comprising a single chain antibody or a VHH nanobody.

3. The therapeutic agent of claim 1 , comprising a protein, an aptamer, a peptide nucleic acid (PNA), a nanoparticle, or a cell that expresses or secretes said one or more molecules.

4. The therapeutic agent according to any one of claims 1 to 3, comprising a protein, preferably a protein comprising an antibody, more preferably a protein comprising a single chain antibody or a VHH nanobody.

5. The therapeutic agent according to any one of claims 1 to 3, comprising a nanoparticle, preferably a lipid nanoparticle (LNP), a dendrimer, a ribonucleoprotein (RNP), or an extracellular vesicle, preferably an exosome or a microvesicle.

6. The therapeutic agent of any one of claims 1 to 3, comprising SEQ ID NO: 1, 3, 5, 54, 56, 58, 60, 62, 64, 66, 68, 71, 73, 216, 218, 220, 223, or 224, or a protein having at least 80% identity to SEQ ID NO: 1, 3, 5, 54, 56, 58, 60, 62, 64, 66, 68, 71, 73, 216, 218, 220, 223, or 224.

7. The therapeutic agent according to any one of claims 1 to 3, comprising a cell having one or more molecules expressed on the surface of the cell.

8. the one or more molecules expressed on the surface of the cells comprise an expressed transmembrane protein, and the cells comprise graft cells, optionally the transmembrane protein may bind to CD45, CD148, or CD43, and optionally wherein said CD45, CD148, or CD43 is present on the surface of a cytotoxic cell, preferably a T cell or a natural killer (NK) cell, and optionally The therapeutic agent of claim 7, wherein the transmembrane protein retains CD45, CD148, or CD43 in the developing immunological synapse on the surface of the cytotoxic cell, thereby preventing the formation of a functional immunological synapse.

9. 1. A protein complex capable of preventing cytotoxic cell-induced lysis, comprising: an engager comprising SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, or a protein having at least 80% identity to SEQ ID NO: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224; and CD45, CD148, or CD43 proteins expressed on the surface of T cells or NK cells A protein complex comprising:

10. The therapeutic agent according to any one of claims 1 to 3 for use as a medicine.

11. The therapeutic agent according to any one of claims 1 to 3, for use in the prevention or treatment of one or more conditions of autoimmune diseases, blood cancers, bone marrow failure syndromes, inherited immune disorders, hemoglobinopathies, neurological disorders, and graft-versus-host disease.

12. A therapeutic agent according to any one of claims 1 to 3 for promoting escape from T cell-mediated lysis, for use in the prevention or treatment of one or more of psoriasis and vitiligo.

13. Used to treat graft-versus-host disease, The therapeutic agent according to any one of claims 1 to 3, wherein the graft is exposed to the therapeutic agent to prevent binding of cytotoxic cells.

14. 10. Use of the therapeutic agent according to claim 1 for producing xenogeneic cells for transplantation, the use comprising protecting the xenogeneic cells to be transplanted with the therapeutic agent.

15. The use of claim 14, wherein the therapeutic agent is bound to the surface of the xenogeneic cells prior to transplantation.

16. A recombinant protein comprising: (i) a signal peptide, (ii) an antibody heavy chain, (iii) a first linker, (iv) an antibody light chain, (v) optionally, a second linker, (vi) a stalk, (vii) a transmembrane region, and (viii) optionally, an intracellular region; a second linker connecting the light chain to the stalk, or a single chain antibody, preferably a single chain antibody that specifically binds to CD45, CD148, or CD43, or (i) to (vii) are each present and connected in order from the amino terminus to the carboxyl terminus of the protein; the signal peptide is an IL2 signal peptide, said first linker comprises an SGGGG motif and / or varies in length from 5 to 60, preferably from 10 to 50, more preferably from 20 to 45 amino acids, said second linker, if present, varies in length from 5 to 60, preferably from 5 to 40, more preferably from 7 to 15 amino acids; the stalk is at least 8 and no more than 200 amino acids in length; and A recombinant protein wherein the transmembrane region is derived from CD34, CD45, CD28, and / or CD8a.

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