Modulators for immune evasion mechanisms in universal cell therapy

Abstract

Therapeutic agents that can place bulky proteins, such as CD45, CD148, and CD43, at the center of the cellular interface between graft cells and CD45-positive host effector cells (e.g., T cells, NK cells, B cells, or dendritic cells) are disclosed, as are methods of their use and products made with such therapeutic agents. The therapeutic agents prevent or inhibit the formation of functional immunological synapses (including physiological SMACs). They also result in the continuous dephosphorylation of signaling pathways.

Description

[Background technology]

[0001] Cell therapy is a remarkable achievement of modern science currently used to replace damaged tissues and / or organs, and appears promising for many diseases, including diabetes, retinitis pigmentosa, Parkinson's disease, hematological cancers including myocardial infarction, lymphoma and leukemia, bone marrow failure syndromes including anemia and cytopenia, hereditary immune disorders including Wiscott-Aldrich syndrome (WAS) and severe combined immunodeficiency (SCID), abnormal hemoglobin disorders including thalassemia, sickle cell anemia and congenital dysplasia, hereditary metabolic disorders including lysosomal storage disorders, galactosemia, phenylketonuria and glycogen storage disorders, neurological disorders including neuromyelitis optica, cartilage replacement including knee replacement, and Crohn's disease. Like organ transplantation, cell therapy also faces challenges of limited donor availability and immune rejection. This necessitates the development of mechanisms that make cells immunely privileged. Immune privileged cells not only enable the creation of "off-the-shelf" cell products, but can also lead to the creation 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 these fields were created. The lack of universal cells generally limits off-the-shelf therapies and reduces many therapies to tight tissue matching between donor and recipient. Immunosuppressants are administered with significant side effects in almost all cases.

[0003] An obvious side effect of immunosuppressant administration is an increased general susceptibility to infections and cancer. Commonly used immunosuppressants include cyclosporine, azathioprine, anti-lymphoblast, anti-thymocyte globulin, muromonab-CD3, and porcine anti-lymphoblast globulin (P-ALG). Cyclosporine is known to cause nephrotoxicity, hepatotoxicity, hyperkalemia, hypertension, tremors, gingival hypergrowth, and hirsutism. Azathioprine suppresses bone marrow suppression, leading to leukopenia. Anti-lymphoblast and anti-thymocyte globulins are exogenous antibodies that can cause allergic reactions, such as fever, chills, and hypotension. The initial side effects of monoclonal antibodies (muromonab-CD3, OKT3) are similar to those of P-ALG. These include high fever, chills, headache, rigidity, 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] This technical field includes many examples of attempts to produce cells compatible with any recipient. The most common approach is beta-2 microglobulin (B2M) interference, which eliminates the surface expression of all class I molecules but leaves the 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-stranded dimers (fused to B2M) or trimers (fused to B2M and peptide antigens) without the surface expression of any HLA-A, B, or C. These HLA-manipulated PSCs and their differentiated derivatives are CD8 + It is not recognized as an allogene by T cells, does not bind to anti-HLA antibodies, and is resistant to NK-mediated lysis. (Gornalusse) 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 US A. 1992;89(23):11381-5. Such approaches are genetic engineering approaches that 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 and lead to suboptimal cell products. Furthermore, the cell products are generated through multiple gene editing steps consisting of simultaneous knockout of all HLA class I molecules and knock-in of HLA-E B2M fusion proteins. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] CRISPR-Cas9 and other gene-editing technologies have ignited a race to create “off-the-shelf” donor cells invisible to the immune system. Common approaches to creating such cells involve the manipulation of genes required for immune recognition, particularly HLA class I and II proteins. Other approaches leverage knowledge of immune-cloaking strategies used by certain bacteria, viruses, parasites, fetal cells, 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 leading to cell and tissue rejection are also linked to autoimmune diseases. The need for safe and effective universal cells remains in this field. [Means for solving the problem]

[0006] Many pathogenic and non-pathogenic microorganisms have formed the immune systems according to the present invention, and as such, they themselves have evolved and mastered immune evasion, particularly in chronic infections. Epstein-Barr virus (EBV) is one example of such an immune system evader. We have found that it is possible to leverage immune evasion mechanisms evolved by various pathogens that grant immune privilege to other pathogens. Human cytomegalovirus (HCMV) inhibits T cell activity through the engagement of the UL11 protein (Figure 27) with CD45, resulting in interference with proximal signaling required for T cell activation and / or development. Similarly, the E3 protein (Figure 21) from adenoviruses engages with CD45 and inhibits NK and T cells. We have shown that graft rejection can be avoided by expressing molecules that bind to CD45 on the surface of graft cells through genetic modification (Figures 7-12). Following the same procedure, the inventors also assembled a single-chain monoclonal antibody against CD45 (a-CD45-sc) (Figure 24). Cells modified to express the CD45 engager are reported here for their immune evasion properties. For this purpose, the cytotoxicity of NK and T cells against target cells expressing UL11, E3.49K, or a-CD45-sc or GFP (as a control) was tested and compared (Figures 7-12).

[0007] CD45 is a transmembrane protein tyrosine phosphatase (PTPase) expressed on nucleated cells. It possesses a large, heavily glycosylated extracellular domain and a tandem intracellular phosphatase domain. CD45 covers approximately 10% of the surface area of ​​B and T cells, where it regulates cell development and activation by controlling membrane-proximal signaling. After cell synapse formation, CD45 dephosphorylates inhibitory tyrosine in the tails of SRC family kinases, enabling "open" non-inhibitory conformation. "Open" SRC family kinases achieve increased kinase activity through autophosphorylation on their own kinase domain activation loops. Activated SRC family kinases further phosphorylate protein molecules containing immune receptor tyrosine-based activation motifs (ITAMs) and SYK family kinases, resulting in signal transduction, propagation, and amplification. In successful cellular immune responses, CD45 is excluded from immune synapses and returned to the synapse only upon termination of the response. CD45 dephosphorylates activated 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 kinase. 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 endogenous catalytic activity in the membrane proximal domain. The membrane proximal extracellular domain 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) produces transcripts of variable length. Human CD45 may be a result of alternative exon use and can produce ABC, AB, BC, B, and O isoforms. The shortest product, in which all three exons (A, B, and C) are deleted, is called CD45RO, while the longest product, which contains all of these exons, is called CD45RABC.Different isoforms are used as developmental and activation markers in various lymphocytes. CD45RO is the conserved domain that is targeted among all isoforms.

[0008] CD148 is a receptor tyrosine phosphatase with a large, heavily glycosylated fibronectin extracellular and intracellular catalytic domain. Along with the hematopoietic line, CD148 is expressed in vascular and glandular endothelial cells, where it negatively regulates cell proliferation and transformation. Loss of CD148 has been observed in cancer cell lines, and reexpression resulted in suppression of tumor growth both in vitro and in vivo. CD148 dephosphorylates numerous 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 mucinous protein with a large extracellular domain and a small spherical intracellular domain, expressed on hematopoietic cells including stem cells, T cells, monocytes, granulocytes, NK cells, and platelets. The extracellular domain of CD43 promotes adhesion through interactions with E-selectin, galectin-1 and galectin-3, siglec-1, M-ficolin, integrins, cell surface nucleolins, and ICAM-1 (intercellular adhesion molecule type 1). The conserved intracellular domain is involved in signaling that mediates connectivity to the cytoskeleton through binding to ezrin, radixin, and moesin (ERM) proteins, while CD43 possesses a proline-rich sequence similar to the SH3-binding consensus and a nuclear localization signal (NLS) that explains CD43's nuclear localization.

[0010] B cell receptors (BCRs) are membrane-bound immunoglobulins with a short 3-amino acid intracellular domain. BCRs are composed of two identical heavy chains and two light chains. The extracellular domain has the ability to specifically recognize and bind to antigens. BCRs are composed of Igα(algebraic) chains. It lacks intracellular signaling compensated for by two associated ITAMs containing Igβ (beta) and β. After successful binding to the antigen, BCRs transmit signals leading to B cell activation and maturation. After class switching, BCRs are switched from membrane-bound to release form and are called antibodies.

[0011] Immunosynapses are interfaces between target cells and lymphocytes, also known as supramolecular activation clusters (SMACs) due to the accumulation of activating and regulatory molecules (Figures 1A-1E, left). Prior to immunosynapse formation, molecules are probabilistically distributed (Figure 1A, left). With 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, in turn, results in the activation of LCK. Finally, as CD45 is pushed to the periphery, co-receptors ligate, resulting in the formation of a mature synapse (Figure 1D, left). This interface, or SMAC, consists 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 a peripheral SMAC (pSMAC) consisting of LFA-1, ICAM-1, and Tallinn's adhesion ring, followed by the outermost circle called the distal SMAC (dSMAC), which consists of glycoproteins including CD45, CD43, and CD148.

[0012] In the system of the present invention, the formation and structural sequencing of immunological synapses is disrupted by implanting a bulky protein, such as CD45, in the center of the cell interface between graft cells and cytotoxic cells, such as T cells or NK cells (Figures 1A-E, right side; Figures 2A-2D). This not only prevents the formation of physiological SMACs (in the case of graft cell-T cell interactions), but also results in the sequential dephosphorylation of signaling pathways. It also results in interference with TCR / CD3 / MHCp, and cells may not even reach close enough to engage with TCR / CD3 / MHCp.

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

[0014] In embodiments, the present invention relates to CD45, CD148, or CD43 These cells form functional immunological synapses with cytotoxic cells. Modulate ability doThe therapeutic agent comprises one or more molecules or cells configured to prevent cytotoxicity. In embodiments, the therapeutic agent may comprise proteins, aptamers, peptide nucleic acids (PNAs), nanoparticles, or cells expressing or secreting one or more of these molecules. In embodiments, the therapeutic agent may comprise proteins, preferably proteins containing antibodies, more preferably proteins containing single-chain antibodies or VHH nanobodies. In embodiments, the therapeutic agent may comprise nanoparticles, preferably lipid nanoparticles (LNPs), dendrimers, or ribonucleoproteins (RNPs). In embodiments, the therapeutic agent may comprise extracellular vesicles, preferably exosomes or microvesicles. In embodiments, the therapeutic agent may comprise cells, preferably eukaryotic cells, more preferably avian or mammalian cells, such as mouse, pig, cat, dog, cat, or sheep cells, most preferably human cells. In embodiments, the therapeutic agent may comprise hematopoietic cells, stem cells, lymphoid cells, bone marrow cells, erythrocytes, or platelets. In embodiments, the therapeutic agent may comprise one or more excipients or additives, preferably fillers, bulking agents, diluents, wetting agents, solvents, emulsifiers, preservatives, absorption enhancers, sustained-release matrices, salts, buffers, starches, sugars, microcrystalline cellulose, granulators, 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 one or more combinations thereof. In embodiments, the therapeutic agent may be administered orally, cutaneously, enterally, or parenterally. In embodiments, the therapeutic agent may be delivered by injection (e.g., direct or systemic injection into diseased tissue), patch or other transdermal delivery device, or perfusion. In embodiments, the therapeutic agent may comprise components of viral or bacterial origin, preferably ULL or E3 / 49k, or fragments thereof. In embodiments, the therapeutic agent may include, for example, a viral or bacterial component that does not contain ULL protein or its fragments, or E3 / 49k protein or its fragments.In embodiments, the therapeutic agent may contain a protein having at least 80% identity to SEQ ID NOs: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, or to SEQ ID NOs: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224. In embodiments, the therapeutic agent may contain cells having one or more molecules expressed on the surface of the cells. In embodiments, the one or more molecules expressed on the surface of the cells include expressed transmembrane proteins, and the cells include graft cells. In embodiments, the transmembrane proteins 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 retain CD45, CD148, or CD43 in developing immunological synapses on the surface of cytotoxic cells, thereby interfering with the formation of functional immunological synapses.

[0015] In another embodiment, the present invention comprises a protein complex capable of preventing cytotoxic cell-induced lysis, comprising an engager containing a protein having at least 80% identity to SEQ ID NOs: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, or to SEQ ID NOs: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224; and a protein complex comprising a CD45, CD148, or CD43 protein expressed on the surface of a T cell or NK cell.

[0016] In another embodiment, the present invention includes a method for producing a composition for disrupting a functional immunological synapse, comprising the step of expressing one or more molecules on the surface of a first cell, wherein the one or more molecules are 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 and second cells.

[0017] In another embodiment, the present invention includes a method of promoting escape from NK-mediated lysis, which includes administering the above-described therapeutic agent to a subject that needs it. In an embodiment, the method may include inhibition or interference of NKG2D that binds to MICA, MICB, and / or ULBP. In an embodiment, the method includes interference with activating NK cell receptors selected from members of the human killer immunoglobulin-like receptor (KIR) family, CD94-NKG2C / E / H heterodimeric receptors, NKG2D, natural cytotoxicity receptors such as NKp30, NKp44, and NKp46, nectin / nectin-like binding receptor DNAM-1 / CD226 and CRTAM, receptors expressed by natural killer (NK) cells that regulate their activation, SLAM family receptors (including 2B4 / CD244, CRACC / SLAMF7, and NTB-A / SLAMF6), in addition to Fc gamma RIIIA / CD16a, CD27, CD100 / semaphorin 4D, and CD160. In an embodiment, the subject may be at risk of or suffering from one or more of the following conditions: autoimmune diseases, hematologic cancers including lymphoma and leukemia; myelodysplastic syndromes including anemia and cytopenia; hereditary immunodeficiencies including WAS and SCID; abnormal hemoglob inopathies 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 of promoting escape from T cell-mediated lysis, which includes administering the above-described therapeutic agent to a subject that needs it. In an embodiment, the method may include inhibition or interference of the T cell receptor that binds to MHC peptides. In an embodiment, the subject that needs it may be at risk of or suffering from one or more of psoriasis and vitiligo.

[0019] In another embodiment, the present invention provides a method for interfering with the formation of a functional immunological synapse by situally implanting CD45 on the surface of a CD45-expressing cell, comprising treating the CD45-expressing cell with an agent having affinity for the membrane-proximal region of the extracellular domain of CD45, thereby situally implanting CD45 against other membrane proteins expressed on the cell surface necessary for the formation of a functional immunological synapse.

[0020] In another embodiment, the present invention includes a non-autologous cell having an engager on its surface and configured to avoid synapse formation with one or more host cytotoxic cells. In the embodiment, the host cytotoxic cell is a natural killer cell, a T cell, or a macrophage. In the embodiment, the cytotoxic cell is a T cell, preferably a gamma-delta T cell, CD8 + T cells, CD4 + These are T cells, or mucosal-associated invariant T cells. In embodiments, non-autologous cells are not genetically modified. In embodiments, non-autologous cells may be treated with an engager.

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

[0022] In another embodiment, the present invention includes a method for preventing rejection of a solid organ or organoid graft, comprising transducing or transfecting cells of a solid organ or organoid with a gene to prevent or inhibit the binding of cytotoxic cells to cells of the solid organ or organoid graft. In embodiments, the gene may encode an engager, which may be expressed in an amount or density effective to inhibit the formation of functional immunological synapses upon exposure of the solid organ or organoid to cytotoxic cells.

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

[0024] In another embodiment, the present invention comprises 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. In an embodiment, the recombinant protein may include a second linker linking 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 linked in the order from the amino terminus to the carboxyl terminus of the protein. In an embodiment, the signal peptide may be an IL2 signal peptide. The first linker may contain an SGGGG motif and / or its length may vary between 5 and 60, preferably 10 and 50, more preferably 20 and 45 amino acids; if a second linker is present, its length may vary between 5 and 60, preferably 5 and 40, more preferably 7 and 15 amino acids; the stalk may have a length of at least 8 and no more than 200 amino acids, and the transmembrane region may originate from CD34, CD45, CD28, and / or Cd8a.

[0025] In another embodiment, the present invention includes cells containing an engager and an exogenous suicide gene.

[0026] In another embodiment, the present invention includes a first cytotoxic cell expressing membrane-bound CD45, CD148, and / or CD43, which may be treated to prevent the formation of functional immunological synapses with a second cytotoxic cell expressing membrane-bound CD45, CD148, and / or CD43. In the embodiment, the cytotoxic cell may be a natural killer cell, a T cell, or a macrophage.

[0027] In another embodiment, the present invention includes a graft treated to prevent the binding of cytotoxic cells, wherein the treatment comprises exposing the graft to a therapeutic agent as described above.

[0028] In another embodiment, the present invention includes a method for controlling inflammation, comprising administering mRNA or DNA encoding an engager to a target requiring it, thereby modulating the formation of functional immunological synapses and controlling 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 an engager to reduce the cytotoxic cell response to transplantation. In embodiments, the use may be carried out in the absence of HLA-I and / or HLA-II knockout or knockdown. In embodiments, the use may be carried out in combination with HLA-1 and / or HLA-II knockout or knockdown.

[0030] In another embodiment, the present invention includes cells comprising a surface-bound engager and a chimeric antigen receptor (CAR). In this embodiment, the CAR comprises a-CD38CAR (SEQ ID NO: 218) or a variant thereof having at least 80% identity thereto. In this 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 present invention includes an anti-CD45, anti-CD148, or anti-CD43 engager comprising a transmembrane domain configured on the surface of a cell. In an embodiment, the present invention includes an engager comprising a membrane-bound antibody, nanobody, or single chain against CD45, CD43, or CD148.

[0032] In another embodiment, the present invention includes a vector or plasmid for generating 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 an embodiment, the present invention includes a vector or plasmid encoding a membrane-bound antibody, nanobody, or single-strand antibody against CD45, CD148, or CD43. [Brief explanation of the drawing]

[0033] [Figure 1A] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to mature immune synapses. [Figure 1B] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to mature immune synapses. [Figure 1C] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to mature immune synapses. [Figure 1D]Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to mature immune synapses. [Figure 1E] Figures 1A-1E are snapshots showing the stages of supramolecular activation cluster (SMAC) formation that lead to mature immune synapses. [Figure 2A] Figure 2A shows the immunological synapses between host T cells and graft cells. Engagement of the host TCR with the donor MHC-peptide complex leads to graft necrosis. [Figure 2B] Figure 2B illustrates the interaction between host T cells and graft cells expressing a novel engager that keeps CD45 centrally located at the synapse. This leads to non-lethal graft formation and a lack of functional immunological synapse formation. [Figure 2C] Figure 2C shows the immunological synapses between host NK cells and graft cells. Engagement of the host-activated receptor with the recipient ligand leads to graft necrosis. [Figure 2D] Figure 2D illustrates the interaction between host NK cells and graft cells expressing a novel engager that keeps CD45 centrally located at the synapse. This leads to non-lethal graft formation and a lack of functional immunological synapse formation. [Figure 3] Figure 3 is a map of plasmid LeGO-iG2-UL11. [Figure 4] Figure 4 is a map of plasmid LeGO-iG2-E3.49k. [Figure 5] Figure 5 is a map of the plasmid LeGO-iG2-A-CD45-SC. [Figure 6] Figure 6 shows the generation of a stable cell line. [Figure 7]Figure 7 is a bar graph showing the inhibition of cell lysis in cells transformed with a-CD45-sc, E3.49K, or UL11 (controls are untransformed). The y-axis shows the percentage of specific lysis 51Cr release in K562 cells incubated with PBMCs. The effector:target (E:T) ratio is shown below the bar groupings. [Figure 8] Figure 8 is a bar graph showing the inhibition of cell lysis in cells transformed with a-CD45-sc, E3.49K, or UL11 (controls are untransformed). The y-axis shows the percentage of specific lysis 51Cr release in NK92 cells and incubated K562 cells; the E:T ratio is shown below the bar groupings. [Figure 9] Figure 9 is a line graph showing the inhibition of cell lysis in K562 cells transformed with a-CD45-sc, E3.49K, or UL11 (controls were untransformed) upon exposure to PBMC cells. The y-axis represents the percentage of specific lysis 51Cr release in K562 cells incubated with PBMCs; the x-axis represents the E:T ratio. [Figure 10] Figure 10 is a line graph showing the inhibition of cell lysis in K562 cells transformed with a-CD45-sc, E3.49K, or UL11 (controls were untransformed) upon exposure to NK92 cells. The y-axis represents the percentage of specific 51Cr release in K562 cells incubated with NK92 cells; the x-axis represents the E:T ratio. [Figure 11] Figure 11 is a line graph showing predictive data on the inhibition of cell lysis in RPMI88226 cells transformed with a-CD45-sc, E3.49K, or UL11 (controls were untransformed) upon exposure to T cells. The y-axis points to the percentage of specific lysate 51Cr release; the x-axis shows the E:T ratio. [Figure 12]Figure 12 is a line graph showing data on the inhibition of cell lysis in CD34-differentiated T-like cells transformed with a-CD45-sc, E3.49K, or UL11 (controls were untransformed) upon exposure to CD8+ T cells. The y-axis represents the percentage of specific lysis 51Cr release; the x-axis represents the E:T ratio. [Figure 13] Figure 13 is a map of the plasmid LeGO-iG2-a-CD45-(M)-VHH1. [Figure 14] Figure 14 is a 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] Figure 17 is a map of the plasmid LeGO-iG2-mVHH1-E3TM. [Figure 18] Figure 18 is a map of the plasmid LeGO-iG2-mVHH2-E3TM. [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 schematic representation of E3.49K (sequence number 3). [Figure 22] Figure 22 is a schematic representation of E3.49K.R1 (sequence number 66). [Figure 23] Figure 23 is a schematic representation of E3.49K.R3 (Sequence ID 68). [Figure 24] Figure 24 is a schematic representation of a-CD45-sc (sequence number 5). [Figure 25] Figure 25 is a schematic representation of m-VHH1-E3-TM (Sequence ID 220). [Figure 26] Figure 26 is a schematic representation of m-VHH2-E3-TM (Sequence ID 222). [Figure 27] Figure 27 is a schematic representation of UL11 (sequence number 1). [Figure 28] Figure 28 is a schematic representation of a-CD38CAR (sequence number 218). [Figure 29] Figure 29 is a schematic representation of a-CD19CAR (sequence number 216). [Figure 30] Figure 30 is a line graph showing the cytolysis of target cells by NK92 cells expressing a-CD45-sc. [Figure 31] Figure 31 is a line graph showing the cytolysis of target cells by TALL-104 cells expressing a-CD45-sc. [Figure 32] Figure 32 is an experimental flowchart that was followed for the in vivo experiment. [Figure 33] Figure 33 is a compilation of IVIS images of RPMI-8226 cells transduced with luciferase and CD45 engagers, treated with PBMCs and daratumumab. Although the same dose of RPMI8226 cells is administered, a higher tumor burden is observed compared to those in Figure 34. [Figure 34] Figure 34 is a compilation of IVIS images of RPMI-8226 cells transduced with luciferase (but without the CD45 engager) and treated with PBMCs and daratumumab. Minimal controlled residual disease is observed. [Figure 35] Figure 35 is a line graph showing the effect of a-CD45-sc on K562 cells after exposure to PBMCs. Figure 35 shows IVIS imaging analysis in K562 tumor-carrying mice compared to K562 cells with CD45 engagers. All mice depicted were given 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-carrying mice compared to SKOV3 cells with CD45 engagers. All mice depicted were given PBMCs and trastuzumab, except for the control group which was given PBMCs only. Each line represents one mouse. [Figure 37] Figure 37 is a schematic diagram of mRNA loading onto EVs. [Figure 38] Figure 38 is a line graph showing arthritis scores after therapeutic EV injection. A higher score indicates a higher degree of malignancy. Each limb was scored using a scale of 0–4 based on the increase in the level of erythema and swelling. [Figure 39A] Figures 39A and 39B are bar graphs showing TNFα (pg / 100μg protein) and IL1b (pg / 100μg protein) secretion in an arthritis model after therapeutic EV injection. [Figure 39B] Figures 39A and 39B are bar graphs showing TNFα (pg / 100μg protein) and IL1b (pg / 100μg protein) secretion in an arthritis model after therapeutic EV injection. [Figure 40] Figure 40 is a schematic flowchart illustrating EV production / isolation and purification of therapeutic EVs. [Figure 41] Figure 41 is a line graph showing the cell lysis of target cells (RPMI8226) by NK92 cells co-expressing α-CD45-sc and α-CD38CAR, as evaluated by the 51Cr release assay. [Figure 42] Figure 42 is a line graph showing the cell lysis of target cells (CD38KO RPMI8226) by NK92 cells co-expressing α-CD45-sc and α-CD38CAR, as evaluated by the 51Cr release assay. [Figure 43] Figure 43 is a bar graph showing the degranulation of target cells (Raji and Jurkat) by NK92 cells co-expressing a-CD45-sc and a-CD19CAR. [Figure 44] Figure 44 shows a co-culture of live target cells (K562) with NK92 co-expressing α-CD45-sc and α-CD38CAR. Dead cells appear bright, while effector cells appear dark. This is a microscopic representation of what is demonstrated in Figures 42 and 43. [Modes for carrying out the invention]

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

[0035] Introduction

[0036] The differentiation ability of pluripotent stem cells, such as embryonic stem cells (ESCs), makes it possible for these cells to provide an unlimited supply of any cell type for transplantation. ESCs were expected to provide “off-the-shelf” cell therapies 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) provided a solution to generate pluripotent cells from a patient and then differentiate them into the cell type then required. iPSC generation, gene repair, and differentiation, as well as validation of their therapeutic and safety for individual patients, are not affordable in terms of convenience and cost. Despite immune rejection remaining a critical issue in the field of cell therapy, remarkable progress has been made in the morphology 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] Sequential immunosuppression with cyclosporine and cyclophosphamide has been the only option in organ transplantation and autoimmune treatment. Furthermore, cyclophosphamide and cyclophosphamide are used to create an environment in which donor cells can be retained for a period of approximately two weeks in the context of donor lymphocyte infusion, chimeric antigen receptor modified T cell therapy, and other genetically modified cell infusions. The use of fludarabine treatment regimens has been utilized for transient host lymphocyte depletion. It is also used in alternative therapies for extended in vivo expression of gene therapy vectors. In cell transplantation, to prevent host T cell-mediated rejection of allogeneic grafts, donor cell HLA was knocked out as a possible host CD8+ T cell (HLA class I knockout) and CD4+ T cell (HLA class II knockout) mediated immune evasion strategy. Simultaneously, non-classical HLA expression was forced in these cells to prevent NK cell-mediated cytotoxicity. Similarly, CTLA4Ig was used to prevent T cell CD28 co-receptor ligation and thus immune response against donor cells, and CD40 mAbs were used to attenuate APC and B cell function. In some studies, viral proteins redirected to intracellular signaling and antigen processing have been utilized to prevent immune responses. For example, ICP4, a cytosolic protein from HSV, inhibits TAP-mediated transport of peptides to the endoplasmic reticulum (ER), while HCMV proteins US11 / 2 lead to MHC-I degradation, US3 retains MHC-I in the ER, and US6 blocks TAP.

[0038] The inventors aim to utilize an immune evasion method used by viruses that targets direct extracellular, intercellular trans-interactions with regulatory proteins of T cells and NK cells. UL11 is a member of the RL11 protein family, expressed on the surface of CMV-infected cells, and binds to CD45 on leukocytes (Figure 3, 27). CD45, a protein tyrosine phosphatase, is a key regulator in T cell antigen receptor (TCR) signaling. CD45 activates SRC family kinases by removing their C-terminal inhibitory phosphorylation. Activated SRC family kinases phosphorylate ITAM 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 blocks both T cell activation and development by binding to CD45 and blocking downstream signaling.

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

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

[0041] CD148 is a receptor tyrosine phosphatase with a large, heavily glycosylated fibronectin extracellular domain and intracellular catalytic domain. Along with the hematopoietic lineage, CD148 is also expressed in vascular and glandular endothelial cells, where it negatively regulates cell proliferation and transformation. Loss of CD148 has been observed in cancer cell lines, and reexpression resulted in suppression of tumor growth both in vitro and in vivo. CD148 dephosphorylates numerous 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 mucinous protein with a large extracellular domain and a small spherical intracellular domain, expressed on hematopoietic cells including stem cells, T lymphocytes, monocytes, granulocytes, NK cells, and platelets. The extracellular domain of CD43 promotes adhesion through interactions with E-selectin, galectin-1 and galectin-3, siglec-1, M-ficolin, integrins, cell surface nucleolins, and ICAM-1 (intercellular adhesion molecule type 1). The conserved intracellular domain is involved in signaling that mediates connectivity to the cytoskeleton through binding to ezrin, radixin, and moesin (ERM) proteins. CD43 has a proline-rich sequence similar to the SH3 binding consensus and a nuclear localization signal (NLS) that explains CD43's nuclear localization.

[0043] B cell receptors (BCRs) are membrane-bound immunoglobulins with a short, three-amino acid intracellular domain. BCRs are composed of two identical heavy chains and two light chains. The extracellular domain has the ability to specifically recognize and bind to antigens. Lacking intracellular signaling, this Igα chain and Igβ chain It is compensated for by two associated ITAMs containing [the substance]. After successful binding to the antigen, the BCR transmits signals that lead to B cell activation and maturation. After class switching, the BCR switches from a membrane-bound form to a release form called an antibody.

[0044] Referring to Figure 2A, the figure shows the formation of synapses between T cells and target cells in the absence of the present invention, which leads to target cell lysis. In Figure 2B, physiological synapses are prevented and lysis does not occur using the engager of the present invention.

[0045] Referring to Figure 2C, the figure shows the formation of synapses between NK cells and target cells in the absence of the present invention, which leads to target cell lysis. In Figure 2D, physiological synapses are prevented and lysis does not occur using the engager of the present invention.

[0046] While data presented by the present invention show that forced retention of CD45 in immunosynapses via engagers prevents cell lysis by cytotoxic cells (Figures 7-12), inhibition of cytotoxic cells through CD43 and CD148 immunosynaptic retention can also prevent lysis. Engagers may be molecules used to interfere with the binding of CD45, CD43, and CD148. “Engagers” are molecules or groups of molecules that can bind to CD45, CD43, or CD148, thereby inhibiting or preventing the formation of functional immunological synapses. “Functional immunological synapses” are immunosynapses that can be formed between CD45, CD148, or CD43-positive cells and graft cells, including non-autologous cells. The inventors have used single-chain, single-domain, and antibody effectors. Those skilled in the art can identify other engagers using the teachings disclosed herein.

[0047] The engager should be present in sufficient quantity to bind to CD45, CD43, or CD148. We have shown that it is possible to modulate or shut down the NK or T cell response to exogenous cells, as will be shown in more detail below. In certain embodiments, the compositions and methods disclosed herein are non-agonist.

[0048] In embodiments, variants of the amino acid sequences disclosed herein are also envisioned. 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 with one or more of the disclosed amino acid sequences. In certain preferred embodiments, an exemplary amino acid sequence may be an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more of the disclosed amino acid sequences. In embodiments, the variant amino acid sequence retains the functions attributed herein (e.g., the ability to bind to CD45, CD43, or CD148 and / or the ability to prevent or inhibit the formation of functional immunological synapses and / or the ability to confer immune evasion and / or the ability to prevent cytotoxicity).

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

[0050] In embodiments, the engager may include, for example, an amino acid sequence in the amino acid sequence of SEQ ID NOs. 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 are deleted, substituted, inserted, and / or added, and which has activity to bind to CD45 and / or inhibit or prevent the formation of a functional immunological synapse. In a preferred embodiment, the engager disclosed herein is an amino acid sequence of SEQ ID NOs: 1, 3, 5, 64, 66, 68, 71, 73, 220, 223, or 224, for example, 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, 1-2 The protein sequence comprises an amino acid sequence having one amino acid residue deleted, substituted, inserted, and / or added, and having activity that inhibits or prevents the formation of a functional immunological synapse.

[0051] The inventors perform assays for competitive and deletion mutants of antibodies to determine the binding sites of the engagers. Immunoprecipitation is used to identify interacting motifs. Top candidates are collected for further experiments.

[0052] All three CD45 engagers (E3.49K, UL11, and α-CD45-sc) bind to all isoforms of CD45, suggesting interaction with the membrane proximal region, including fibronectin-III and cysteine-rich domains. Immunoprecipitation studies revealed physical interactions between CD45 and E3.49K, UL11, or anti-CD45-sc, and Ab competition experiments and deletion mutations showed that E3.49K, UL11, and α-CD45 -sc further supports the concept that it primarily interacts with the membrane-proximal region of CD45, which is common to all isoforms.

[0053] This invention also envisions the use of aptamers directed to CD45, CD43, and CD148. Aptamers are short chains of nucleic acids, proteins, or other properties that, similar to antibodies, 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 that consist of either nucleic acids, proteins, or other molecules that can specifically bind to the target molecules CD45 and / or CD148 and / or CD43. These aptamers may be naturally occurring or de novo synthesized. 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 may be used in the absence of HLA-I and HLA-II knockout or knockdown strategies. However, it is also conceivable that combinations of HLA class-I (e.g., B2M) and / or HLA class-II (e.g., CIITA) with CD45 / CD148 / CD43 engagers may 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. [Examples]

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

[0057] The inventors initially began by demonstrating that CD45 content in immunological synapses can be modulated using UL11 and E3.49K.

[0058] material and method

[0059] A vector was created incorporating the HCMV-M (Merlin strain; HHV5) protein UL11 sequence downloaded from uniprot. The UL11 sequence is shown below as Sequence 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(Sequence ID 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 to generate lentiviral particles. Transduction was performed into K562 and RPMI82261 cells using the viral particles, and these cells were grown in RPMI 1640 medium supplemented with 10% FBS. Transduced cells were magnified and sorted for GFP expression. Sorted cells were magnified and subjected to killing assays and degranulation assays. This was done for UL11 and E3.49K to generate 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] The following Sequence ID No. 2 is UL11, codon-optimized for human cells.

[0064] (Sequence 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 Sequence 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 (Sequence ID 3)

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

[0068] Sequence ID 4

[0069]

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

[0071] Using the method described in Example 1, a single chain recognizing CD45 was designed as shown below to obtain the plasmid LeGO-iG2-a-CD45-sc shown in Figures 5 and 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 anti-CD45 antibodies that has a stalk and transmembrane region linked 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 region is the heavy chain, the underlined uppercase region is the linker, the ununderlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane region. myrmqllscialslalvtns qvqlvesggglvqpggslklscaasgfdfsrywmswvrqapgkglewigeinptsstinftpslkdkvfisrdnakntlylqmskvrsedtalyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGSGTKLEIK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 5)

[0073] First, cDNA was generated using a single-chain (SC) stalk and CD34 transmembrane region along with an IL-2 signal peptide, VH, linker, VL, and linker. Sequence ID 6 below encodes the plasmid LeGO-iG2-a-CD45-sc.

[0074] In the sequence below, the underlined lowercase regions are the IL2 signal peptide, the lowercase regions are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chains, the bold uppercase regions are the stalks, and the bold underlined regions are the CD34 transmembrane domains.

[0075] atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt caggttcagctggtggaatcaggaggtggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcgatttcagtagatactggatgagttgggtccggcaggctccagggaaagggctagaatggattggagagattaatccaactagcagtac gataaactttacgccatctctaaaggataaagtcttcatctccagagacaacgccaaaaatacgctgtacctgcaaatgagcaaagtgagatccgaggacacagccctttattactgtgcaagagggaactactataggtacggagatgctatggactactggggtcaag gaacctcagtcaccgtgagcaagatc TCTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGCTCGGGTGGTGGTGGGTCGGGCGGCGGCGGCTCGAGCGACATCGTGCTGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGACAGAGGGCCACCATCTCATGCAGGGCCAGCAAAGTGTCAGTACATCTGGCTATAGTTATCTGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCAT CCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG AGCTCTGGCTCTGGTTCG CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCGCAGGTGCACACGAGGGGGCTGGACTTC GCCCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTG TAA (Sequence ID 6)

[0076] CD45 single-chain generation Single-chain antibodies are fusion proteins of light and heavy chains joined by a linker. The translation of the CD45 single-chain protein is shown in Sequence ID No. 7 below. The heavy chain is shown in lowercase, and the light chain in uppercase. The linker is shown in underlined uppercase.

[0077] In the array below, lowercase letters represent heavy chains, underlined uppercase regions represent linkers, and ununderlined uppercase regions represent light chains. qvqlvesggglvqpggslklscaasgfxfsrywmsxvrqapgkglewigeinptsstinxtpslkdkvfisrdnakntlylqmskvrsedtaxyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGSGGGGSGGGGSGGGGSS DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK SSGSGS (Sequence ID 7)

[0078] The heavy chain is coded by sequence number 8. QVQLVESGGGLVQPGGSLKLSCAASGFXFSRYWMSXVRQAPGKGLEWIGEINPTSSTINXTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAXYYCARGNYYRYGDAMDYWGQGTSVTVSKI (Sequence ID 8)

[0079] The light chain is coded by sequence number 9. DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK (Sequence ID 9)

[0080] Stork

[0081] A stalk is a structural domain between a single-chain antibody and the cell's outer membrane. This, or a portion thereof, is sometimes referred to as a hinge or spacer. The stalk helps to position the antibody region at a desired location outside the cell membrane. The stalk is preferably 8 to 200 amino acids long. The stalk needs to protrude from the cell membrane surface but should not be long enough to fold. This stalk is fused to the single-chain antibody, which then binds to the transmembrane domain. In this example, the inventors utilized 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 5'3' frame 1, sequence number 10. cccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgccc ctaggaaaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO: 10)

[0083] Sequence ID 10 encodes the protein of Sequence ID 11 below. PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKP(Sequence ID 11)

[0084] Stork included the underlined Homo sapiens CD8a sequence shown below in sequence number 12 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(Sequence ID 12)

[0085] The underlined stalk region shown above is designated as Sequence ID 13 and is shown below: PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (Sequence ID 13)

[0086] CD8a

[0087] The CD8a nucleotide sequence is shown below as sequence number 14. The underlined region codes for Stork. Nucleotide sequence (708 nt): ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAGCCAGTTCCGGGTGTCGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTGGAGCTGAAGTGCCAGGTGCTGCTGCTGTCCAACCCGACGTCGGGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCCAGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCAAGGCG GCCGAGGGCTGGACACCCAGCGGTTCTCGGGCAAGAGGTTGGGGACACCTTCGTCCTCACCCTGAGCGACTTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCCCTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAG CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC TGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGGTCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACGTCTAA (SEQ ID NO: 14)

[0088] CD8a Stork is encoded by the polynucleotide shown in SEQ ID NO: 15 below: CCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC (SEQ ID NO: 15)

[0089] CD8a translation (235aa): MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV(Sequence ID 16)

[0090] The inventors used the underlined portion of Sequence ID No. 16 as the stalk and the transmembrane shown in Sequence ID No. 17. PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(Sequence ID 17)

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

[0092] The cDNA with the CD28 nucleotide sequence is shown below as Sequence ID No. 19: ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGC AAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGC AAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 19)

[0093] In the final construct, the underlined portion of sequence number 19 is shown below as sequence number 20, which serves as the stalk portion. KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(Sequence ID 20)

[0094] transmembrane region

[0095] The transmembrane region is useful for anchoring the stalk / protein to the cell. The transmembrane region was taken from CD34 FASTA, and its protein sequence is shown below as Sequence 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 (Sequence ID 21)

[0097] The inventors used the following sequence, taken from the underlined portion of Sequence ID No. 20, as a transmembrane sequence: LIALVTSGALLAVLGITGYFL (Sequence ID No. 22).

[0098] The protein of sequence number 21 is encoded by the cDNA of sequence number 23 below. ATGCTGGTCCGCAGGGGCGCGCGCGCAGGGCCCAGGATGCCGCGGGGCTGGACCGCGCTTTGCTTGCTGAGTTTGCTGCCTTCTGGGTTCATGAGTCTTGACAACAACGGTACTGCTACCCCAGAGTTACCTACCCAGGGAACATTTTCAAATGTTTCTACAAATGTATCCTACCAAGAAACTACAACACCTAGTACCCTTGGAAGTACCAGCCTGCACCCTGTGTCTCAACATGGCAAT GAGGCCACAACAAACATCACAGAAACGACAGTCAAATTCACATCTACCTCTGTGATAACCTCAGTTTATGGAAACACAAACTCTTCTGTCCAGTCACAGACCTCTGTAATCAGCACAGTGTTCACCACCCCAGCCAACGTTTCAACTCCAGAGACAACCTTGAAGCCTAGCCTGTCACCTGGAAATGTTTCAGACCTTTCAACCACTAGCACTAGCCTTGCAACATCTCCCACTAAACCCTATACATCATCTTCTCCTATCCTAAGTGACATCAAGGCAGAAATCAAATGTTCAGGCATCAGAGAAGTGAAATTGACTCAGGGCATCTGCCTGGAGCAAAATAAGACCTCCAGCTGTGCGGAGTTTAAGAAGGACAGGGGAGAGGGCCTGGCCCGAGTGCTGTGTGGGGAGGAGCAGGCTGATGCTGATGCTGGGGCCCAGGTATGCTCCCTGCTCCTTGCCCAGTCTGAGGTGAGGCCTCAGTGTCTACTGCTGGTCTTGGCCAACAGAACAGAAATTTCCAGCAAACTCCAACTTATGAAAAAGCACCAATCTGACCTGAAAAAGCTGGGGATCCTAGATTTCACTGAGCAAGATGTTGCAAGCCACCAGAGCTATTCCCAAAAG ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTGATGAATCGCCGCAGCTGGAGCCCCACAGGAGAAAGGCTGGGCGAAGACCCTTATTACACGGAAAACGGTGGAGGCCAGGGCTATAGCTCAGGACCTGGGACCTCCCCTGAGGCTCAGGGAAAGGCCAGTGTGAACCGAGGGGCTCAGGAAAACGGGACCGGCCAGGCCACCTCCAGAAACGGCCATTCAGCAAGACAACACGTGGTGGCTGATACCGAATTGTGA (SEQ ID NO: 23)

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

[0100] ACCCTGATTGCACTGGTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACTGGCTATTTCCTG(Sequence No. 24)

[0101] Transmembrane domains from other proteins (membrane-bound) can also be utilized, such as the transmembrane domain of CD34. There appear to be no restrictions 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 listed below.

[0102] CD45

[0103] The transmembrane region of CD45 is underlined in the protein of Sequence 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(Sequence ID 25)

[0104] CD45 transmembrane domain

[0105] >sp|P08575|578~598

[0106] ALIAFLAFLIIVTSIALLVVL (Sequence ID 26)

[0107] CD45 DNA sequence ATGACCATGTATTTGTGGCTTAAACTCTTGGCATTTGGCTTTGCCTTTCTGGACACAGAAGTATTTGTGACAGGGCAAAGCCCAACACCTTCCCCCACTGGATTGACTACAGCAAAGATGCCCAGTGTTCCACTTTCAAGTGACCCCTTACCTACTCACACCACTGCATTCTCCGCAAGCACCTTTGAAAGAGAAAATGACTTCTCAGAGACCACAACTTCTCTTAG GCACTGATAGCATTTCTGGCATTTCTGATTATTGTGACATCAATAGCCCTGCTTGTTGTTCTC TACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTTAGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGATTGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTATAAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATTATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCACAAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGA

[0108] CD28

[0109] The transmembrane region of CD28 is underlined in the protein of sequence number 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(Sequence ID 28)

[0110] CD28 transmembrane domain >sp|P10747|153~179 FWVLVVVGGVLACYSLLVTVAFIIFWV (Sequence ID 29)

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

[0112] The underlined region in Sequence ID No. 30 is the transmembrane domain that encodes Sequence ID No. 29.

[0113] CD8a

[0114] The protein sequence of CD8a is shown in Sequence 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(Sequence ID 31)

[0115] CD8a transmembrane domain

[0116] >sp|P01732|183~203

[0117] IYIWAPLAGTCGVLLLSLVIT(Sequence ID 32)

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

[0119] The underlined region in Sequence ID No. 33 encodes the transmembrane region of the protein.

[0120] Signal peptide

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

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

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

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

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

[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 (Sequence ID 37)

[0127] IFN gamma signal peptide >sp|P01579|1~23 MKYTSYILAFQLCIVLGSLGCYC (Sequence ID 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 ELCDDDPPEIPHATFKAMA YKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI(Sequence ID 40)

[0131] CD25 signal peptide

[0132] >sp|P01589|1~21

[0133] MDSYLLMWGLLTFIMVPGCQA(Sequence ID 41)

[0134] IL2Ra DNA sequence. The DNA encoding the IL2Ra signal peptide is underlined in sequence number 42. ATGGATTCATACCTGCTGATGTGGGGACTGCTCACGTTCATCATGGTGCCTGGCTCGCCAGGCA(Sequence ID 42)

[0135] The a-CD45-sc translation is shown as sequence number 43.

[0136] In the array below, the bold lowercase letters represent heavy links, the underlined uppercase letters represent linkers, and the ununderlined bold uppercase letters represent light links.

[0137] CD45 V H V L qvqlvesggglvqpggslklscaasgfxfsrywmsxvrqapgkglewigeinptsstinxtpslkdkvfisrdnakntlylqmskvrsedtaxyycargnyyrygdamdywgqgtsvtvski SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLL IYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTXGSGTKLEIK SSGSGS (Sequence No. 43)

[0138] Homo sapiens CD8a molecule (CD8A), transcript variant 4, non-coding RNA Array ID: NR_027353.1 Length: 2621 Number of matches: 1 Related Information Gene - Details of related genes UniGene-clustered expression sequence tags GEO Profile - Microarray Expression Data PubChem Bioassay - 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 Array ID: NM_006139.4 Length: 4721 Number of matches: 1 Related Information Gene - Details of related genes PubChem Bioassay - Bioactivity Screening Genome Data Viewer - Context of Aligned Genomes Range 1: 395~514 GenBankGraphics

[0141] [Table 2]

[0142] a-CD45-sc; Additional Engagers

[0143] The inventors have created an additional engager: anti-CD45(9.4) single chain. This is derived from human HIB-10508=9.4=IgG2a=mouse anti-human CD45.

[0144] The protein sequence is shown in Sequence ID No. 54 below. The underlined lowercase region is the IL2 signal peptide, the lowercase region is the heavy chain, the underlined uppercase region is the linker, the ununderlined 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 (Sequence No. 54)

[0146] This results in an anti-CD45(9.4) single-strand codon-optimized cDNA, shown as Sequence ID No. 55 below. ATGTACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCCAGGTGCAGCTGCAGCAGCTGGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATG

[0147] The inventors have created anti-CD45 (GAP8.3) monochains from light and heavy chains. The light and heavy chain sequences were obtained from GAP8.3 hybridoma (ATCC® HB-12®) = IgG2a, kappa = immunoglobulin; monoclonal antibody; against human leukocytes (monocytes, lymphocytes, granulocytes); against CD45.

[0148] In the sequence below (SEQ ID NO: 56), the underlined lowercase segments are the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are the CD34 transmembrane domain. myrmqllscialslalvtns evqlqlqqsgpelvktgasvkisckasgysftgyfihwvkqshgkslewigyiscyngatsynqkfkgkatftvdtssstaymqfnsvtsedsavyycvrnyygnldamdywgqgtsvtvssa SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKILIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPRTFGGGTKLEIKRADAAQTCI SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 56)

[0149] Anti-CD45 (GAP8.3) single-chain codon optimization cD for the protein of sequence number 56 NA is represented as sequence number 57 below.

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

[0151] In the sequence below (SEQ ID NO: 58), the underlined lowercase segments are the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are CD34. myrmqllscialslalvtns qvqlkesgpglvkpsltlsltctvsgfslnsygviwvrqppgkglewlgvkwgygntnynsalksrlninrdtsksqvflkmdnvqtedtamyfcarsrfnyggpldywgqgvmvtvssa SGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSS DIVLTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRADAAPTVS SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL ( Sequence ID 58)

[0152] Codon-optimized anti-CD45(M1) for human cell expression.

[0153] Anti-CD45(4B2) single-chain 4B2 (ATCC(registered trademark) HB-196(trademark)) = Mouse anti-human CD45.

[0154] In the sequence below (SEQ ID NO: 60), the underlined lowercase segments are the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are CD34. myrmqllscialslalvtns qvqlkesgaelarpgasvkmsckasgytftsytmqwvkqrpgqglewigyinpssgyikynqkfkdkvtltadkssttaymqlsrltsedsavyycarrgsyffdfwgqgtsvtvssa SGGGGSGGGGSGGGGSGGGGSGGGGSS DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPFTFGGGTKLEVKRADAAPTVS SSGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 60)

[0155] Codon-optimized anti-CD45(4B2) 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 Monoclonal antibody, and construction of a mouse / human chimeric antibody. J Biochem. 1996 Sep;120(3):657-62. doi: 10.1093 / oxfordjournals.jbchem.a021462. PMID: 8902633. Anti-CD3 (OKT3) monochain taken from this antibody. In the sequence below (SEQ ID NO: 62), the underlined lowercase segments are the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are CD34.

[0157] myrmqllscialslalvtns qvqlqqsgaelarpgasvkmsckasgytftrytmhwvkqrpgqglewigyinpsrgytnynqkfkdkatlttdkssstaymqlssltsedsavyycaryyddhycldywgqgttltvssak SGGGGSGGGGSGGGGSGGGGSGGGGSS QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRS SGSGS TGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 62)

[0158] A codon-optimized anti-CD3 (OKT3) monochain for human cell expression is shown below as Sequence ID No. 63.

[0159] The anti-CD148 single chain was taken from sequence AB1 of U.S. Patent Application Publication No. 2005 / 0287,140(A1). In the sequence below (Sequence ID 64), the underlined lowercase segments are the IL2 signal peptide, the lowercase letters are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are the transmembrane domain of CD34.

[0160] myrmqllscialslalvtns evqllesggglvqpggslrlscaasgftfssyamswvrqapgkglewvsaisgsggstyyadsvkgrftisrdnskntlylqmnslraedtavyycargrtevatpgaywgqgtmvtvssa SGGGGSGGGGSGGGGSGGGGSGGGGSS QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAVTGLQAEDEADYYCQSYDSSLSDVFGGGTKLTVLSGSGSGSTGPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 64)

[0161] A codon-optimized anti-CD148 single strand for human cell expression is shown as cDNA in Sequence ID No. 65 below.

[0162] E3.49K R1 mutant

[0163] E3.49K R1 mutants were created by deleting a portion of the extracellular region of E3.49k taken from Uniprot (Figures 15 and 22). In the following sequence (SEQ ID NO: 66), the underlined lowercase segment is the E3.49K signal peptide, and the lowercase letters are This is the R1 domain, where the underlined capitalized region is the linker, the ununderlined capitalized region is the proximal extracellular region of E3.49K, the bold underlined region is the transmembrane region of E3.49K, and the subsequent bold capitalized region is the intracellular region of E3.49K.

[0164] mntvirivllsllvafsqagfhtinatwwanitlvg ppdtpvtwydtqglwfcngsrvknpqirhtcndqnltlihvnktyertymgynrqgtkkedykvvvi GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (Sequence ID 66)

[0165] E3.49K R1

[0166] The codon-optimized E3.49K R1 for human cell expression is shown in the cDNA of Sequence 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 ununderlined uppercase region is the proximal extracellular region of E3.49K, the bold underlined region is the transmembrane region of E3.49K, and the subsequent bold uppercase region is the intracellular region of E3.49K.

[0168] atgaacacggtgatccgcatagtccttctgtctctgctggtggctttctcccaggccggcttccacacaatt aatgccacctggtgggctaacattactctcgtaggccccccggatacccccgtgacttggtacgacactcagggtctgtggttctgtaacgggagtcgagtgaaaaatcctcaaattc gccatacctgtaacgaccaaaatctgaccttgatccacgtgaacaagacatacgagcgtacatatatgggctacaataggcagggtacaaagaaagaggactataaagtggtagtgatt GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA(Sequence No. 67)

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

[0170] In the sequence below (SEQ ID NO: 68), the underlined lowercase region is the E3.49K signal peptide, the underlined uppercase region is the linker, the lowercase region is the R3 domain, the ununderlined uppercase region is the proximal extracellular region of E3.49K, the bold underlined region is the transmembrane region of E3.49K, and the subsequent bold uppercase region is the intracellular region of E3.49K. mntvirivllsllvafsqagfhtinatwwanitlvGGGGS vtvtagsnltlvgpkaegkvtwfdgdlkrpcepnyrlrhecnnqnltlinvtkdyegtyygtndkdegkryrvkvNTTNS QSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (Sequence ID 68)

[0171] The codon-optimized cDNA for human cell expression of E3.49K-Ig-R3 is shown below as sequence number 69. atgaacacggtgatccgcatagtccttctgtctctgctggtggctttctcccaggccggcttccacacaattaatgccacctggtgggctaacattactctcgtaGGCGGCGGCGGCAGC gtgacagtaactgctggaagtaacctgaccctcgtggggcccaaggcggaggggaaagtaacctggttcgacggcgatctaaaacgcccctgtgaaccaaactacagacttagacacgaatgcaacaaccagaacctgactctgattaacgtgaccaaggactacgaaggaacatac tacgggacgaatgataaggatgagggaaaacggtaccgggttaaggttAACACCACAAACTCCCAGAGTGTCAAAATTCAGCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA(Sequence No. 69)

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

[0173] Example 3: Creation of VHH-nanobodies

[0174] Nanobodies are single-monomer variable antibody domains that selectively bind to specific antigens, similar to 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 given below is specific to mouse CD45. Codon optimization was performed using CLC Main Workbench as described above.

[0175] VHH generation method

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

[0177] After immunization, peripheral blood lymphocytes are isolated by centrifugation using a Ficoll discontinuous gradient. Total RNA is extracted from peripheral blood lymphocytes, and first-strand cDNA is synthesized from total or poly(A+) RNA using a cDNA synthesis kit. A bacteriophage library is generated from this cDNA. Single-domain antibodies are panned by adding 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 for 1 hour at 37°C. PCR amplification is performed using 2 μL of medium, and positive clones are screened. Positive clones are grown in 10 ml of Luria Bertani medium (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 created codon-optimized cDNA of VHH-nanobody a-CD45-1 (mouse) for human cell expression (by xenotransplanting into mice and transducing into human cells; relative to 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 the LeGO-iG2-a-CD45(M)-VHH-1 portion, as shown in Sequence ID No. 70 below. ATGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCACCCCGGCGACAGCCTGAGACTGAGCTGCGCCGCCAGCGGCAGCGTGTTCAACAGCGCCACCATGGGCTGGTACAGACAGAGCCCCGGCAGCCAGAGAGAGCTGGTGGCCACCATCGTGGTGGGCACCCCCACCTACGCCG ACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACATCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCAACTACAGAGCCACCTACACCAGCGGCTACAGCAGAGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGC (SEQ ID NO: 70)

[0179] VHH-nanobody a-CD45-1 (mouse) protein sequence MAQVQLVESGGGLVHPGDSLRLSCAASGSVFNSATMGWYRQSPGSQRELVATIVVGTPTYADSVKGRFTISRDNAKNIVYLQMNSLKPEDTAVYYCNYRATYTSGYSRDYWGQGTQVTVS (Sequence ID 71)

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

[0181] VHH-nanobody a-CD45-2 (mouse) (DNA sequence) codon-optimized for human cell expression. This is shown in Figure 14 as LeGO-iG2-a-CD45(M)-VHH-2 This is shown as follows. ATGGCCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCAGAGCCTTCAACAGCGCCGCCATGGGCTGGTACAGACAGGCCCCCGGCAGCCAGAGAGAGCTGGTGGCCAGCATCAGCGCCGGCACCGCCAGCTACGCCGACGCCGTGAAGGGCAGATTCACCATCAGCAGAGACTACGCCAAGAACATCATCTACCTGCAGATGAACAGCCTGAAGCCCGACGACACCGCCGTGTACTTCTGCAACTACAGAACCACCTACACCAGCGGCTACAGCGAGGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGC(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] The inventors generated an engager containing a human single-domain / nanobody sequence using the method disclosed above for Example 3. The resulting protein and cDNA sequences are shown in SEQ ID NOs: 74 - 215. These were generated for the inventors by Nanotag.

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

[0186] EVQLVESGGGLVQPGGSLRLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVLSAHHSEDPIS(Sequence ID 74)

[0187] This protein sequence is encoded by the cDNA shown in Sequence ID No. 75 below.

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

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

[0190] EVQLLESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQA PGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT(Sequence ID 76)

[0191] This protein sequence is encoded by the cDNA shown in Sequence ID No. 77 below.

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

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

[0194] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVAVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCAARVRGSTGDFGSWGQGTQVTVSSEPKTPKPQT(Sequence ID 78)

[0195] This protein sequence is encoded by the cDNA shown in Sequence ID No. 79 below.

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

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

[0198] EVQLVESGGGLVETGGSLRLSCAGSGRTFSSRHVGWFRQTPGKEREFVASIRWSGGHTYYADSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT(Sequence ID 80)

[0199] This protein sequence is encoded by the cDNA shown in Sequence ID No. 81 below.

[0200] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTCGAAACTGGGGGTTCTCTGAGACTCTCCTGTGCAGGTTCTGGACGCACCTTCAGTAGCCGGCACGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGAGTTTGTAGCATCCATTAGGTGGAGTGGCGGTCACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACACGGTGGCTCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGCAGCGGCTGTGCGTCTAACTGCGCCTCTCAATTTTGACACCTCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCATCTCCTCAGAACCCAAGACACCAAAACCACAAACT(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(Sequence ID 84)

[0207] This protein sequence is encoded by the cDNA shown in Sequence ID No. 85 below.

[0208] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTGGATTCACCTTCAGTATCTACGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCGCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGTCACAAGTTATGCAGACTCCGTGAAGGACCGATTCACCATCTCCAGAGATAACGCCAGGAGC ACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 85)

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

[0210] EVQLVESGGGLVQAGGSLKLSCAASGRTLTYYTAWFRQAPGKEREFVASLGWSGDVTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCNVMQAWGQGTQVTVSSEPKTPKPQT(Sequence ID 86)

[0211] This protein sequence is encoded by the cDNA shown in Sequence ID No. 87 below.

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

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

[0214] EVQLLESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT(Sequence ID 88)

[0215] This protein sequence is encoded by the cDNA shown in Sequence ID No. 89 below.

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

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

[0218] EVQLLESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGHTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREDLYDVWGQGTQVTVSSEPKTPKPQT(Sequence ID 90)

[0219] This protein sequence is encoded by the cDNA shown in Sequence ID No. 91 below.

[0220] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCCATTAGGTGGAGTGGCGGTCACACATACTATGCAGACTCCGTGAAGGGCCGATTCAC CATCTCCGGAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGACCTCTATGATGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 91)

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

[0222] EVQLQESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPIS(Sequence ID 92)

[0223] This protein sequence is encoded by the cDNA shown in Sequence ID No. 93 below.

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

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

[0226] EVQLEESGGGLVQAGDSLRLSCACSERAYRNRLLGWFRQAPGKEREFVANIRPIDSASDYAGSVKGRFTISRDIAKRTVYLQMNSLKPEDTAVYYCASTYMFDSVREDEYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 94)

[0227] This protein sequence is encoded by the cDNA shown in Sequence ID No. 95 below.

[0228] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCTTGCTCTGAACGCGCCTATAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAATTTGTAGCAAATATCAGACCCATTGATAGCGCTCCGATTATGCAGGCTCCGTGAAGGGCCGAT TCACCATCTCTAGAGACATCGCCAAGAGAACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGTCCGGGAGGATGAATATGACTACTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 95)

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

[0230] EVQLVESGGGLVQAGGSLRLSCVVSGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTISSEPKTPKPQT(Sequence ID 96)

[0231] This protein sequence is encoded by the cDNA shown in Sequence ID No. 97 below.

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

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

[0234] EVQLLESGGGLVQAGGSLRLSCVASGRGFSRYDMGWFRQASGKEREFVAAISWSNSTTAYADSVKGRFAISRDNNKNMVYLQMNSLKPEDTAVYYCAARVRGSTGDFGSWGQGTQVTVSSEPKTPKPQT(Sequence ID 98)

[0235] This protein sequence is encoded by the cDNA shown in Sequence ID No. 99 below.

[0236] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGGGGCTTCAGTAGGTATGACATGGGCTGGTTCCGCCAGGCTTCAGGGAAGGAGCGTGAGTTTGTAGCAGCAATTAGCTGGA GTAATAGTACCACGGCCTATGCAGACTCCGTGAAGGGCCGATTCGCCATCTCAAGAGACAACAACAAGAATATGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCGTGTATTACTGTGCAGGAGGGTACGCGGCAGCACAGGGGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 99)

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

[0238] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYNYWGQGTQVTVSSEPKTPKPQT(Sequence ID 100)

[0239] This protein sequence is encoded by the cDNA shown in Sequence ID No. 101 below.

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

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

[0242] EVQLLESGGGLVQAGGSLRLSCVSSGDSISGVVVRWYRQVPGKQREWIGGIGTSDNPEYADSVWGRFVLSRDNAGSRVNLQMNNLKLEDTATYYCNAVHKWGPGTQVTVSSEPKTPKPQ(Sequence ID 102)

[0243] This protein sequence is encoded by the cDNA shown in Sequence ID No. 103 below.

[0244] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCCTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAAGTTCTGGAGACAGTATCAGTGGAGTGGTCGTCCGTTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTGGATCGGAGGTATTGGTACTAGTGATAACCCAGAATATGCG GACTCCGTCTGGGGCCGATTCGTCCTCTCCAGAGACAATGCCGGGAGCCGCGTAAATCTGCAAATGAACAACCTGAAACTTGAGGACACGGCCACCTATTACTGCAATGCAGTGCACAAATGGGGCCCGGGTACCCAGGTCACCGTCTCTTCTGAACCCAAGACACCAAAACCACAAAC( (Sequence No. 103)

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

[0246] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAVMSWVRQAPGKEREFVASIRWSGGNTYYADSVKGRFTITGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSEPKTPKPQT(Sequence ID 104)

[0247] This protein sequence is encoded by the cDNA shown in Sequence ID No. 105 below.

[0248] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCGTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCA CCATCACCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 105)

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

[0250] EVQLEESGGGLVQAGGSLRLSCAASGRTFSSYRLGWFRQAPGKEREFVAGWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 106)

[0251] This protein sequence is encoded by the cDNA shown in Sequence ID No. 107 below.

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

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

[0254] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT(Sequence ID 108)

[0255] This protein sequence is encoded by the cDNA shown in Sequence ID No. 109 below.

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

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

[0258] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 110)

[0259] This protein sequence is encoded by the cDNA shown in Sequence ID No. 111 below.

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

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

[0262] EVQLEESGGGLVQAGDSLRLSCVVSGSISSIYAMGWVREDPGKERVVVAGINSGAIRWYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 112)

[0263] This protein sequence is encoded by the cDNA shown in Sequence ID No. 113 below.

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

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

[0266] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSAEPKTPKPQT(Sequence ID 114)

[0267] This protein sequence is encoded by the cDNA shown in Sequence ID No. 115 below.

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

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

[0270] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNAVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 116)

[0271] This protein sequence is encoded by the cDNA shown in Sequence ID No. 117 below.

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

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

[0274] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 118)

[0275] This protein sequence is encoded by the cDNA shown in Sequence ID No. 119 below.

[0276] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTCAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 119)

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

[0278] EVQLEESGGGLVETGDSLRLSCSASGGGFSFNAIGWYRQGPGKGRELVAAGTSGSTTYYAPSVKGRFIFSRDSAKNTVYLQMNNLNPEDTAIYYCATPALGQMEYDVVSGDGLAHWGKGTLVIVSSAHHSEDPNS (Sequence ID 120)

[0279] This protein sequence is encoded by the cDNA shown in Sequence ID No. 121 below.

[0280] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCCTGGTGGAGACTGGGGATTCTCTGAGACTCTCCTGCTCTGCCTCTGGGGGCGGTTTTAGTTTCAATGCCATAGGCTGGTACCGGCAGGGGCCGGGAAAGGGGCGCGAATTGGTCGCAGCAGGTACTAGTG GAAGTACCACATATTACGCGCCCTCTGTGAAGGGCCGATTCATCTTCTCCAGAGACAGTGCCAAAAACACCGTCTATCTGCAAATGAACAACCTGAACCCTGAAGACACGGCCATCTATTATTACTGTGC CACACCGGCACTTGGACAAATGGAGTATGACGTAGTGAGCGGCGACGGCTTGGCCCACTGGGGCAAAGGGACCCTGGTCATCGTCTCTTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 121)

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

[0282] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 122)

[0283] This protein sequence is encoded by the cDNA shown in Sequence ID No. 123 below.

[0284] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGTGAGG GGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 123)

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

[0286] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNHVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 124)

[0287] This protein sequence is encoded by the cDNA shown in Sequence ID No. 125 below.

[0288] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCACGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTG TCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTC TACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAACCACAAACT (SEQ ID NO: 125)

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

[0290] EVQLVESGGGLVQPGGSLRLSCATSGLTNPERRLAWFRQEPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYYCAARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT(Sequence ID 126)

[0291] This protein sequence is encoded by the cDNA shown in Sequence ID No. 127 below.

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

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

[0294] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYNYWGQGTQVTVSSEPKTPKPQT(Sequence ID 128)

[0295] This protein sequence is encoded by the cDNA shown in Sequence ID No. 129 below.

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

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

[0298] EVQLVESGGGLVQAGDSLTLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 130)

[0299] This protein sequence is encoded by the cDNA shown in Sequence ID No. 131 below.

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

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

[0302] EVQLEESGGGSVQAGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 132)

[0303] This protein sequence is encoded by the cDNA shown in Sequence ID No. 133 below.

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

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

[0306] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARS TLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 134)

[0307] This protein sequence is encoded by the cDNA shown in Sequence ID No. 135 below.

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

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

[0310] EVQLLESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS(Sequence ID 136)

[0311] This protein sequence is encoded by the cDNA shown in Sequence ID No. 137 below.

[0312] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTTTTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCTTCTATTAGGTGGAGTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACC ATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 137)

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

[0314] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNAKNMVYLQMDNIKPEDTARYFCASSYTFSSVREDDYDYWGQGTQVTVLSAHHSEDPIS (Sequence ID 138)

[0315] This protein sequence is encoded by the cDNA shown in Sequence ID No. 139 below. .

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

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

[0318] EVQLVESGGGLVQAGGSLRLSCAASGRTVSRYDMGWFRQAPGAERVVVAISWSGGSTYYVDSVKGRFTMSRDNSKNTVYLQMNSLKPEDTAVYYCAVRTERSSLDFHSWGQGTQVTVSSEPKTPKPQT(Sequence ID 140)

[0319] This protein sequence is encoded by the cDNA shown in Sequence ID No. 141 below.

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

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

[0322] EVQLEESGGGLVQAGDSLRLSCAASERAYRNRLLGWFRQVPGKEREFVAWIRPIDSSTNYADSVKGRFTISRDNDKNTVYLQMDNMKPEDTALYYCASTYYYSSIREDDYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 142)

[0323] This protein sequence is encoded by the cDNA shown in Sequence ID No. 143 below.

[0324] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGTT CCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCATTGATAGCAGCACAAATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGATAACGACAAGAACACGGTGTATTTGCAAATGGACAATATGAAAC CTGAGGACACGGCCCTCTATTATTGTGCGTCCACATACTACTACAGTAGTATCCGGGAGGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 143)

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

[0326] EVQLVESGGGLVQAGGSLRLSCAASGRAFSNRALGWFRQAPGKEREFVAWIRGIGSSTNYAGSVQGRFTISRDNAKNTLYLQMDKLKPEDTAVYYCASTYMFDSVREDEYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 144)

[0327] This protein sequence is encoded by the cDNA shown in Sequence ID No. 145 below.

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

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

[0330] EVQLQESGGGLLQTGDSLRLACEASEIVVENYVMAWFRQAPGKEREWLARIIWNTGGTHLQEFVKGRLTISRDIAKKTVYLQMNSLKPEDTAVYYCAGGSFDAIADPFSARRYGFWGQGTQVTVSSEPKTPKPQT(Sequence ID 146)

[0331] This protein sequence is encoded by the cDNA shown in Sequence ID No. 147 below.

[0332] GAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGCTGCAGACTGGGGACTCACTGAGACTCGCCTGTGAAGCCTCTGAAATCGTCGTCGAAAATTATGTCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTG GCTAGCGCGTATTATTTGGAATACCGGTGGCACACATCTTCAAGAATTTGTGAAGGGCCGACTCACCATCTCTAGAGACATCGCCAAGAAAACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTT ATTACTGTGCCGGTGGAAGTTTTGACGCTATAGCCGATCCCTTCTCGGCCCGCCGGTATGGGTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 147)

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

[0334] EVQLQESGGGLVQAGGSLRLSCVSSGDSISGVVVRWYRQVPGKQREWIGGIGTSDNPEYADSVWGRFVLSRDNAGSRVNLQMNNLKLEDTATYYCNAVHKWGPGTQVTVSSEPKTPKPQT(Sequence ID 148)

[0335] This protein sequence is encoded by the cDNA shown in Sequence ID No. 149 below.

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

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

[0338] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKEREFVAWIRGIGGSTHYAGSVEGRFTISRDSAKNTLYLQMDNVKPEDTAVYYCASTYMFDSVREDEYDYWGQGTEVTVSSAHHSEDPNS(Sequence ID 150)

[0339] This protein sequence is encoded by the cDNA shown in Sequence ID No. 151 below.

[0340] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGTGGATTAGAGGCATCGGTGGCAGCACACATTATGCAGGCTCCGTGGAGGGCCGAT TCACCATCTCCAGAGACAGCGCCAAGAACACGTTGTATCTACAGATGGACAACGTGAAACCCGAGGACACGGCCGTTTATTATTGTGCGTCCACATACATGTTCGATAGTGTCCGGGAGGATGAATATGACTACTGGGGCCAGGGACCGAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 151)

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

[0342] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDSAKNTLYLQMDNVKPEDTAVYYCASTYMFDSVREDEYDYWGQGTEVTVSSEPKTPKPQT(Sequence ID 152)

[0343] This protein sequence is encoded by the cDNA shown in Sequence ID No. 153 below.

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

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

[0346] EVQLEESGGGLVQTGGSLRLSCAASGGTFSSYVMGWFRQAPGKEREFVAWIRPIDSSTNYADSVKGRFTISRDDAKNSLYLQMDNMKPEDTALYYCASTYYYSSIREDDYDYWGRGTQVTVLSAHHSEDPNS(Sequence ID 154)

[0347] This protein sequence is encoded by the cDNA shown in Sequence ID No. 155 below.

[0348] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTACAGACCGGGGGATCTTTGAGACTCTCCTGTGCAGCCTCTGGCGGCACCTTCAGTAGCTATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTGGCATGGATCAGACCCATTGATAGCAGCACAAATTATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCTAGGGATGACGCCAAGAACTCGCTGTATCTGCAAATGGACAATATGAAACCTGAGGACACGGCCCTCTATTATTGTGCGTCCACATACTACTACAGTAGTATCCGGGAGGATGACTATGACTACTGGGCCGGGGGGACCCAGGTCACCGTCTTGTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 155)

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

[0350] EVQLVESGGGLVQPGGSLRLSCATSGFTFSNNVMSWVRQA PGKGPERVAVIGSVGGTTGATSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 156)

[0351] This protein sequence is encoded by the cDNA shown in Sequence ID No. 157 below.

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

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

[0354] EVQLVESGGGLVQARGSLRLSCVASGRTLTYYTGWFRQAPGKEREFVASFAWSGGNTYYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 158)

[0355] This protein sequence is encoded by the cDNA shown in Sequence ID No. 159 below.

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

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

[0358] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 160)

[0359] This protein sequence is encoded by the cDNA shown in Sequence ID No. 161 below. .

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

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

[0362] EVQLEESGGGLVQAGDSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 162)

[0363] This protein sequence is encoded by the cDNA shown in Sequence ID No. 163 below.

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

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

[0366] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 164)

[0367] This protein sequence is encoded by the cDNA shown in Sequence ID No. 165 below.

[0368] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTAACAGCGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAATTATCGGCAGTGTCGGAGGTACCTCAGGTGTCACAAGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCACCAGAGATAACGCCAGGAGCACGC TGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 165)

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

[0370] EVQLLESGGGLVQAGDSLRLSCTQSGRTFSRYAIGWFRQAPGKEREFVASIRWSGGHTYYADSVKGRFTISKDNAKDTVYLQMNSLKPEDTAVYYCAGGSFDAIADPFSARRYGFWGQGTQVTVSSAHHSEDPIS (Sequence ID 166)

[0371] This protein sequence is encoded by the cDNA shown in Sequence ID No. 167 below.

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

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

[0374] EVQLEESGGGLVQAGGSLRLSCAASGRTLTYYTGWFRQAPGKEREFVASFAWMGDNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCAALRFWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS (Sequence ID 168)

[0375] This protein sequence is encoded by the cDNA shown in Sequence ID No. 169 below.

[0376] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTATTATACTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTTTTGCGTGGATGGGTGATAACACATACTACGCTGACTCCGTGAAGGGCCGGTTCACCATCTCCGGCGACAACGCCAAGAACACGGTGTATCTG CAAATGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCATTAAGATTTTGGACTACTACACCGCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 169)

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

[0378] EVQLVESGGGLVQAGDSLRLSCAASGLTNPERRLAWFRQAPGKEREFVASIRWSGGPNTHYGDSVKGRFTISRDNGKNTVALQMNNLKPEDTAVYFCAAAVRLTAPLNFDTSYDYWGQGTQVTISSEPKTPKPQT(Sequence ID 170)

[0379] This protein sequence is encoded by the cDNA shown in Sequence ID No. 171 below.

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

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

[0382] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 172)

[0383] This protein sequence is encoded by the cDNA shown in Sequence ID No. 173 below.

[0384] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACCAAGTCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGCGGGTCTCAGTTATCGGCAGTGTCGGAGGTGCCACAGGTGCCACAAGTTATGCAGACTCCGT GAGGGGCCGATTCACCATCTCCAGAGATAACGCCAGGAGCACGCTGCATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAGGGGAACGGACTTACTTCTACTCGCGCGAGTGACTACTGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT( (Sequence ID 173)

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

[0386] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 174)

[0387] This protein sequence is encoded by the cDNA shown in Sequence ID No. 175 below.

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

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

[0390] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 176)

[0391] The protein sequence is encoded by the cDNA shown in Sequence ID No. 177 below.

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

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

[0394] EVQLVESGGGLVQAGGSLRLACTASGSDFKRAALGWYRQAPGQERELVAAFNSGGKTYYTDSVKDRFTISRDNAKSTLYLQMNSLKPDDTAMYYCALSRFDYYLPPTQFDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 178)

[0395] This protein sequence is encoded by the cDNA shown in Sequence ID No. 179 below.

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

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

[0398] EVQLVESGGGLVQAGGSLRLSCAASGRTLTFYTGWFRQAPGKEREFVASIRWSGGNTDYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 180)

[0399] This protein sequence is encoded by the cDNA shown in Sequence ID No. 181 below.

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

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

[0402] EVQLVESGGGLVQAGGSLKLSCAASGRTLTYYTAWFRQAPGKEREFVASLGWSGDVTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTATYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPNS (Sequence ID 182)

[0403] This protein sequence is encoded by the cDNA shown in Sequence ID No. 183 below.

[0404] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAAACTCTCCTGTGCAGCCTCCGGACGCACCCTCACTTATTATACTGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCGCTAGGGTGGAGTGGCGATGTCACATACTATGCAGACTCCGTGAAGGGCCGATTCACC ATCTCCGGCGACAACGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTAATAGT (SEQ ID NO: 183)

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

[0406] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 184)

[0407] This protein sequence is encoded by the cDNA shown in Sequence ID No. 185 below.

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

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

[0410] EVQLVESGGGLVQAGDSLKLSCVGSGRTFSSYGLGWFRQAPGKEREFLAHITWTAGGTYHADNVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCAARSSGDWRVERYYDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 186)

[0411] This protein sequence is encoded by the cDNA shown in Sequence ID No. 187 below.

[0412] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAAACTCTCCTGTGTAGGCTCTGGACGCACCTTCAGCAGCTATGGGTTGGGCTGGTTCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTAGCACATATTACCTGGACTGCTGGTGGAACATACCATGCAGACAACGTGAAGGGCCGAT TCACCATCTCCAGAGACGACGCCAAGAATACGGTGTATCTACAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGCACGTTCCTCTGGGGATTGGCGTGTCGAGAGATATTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 187)

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

[0414] EVQLEESGGGLVQPGGSLRLSCATSGFTFSNNVMSWVRQAPGKGPERVAVIGSVGGATGATSYADSVKGRFTITRDNARSTLHLQMNGLKPEDTAMYYCAAETSSGLYYSYDDLQTIDFDSWGQGTQVTVSSAHHSEDPNS (Sequence ID 188)

[0415] This protein sequence is encoded by the cDNA shown in Sequence ID No. 189 below.

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

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

[0418] EVQLVESGGGLVQAGGSLRLSCAASERAFKNRALGWFRQAPGKEREFVASIRWSGGNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSEPKTPKPQT(Sequence ID 190)

[0419] This protein sequence is encoded by the cDNA shown in Sequence ID No. 191 below.

[0420] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGAACGCGCCTTCAAGAACCGTGCACTTGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCATCTATTAGGTGGA GTGGCGGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 191)

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

[0422] EVQLVESGGGLVQAGGSLRLSCAASEFTFSGYWMHWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVRGRFTVSRDDAKNTVYLHMDSLKAEDTAVYYCNVMQAWGQGTQVTVLSAHHSEDPIS (Sequence ID 192)

[0423] This protein sequence is encoded by the cDNA shown in Sequence ID No. 193 below.

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

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

[0426] EVQLVESGGGLVETGGSLRLSCAGSGRTFSSRHVGWFRQTPGKEREWVGSVAWNTGSEYYADSVKGRFTISKDNAKDTVYLQMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGQGTQVTVSSAHHSEDPIS (Sequence ID 194)

[0427] This protein sequence is encoded by the cDNA shown in Sequence ID No. 195 below.

[0428] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTCGAAACTGGGGGTTCTCTGAGACTCTCCTGTGCAGGTTCTGGACGCACCTTCAGTAGCCGGCACGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGAGTGGGTTGGAAGTGTTGCCTGGAACACTGGTAGTGAATATTAT GCAGACTCCGTGAAGGGTCGCTTCACCATTTCCAAGGACAACGCCAAAGACACGGTGTATCTGCAAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCGGCACTTAGATCTTGGACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCAGGGGACCCAG GTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (Sequence ID 195)

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

[0430] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGPERVSVIGSVGGVGGVTSYADSVKGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 196)

[0431] This protein sequence is encoded by the cDNA shown in Sequence ID No. 197 below.

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

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

[0434] EVQLQESGGGLVQPGGSLRLSCAASGFTFSNQVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVLSAHHSEDPNS(Sequence ID 198)

[0435] This protein sequence is encoded by the cDNA shown in Sequence ID No. 199 below.

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

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

[0438] EVQLVESGGGLVQAGGSLRLSCVASGEEDFQPYAMGWFRQAPGKEREYVAATTWNGGRIRYGDSVKGRFTISRDHPKNTITLQMTSLKPDDTAVYYCAARYGTVLLTREDYQHWGRGTQVTVSAAHHSEDPIS (Sequence ID 200)

[0439] This protein sequence is encoded by the cDNA shown in Sequence ID No. 201 below.

[0440] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGCGTAGCCTCTGGAGAGGAGGATTTTCAGCCGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATACGTGGCCGCGACTACATGGAATGGTGGTAGAATAAGATATGGAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACCACCCCAAGAACACGATCACTTTACAAATGACCAGTTTGAAACCCTGACGACACGGCCGTTTATTACTGTGCAGCACGGTACGGTACAGTCCTACTTACACGCGAAGACTATCAACACTGGGGCCGTGGGACCCAGGTCACCGTTTCCGCGGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 201)

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

[0442] EVQLVESGGGLVQAGGSLSLSCAASGRTFSTGAMGWFRQAPGKEREFLARITLIGHGTYYADALKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT(Sequence ID 202)

[0443] This protein sequence is encoded by the cDNA shown in Sequence ID No. 203 below.

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

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

[0446] EVQLLESGGGLVQAGGSLRLLSCAASGFTFSNYAMSWVRQAPGKGPERVSIIGSVGGTSGVTSYADSVKGRFTITRDNARS TLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSAHHSEDPIS (Sequence ID 204)

[0447] This protein sequence is encoded by the cDNA shown in Sequence ID No. 205 below.

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

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

[0450] EVQLVESGGGLVQAGGSLRLSCAASERTVSVYTMGWFRQAPGKEREFVASIRWSGGPNTYYADSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYYCVARDSPCVGNCWYENAGDYEYWGQGTQVTVSSEPKTPKPQT(Sequence ID 206)

[0451] This protein sequence is encoded by the cDNA shown in Sequence ID No. 207 below.

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

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

[0454] EVQLVESGGGLVQPGDSLRLSCAASGFTFSSYAMSWVRQAPGKGPERVSVIGSVGGTTGVTSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSEPKTPKPQT(Sequence ID 208)

[0455] This protein sequence is encoded by the cDNA shown in Sequence ID No. 209 below. .

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

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

[0458] EVQLEESGGGLVQPGGSLRLSCAASGFTFSNSVMSWVRQAPGKGPERVSVIGSVGGATGATSYADSVRGRFTISRDNARSTLHLQMNSLKPEDTAVYYCVKGNGLTSTRASDYWGQGTQVTVSSAHHSEDPIS (Sequence ID 210)

[0459] This protein sequence is encoded by the cDNA shown in Sequence ID No. 211 below.

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

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

[0462] EVQLLESGGGLVQAGDSLRLSCAASERAYRNRLLGWFRQAPGAERVVVAISWSGGSTYYVDSVKGRFTMSRDNSKNTVYLQMNSLKPEDTATYYCAALRFWTTTPQKEGLYDTWGQGTQVTVSSEPKTPKPQT(Sequence ID 212)

[0463] This protein sequence is encoded by the cDNA shown in Sequence ID No. 213 below.

[0464] GAGGTGCAGCTGCTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGAACG CGCCTACAGGAACCGTCTTCTTGGCTGGTTCCGCCAGGCTCCAGGGGCGGAGCGTGTCGTTGTAGCTATTAGCTGGAGCGGTGGTAGTACATACTATGTAGACTCCGTGAAGGGCCGATTCACCATGTCCAGAGACAACAGCAAGAACACGGTGTATCTGCAAA TGAACAGCCTGAAACCCGAGGACACGGCCACTTATTACTGCGCAGCACTTAGATTTTGGACTACAACACCTCAGAAAGAGGGCCTCTATGACACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCCGAACCCAAGACACCAAAACCACAAACT (SEQ ID NO: 213)

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

[0466] EVQLQESGGGSLQTGDSLRLACEASEIVVENYVMAWFRQAPGKEREWLARIIWNTGGTHLQEFVKGREGIGYSVKTSTRTVMNSLKPEDTAIYYCAALRSWTTTPQREVLYDNWGHGTQVTVSSAHHSEDPIS (Sequence ID 214)

[0467] This protein sequence is encoded by the cDNA shown in Sequence ID No. 215 below. GAGGTGCAGCTGCAGGAGTCTGGGGGAGGATCGCTGCAGACTGGGGACTCACTGAGACTCGCTGTGAAGCCTCTGAAATCGTCGTCGAAAATTATGTCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTGGCTAGCGCGTATTATCTGGAATACCGGTGGCACACATCTTCAAGAATTTGTGAAGGGCCGAGAA GGGATCGGCTATAGCGTCAAAACTTCCACCCGCACAGTAATGAACAGCCTGAAACCCGAGGACACGGCCATTTATTACTGCGCAGCACTTAGATCTTGGACTACTACACCTCAGAGGGAGGTCCTCTATGACAACTGGGGCCACGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCTATTAGT (SEQ ID NO: 215)

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

[0469] In the sequence below, the underlined lowercase regions are the IL2 signal peptide, the lowercase regions are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chains, the bold uppercase regions are the stalks, the bold underlined regions are the CD28 transmembrane domain, the capital italics are the CD28 intracellular domain, and the underlined capital italics in bold are the CD3Z intracellular domain. a-CD19CAR mlllvtslllcelphpaflli pdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleit GGGGSGGGGSGGGGS EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTV SSSGSGSG KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMT PRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 216)

[0470] This protein sequence is encoded by the cDNA shown in Sequence ID No. 217 below.

[0471]

[0472] a-CD38CAR

[0473] In the sequence below, the underlined lowercase regions are the IL2 signal peptide, the lowercase regions are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, the bold underlined regions are the CD28 transmembrane domain, the capital italics are the CD28 intracellular domain, and the capital italics in bold are the CD3Z intracellular domain.

[0474] myrmqllscialslalvtns qvqlvqsgaevkkpgssvkvsckafggtfssyaiswvrqapgqglewmgriirflgianyaqkfqgrvtliadkstntaymelsslrsedtavyycagepgredpdavdiwgqgtmvtvss SGGGGSGGGGSGGGGSGGGGSGGGGSS DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKARKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 218)

[0475] This protein sequence is encoded by the cDNA shown in Sequence ID No. 219 below.

[0476] Codon-optimized a-CD38CAR

[0477] ATGTACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTTCGGCGGCACCTTCAGCAGCTACGCCATCAGCTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCAGAATCATCAGATTCCTGGGCATCGCCAACTACGCCCAGAAGTTCCAGGGCAGAGTGACCCTGATCGCCGACAAGAGCACCAACACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCGGCGAGCCCGGCAGAGAGGACCCCGACGCCGTGGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCCAGCCAGGGCATCAGAAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCAGAAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGG CGGCGGCACCAAGGTGGAGATCAAGAGCAGCGGCAGCGGCAGCCCCACCACCACCCCCGCCCCCAGACCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCCGCCGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCCCCAGAAAGATCGAGGTGATGTACCCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGAGAAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGACCCGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCCCCCAGAGACTTCGCCGCCTACAGAAGCAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA (SEQ ID NO: 219)

[0478] m-VHH1-E3-TM

[0479] In the following protein sequence (SEQ ID NO: 220), the lowercase region is anti-mouse CD45 In VHH, the underlined capitalized regions are linkers, the ununderlined capitalized regions are the proximal extracellular region of E3.49K, the bold capitalized underlined regions are the E3.49K transmembrane region, and the bold capitalized regions are the intracellular region of E3.49K. maqvqlvesggglvhpgdslrlscaasgsvfnsatmgwyrqspgsqrelvativvgtptyadsvkgrftisrdnaknivylqmnslkpedtavyycnyratytsgysrdywgqgtqvtvs GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (Sequence ID 220)

[0480] In the DNA sequence below (SEQ ID NO: 221), the lowercase region is anti-mouse CD45 VHH, the underlined uppercase region is the linker, the ununderlined uppercase region is the E3.49K proximal extracellular membrane region, the bold uppercase underlined region is the E3.49K transmembrane region, and the bold uppercase region is the E3.49K intracellular region. This sequence codes for the protein of SEQ ID NO: 220.

[0481] atggcccaggtgcagctggtggagagcggcggcggcctggtgcaccccggcgacagcctgagactgagctgcgccgccagcggcagcgtgttcaacagcgccaccatgggctggtacagacagagccccggcagccgagagagctggtggccaccatcg tggtgggcacccccacctacgccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacatcgtgtacctgcagatgaacagcctgaagcccgaggacaccgccgtgtactactgcaactacagagccacctacaccagcggctacag cagagactactggggccagggcacccaggtgaccgtgagc GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA(Sequence ID 221)

[0482] mVHH2-E3-TM

[0483] In the protein sequence below (SEQ ID NO: 222), the region in lowercase letters is anti-mouse CD45 In VHH, the underlined capitalized regions are linkers, the ununderlined capitalized regions are the proximal extracellular region of E3.49K, the bold capitalized underlined regions are the E3.49K transmembrane region, and the bold capitalized regions are the intracellular region of E3.49K. maqvqlvqsggglvqpggslrlscaasgrafnsaamgwyrqapgsqrelvasisagtasyadavkgrftisrdyakniiylqmnslkpddtavyfcnyrttytsgysedywgqgtqvtvs GGGGS DEGKRYRVKVIPPNTTTNSQSVKIQPYTRQTTPDQEHKFELQFETNGNYDSKIPSTT VAIVVGVIAGFITLIIVFICYICC RKRPRAYNHMVDPLLSFSY (Sequence ID 222)

[0484] In the DNA sequence below (SEQ ID NO: 223), the lowercase region is anti-mouse CD45 VHH, the underlined uppercase region is the linker, the ununderlined uppercase region is the E3.49K proximal extracellular membrane region, the bold uppercase underlined region is the E3.49K transmembrane region, and the bold uppercase region is the E3.49K intracellular region. This sequence codes for the protein of SEQ ID NO: 222.

[0485] atggcccaggtgcagctggtgcagagcggcggcggcctggtgcagcccggcggcagcctgagactgagctgcgccgccagcggcagagccttcaacagcgccgccatgggctggtacagacaggcccccggcagccgagagagctggtggccagcatcagcgccggcaccgccagctac gccgacgccgtgaagggcagattcaccatcagcagagactacgccaagaacatcatctacctgcagatgaacagcctgaagcccgacgacaccgccgtgtacttctgcaactacagaaccacctacaccagcggctacagcgaggactactggggccagggcacccaggtgaccgtgagc GGCGGCGGCGGCAGC GATGAGGGAAAACGGTACCGGGTTAAGGTTATTCCGCCTAACACCACAAACTCCCAGAGTGTCAAAATTCAGCCTTACACCAGGCAGACTACTCCTGACCAGGAACACAAATTCGAATTACAGTTTGAGACTAACGGTAACTATGACTCCAAGATTCCATCTACAACG GTCGCGATCGTAGTGGGCGTGATTGCAGGCTTCATCACATTGATCATCGTGTTCATCTGCTATATCTGCTGT AGGAAGCGCCCTCGGGCGTACAACCACATGGTGGACCCTCTGTTGAGTTTCTCATATTAA(Sequence No. 223)

[0486] a-CD43-sc a-CD43-sc (SEQ ID NO: 224) is a protein for anti-CD43 antibodies that has a stalk and transmembrane region linked 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 region is the heavy chain, the underlined uppercase region is the linker, the ununderlined uppercase region is the light chain, the bold uppercase region is the stalk, and the bold underlined region is the CD34 transmembrane region. myrmqllscialslalvtns evqlqqsgpelvkpgasvrmsctasgytftsyvmhwikqkpgqgldwigyinpynggtqynekfkgkatltsdkssstaymelssltsedsavyycarrtfpyyfdywgqgttltvss SGGGGSGGGGSGGGGSGGGGSGGGGSS DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHAPLTFGAGTKLELK SSGSGS PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP TLIALVTSGALLAVLGITGYFL (Sequence ID 224) In the DNA sequence below (SEQ ID NO: 225), the underlined lowercase regions are the IL2 signal peptide, the lowercase regions are the heavy chain, the underlined uppercase regions are the linkers, the ununderlined uppercase regions are the light chain, the bold uppercase regions are the stalks, and the bold underlined regions are the CD34 transmembrane domain. atgtacagaatgcagctgctgagctgcatcgccctgagcctggccctggtgaccaacagc gaggtgcagctgcagcagagcggccccgagctggtgaagcccggcgccagcgtgagaatgagctgcaccgccagcggctacaccttcaccagctacgtgatgcactggatcaagcagaagcccggccagggcctggactggatcggctacatcaaccccctacaacggcggcacccag tacaacgagaagttcaagggcaaggccaccctgaccagcgacaagagcagcagcaccgcctacatggagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgccagaagaaccttcccctactacttcgactactggggccagggcaccaccctgaccgtgagcagc AGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGC GACGTGCTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGAAGCAGCCAGAGCATCCTGCACAGCAACGGCAACACCTACCTGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG AGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTTCCAGGGCAGCCACGCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG AGCAGCGGCAGCGGCAGC CCCACCACCACCCCCGCCCCCAGACCCCCCACCCCGCCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCCGCCGGCGGCGCCGTGCACACCAGAGGCCTGGACTTC GCCCCCAGAAAGATCGAGGTGATGTACCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCAGCAAGCCC ACCCTGATCGCCCTGGTGACCAGCGGCGCCCTGCTGGCCGTGCTGGGCATCACCGGCTACTTCCTGTAA (Sequence ID 225)

[0487] Example 6. Production of lentiviral vectors

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

[0489] Small-scale manufacturing of VSV-G pseudotype lentiviral vectors

[0490] This method describes the preparation of a VSV-G pseudotyped lentiviral vector in a 6-well plate using a calcium phosphate transfection kit. The amount of viral supernatant prepared 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, 1.5 ml microcentrifuge tube, 5 ml syringe, Trypan Blue, Virkon, M-ytdes.

[0494] plasmid Vector: Dependent on selection Gag-pol plasmid: pMDLg / pRRE Rev plasmid: pRSV-Rev Envelope plasmid: phCMV-VSV-G

[0495] procedure

[0496] Maintenance of 293FT cells

[0497] Maintain the cells by dividing them 1:5 to 1:4 every other day and keep them in T75 or T150 flasks. After thawing the cells, culture them for at least 3 passages, then use 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 culture medium from the flask using 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, resuspend thoroughly, and dissociate all clumps by pipetting up and down several times. - Count cells using Trypan Blue and prepare a cell suspension of 250,000 cells / ml. - Place 2 ml of this suspension into the wells (poly-L-lysine coated 6-well plate). Prepare one well for each vector. - Place the plate in the incubator for at least 7-8 hours.

[0500] After incubation, transfect the cells. Ensure that the cells are adhered, ideally at an optimal density of 80%. If the cells are viable and at the appropriate density, return the plate to the incubator and proceed to prepare the transfection mix. Stop transfection if the density is less than 60%.

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

[0502] - Prepare the plasmid mixture in a microcentrifuge tube as follows (total 4 μg of DNA): - 2 μg of LeGO-iG2 vector (including transgene) - 1 μg of pMDLg / pRRE(Gag / Pol) - 0.75 μg of pRSV-REV(Rev) - 0.25 μg of pH CMV-VSV-G (envelope) - Mix the plasmids and adjust the volume to 54 μl using ddH2O. - Add 6 μl of 2.5 M CaCl2 solution to the DNA mixture. - Place 60 μl of 2X HeBS buffer into separate microcentrifuge tubes. Add the CaCl2 / DNA mixture and vortex. - Allow this mixture to stand at room temperature for 15 minutes. Do not let it stand for longer than 30 minutes, as this may reduce the transfection efficiency. - During these 15 minutes, remove the dish, discard the medium, and add 1 ml of preheated complete growth medium containing 25 μM chloroquine. - After 15 minutes of incubation, add 120 μl of the mixture to the wells by drop, while gently swirling the dish 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: Culture medium change

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

[0505] Day 3: Collection 1 in Shangqing 24 hours after medium change: - Check cells for GFP expression under a UV microscope. Transfection efficiency should be over 90%. - Prepare a 0.45 μm filter, a 5 ml syringe, a 5 ml microcentrifuge tube, and a 1.5 ml tube for each well. - Use a 5 ml syringe to harvest the culture medium from the dish. Apply it to a filter and filter the supernatant into a 5 ml microcentrifuge tube. Filter gently, avoiding the creation of air bubbles and excessive force. Once complete, drop the syringe and filter into Virkon solution. - Transfer a 100 μl aliquot from the filtered supernatant to 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 preheated to at least room temperature, preferably 37°C. Place the cells in the incubator.

[0506] Day 4: Shangqing Collection 2 48 hours after medium change: - Collect the viral supernatant using the same method as the previous day. - Dispose of the plate.

[0507] Example 7: Generation of a cell line

[0508] Using lentiviral particles harvested in previous examples, K562 and RPMI8226 (Figure 6) cells were transduced, sorted, and expanded. The resulting cells were tested in the following manner.

[0509] Preparation of target cells 1. Take a sample from target cells for cell counting. 2. Put 1-2 x 10 into the tube 6 Take individual 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 well. 9. Incubate for 1 hour, shaking the vial every 15 minutes.

[0510] Preparation of effector cells 1. Take a sample from cultured cells for cell counting. 2. Centrifuge the cells and allow them to settle to 0.3 × 10⁻⁶ 6 Resuspend the pellet in warm RPMI + 10% FCS at a concentration of cells / ml. 3. Add 150 μl / well of diluted sample to the first row in a triple-row configuration. 4. In a triple-row system, 100 μl / well of RPMI + 10% FCS is applied to the 2nd to 4th columns and with minimal release. Add it to the three wells. 5. Three 100 μl / well dH2O + 1% Triton X100 solutions for maximum release. Add to the well. 6. Prepare serially diluted solutions in a 1:3 ratio throughout the entire well. 7. Place the plate in the incubator.

[0511] target cell 1. Wash the target cells twice in PBS. 2. Resuspend the 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 with 300g for 3 minutes. 7. Place 20 µl of cell suspension into each well of each of the 96 wells of the LumaPlate. Add it using a pipette. 8. Leave the plate in the chrome hood overnight to dry. Calculate the specific solubility percentage: [(Experimental release - Spontaneous release) / (Maximum release - Spontaneous release)] × 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. These NK cells exhibit both functional maturity and a representative phenotypic signature of blood-derived NK cells, and possess potent antitumor effector function.

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

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

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

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

[0518] Prior to passage, 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 three days.

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

[0520] The cells were centrifuged at 220g for 5 minutes to promote the formation of embryoid body (EB) structures, and then gently incubated at 37°C and 5% CO2 for 3 days.

[0521] The culture medium was changed on days 3, 6, and 9 by removing 70 μL of medium from each well and adding 100 μL of freshly prepared HPDM without the Rock inhibitor.

[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 culture.

[0523] Hematopoietic cell differentiation into NK cells

[0524] In the second phase of NK cell differentiation from iPSCs, hematopoietic progenitor cells were seeded in 4 mL of NK cell differentiation medium (NKDM) supplemented with 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), at a concentration of 32 EB / well in a standard 6-well cell culture plate. This medium consisted of 5 ng / mL IL-3 (PeproTech, 200-03).

[0525] For four weeks, the culture medium was changed twice a week, with IL-3 included only in the first week, and half of the medium being replaced with newly prepared NKDM without IL-3 for the following three weeks.

[0526] After 4 weeks of NK cell differentiation culture, the cells were collected and subjected to phenotypic analysis via flow cytometry, or expanded in CTS OpTmizerTM T Cell Expansion medium (ThermoFisher, A1048501) supplemented with 5% hAB serum (Corning, 35-060-CI), 1% penicillin / streptomycin (Gibco, 15140122), 0.2 mM L-glutamine (Gibco, 25030081), 10 ng / mL of rhIL-15 (Gold Biotechnology, 1110-15), 500 IU / mL of rhIL-2 (Akron Biotech, AK8223), and 25 ng / mL of rhIL-21 (Gold Biotechnology, 1110-21) for 3 to 4 weeks, followed by cytotoxicity and functionality assays.

[0527] Example 8: Test Assay

[0528] Khan FA, Almohazey D, Alomari M, Almofty SA. Isolation, Culture, and Functional Embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) were cultured according to the procedure described in Khan FA, Almohazey D, Alomari M, Almofty SA. Isolation, Culture, and Functional 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 the cell lines was performed in the cytogenetics facility of the laboratory of the present invention using standard protocols.

[0529] The ESC / iPSC cultures were dissociated with dispase and embryoid bodies were generated by plating 5×10 6 cells per well in a low-attachment 6-well plate containing X-VIVO medium with supplements. The medium was changed every 3 days and the culture was maintained for 15 to 20 days.

[0530] Hematopoietic differentiation and genetic modification of ESCs / iPSCs were achieved by electroporation of standard mammalian expression vectors / or resectable lentiviral vectors. 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, NK cells were differentiated by co-culture with OP9 and OP9-DLL1 cells.

[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 strain HS980 was established, cultured on rhLN-521 under defined conditions free of foreign matter, and passaged using a standard protocol. For differentiation, cells were plated on dishes coated with 20 mg / mL of rhLN-111 at a density of 2.4-3104 cells / cm2, using NutriStem hESC XF medium and a Rho kinase inhibitor during the first 24 hours. The culture medium was then replaced with NutriStem hESC XF, which does not contain basic fibroblast growth factor and transforming growth factor b. Starting 6 days after plating, 100 ng / mL of activin A was added to the medium for a total of 5 weeks.

[0533] Normal human CD56+ NK cells and CD8+ T cells were isolated by positive / negative enrichment of blood cells collected from healthy human donors (Miltenyi CliniMACS system). 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 test using chromium release assay

[0535] The sensitivity of target cells to NK cell-mediated lysis was assessed over 4 hours. 51Evaluation was performed by a Cr release assay. 48 hours prior to the assay, NK cells were cultured in NK medium containing IL2. Target cells were cultured at 50 μCi. 51 Label with Cr at 37°C for 2 hours. 51 Cr-labeled cells are plated into each well of a 96-well U-bottom plate. NK cells are added to target cells in different ratios and incubated at 37°C for 4 hours. 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 dried overnight in a hood. After reading the radioactivity, the specific lysis percentage is calculated. A more detailed protocol is shown below. a. Take a sample from K-562 for cell counting. b. Take 1-2 × 10⁶ cells into 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 cultured cells for cell counting. h. Dilute the sample to 0.3 × 10⁶ cells / ml in RPMI + 10% FCS. Total volume 1 ml. i. Label the 96-well plate. j. Add 150 μl / well of diluted sample to the first column in a triple-row container. k. RPMI + 10% FCS in 3 steps with minimal release of 100 μl / well Add it to the three wells. l. Add 100 μl / well of dH2O + 2M HCl to the three wells for maximum release. El. m. Take 50 μl from the first column, add it to the second column, mix, and take 50 μl from the second column. Place it in the third column. 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. Resuspend p cells in 1 ml of 10% RPMI. q. Take a sample for cell counting. r. Calculate the required number of cells. Add 100 μl / well of target cells. t. Incubate for at least 4 hours. Label the u-tube for the gamma counter. v. Take 70 μl of sample / tube. Do so carefully, avoiding cells. The analysis will be performed using a w / gamma counter. x. Incucyte-based cytotoxicity measurement.

[0536] Incucyte-based cytotoxicity measurement IncuCyte was used to measure immune cell-mediated cytotoxicity and the invasion of monotumor spheroids. Spheroids more accurately mimic in vivo conditions than cell monolayers and exhibit several features that determine invasion, such as solid tumor killing and intratumoral cell adhesion, as well as increased cell viability, and diffusion gradients of oxygen, nutrients, and waste products from the outer cellular ring to the inner core. IncuCyte-based measurements of immune cell-mediated cytotoxicity allow for real-time observation.

[0537] procedure: 1. Add 21.5 µl of DMSO to a new vial to create Cytolight Resuspend the Green vial and prepare a 5 mM stock solution. 2. Add 2.8 µl of stock solution to 360 µl of PBS to make a 100x dilution. 3. Place the effector cells into 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 washing solution. 5. Resuspend the pellet in 6 ml of PBS and add 60 µl of 100 × 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 along with the lid inside the incubator to preheat the lid. 8. Add 3.6 ml of 100% FBS to bind to the excess Cytolight reagent. Mix the cells and centrifuge at 400 × g for 5 minutes. Aspirate the supernatant and resuspend the cells in 500 ul of culture medium. 9. Count the cells and adjust the concentration by adding culture medium. 10. Place one vial of CytotoxRed (5 μL) into RT and centrifuge briefly. Prepare a CytotoxRed stock solution by adding 45 μL of PBS to CytotoxRed. 11. 32.5 µl of C in a total volume of SCGM containing 10% FBS in 6.5 ml The active concentration of CytotoxRed is prepared by adding ytotoxRed. 12. Assemble the plate and add 100 µl of Cytotox Red. Add 50 µl of target cells and 50 µl of effector cells or culture medium. 13. Place the plate in Incucyte and count the red cells for 4 hours.

[0538] Cytotoxicity study using CD8+ T cells

[0539] Normal human CD8+ T cells were treated with IFN-γ to target cells (2 x 10⁶ cells per 2 ml). 6 individual CD8+ cells and 5 × 10⁶ 5 The target cells were primed by co-culturing them in a culture medium supplemented with 50 U / ml IL-2 and 25 ng / ml IFN-γ. On day 7, the co-culture was primed with 1.75 × 10⁶ cells. 6 Replenish with fresh IFN-γ treated target cells. On day 14, collect these primed CD8+ T cells by centrifugation and use them in the chromium release assay as described above for NK cells.

[0540] Cell line generation

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

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

[0543] All mouse rearing, breeding, and surgical procedures were approved by the Animal Ethics Committee in Stockholm, Sweden. The mice were purchased from Charles River Laboratories. NSG mice were 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 IL2Rγnull mice engrafted with mobilized human hemopoietic stem cells. They were bred and maintained at the AKM5 animal facility at the Karolinska Institute, Huddinge, Sweden (J. Immunol. 174, 6477-6489 [PubMed: 15879151]).

[0544] The 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 injected subcutaneously with UC or K562 cell lines (1 × 10⁶). All cells were examined and found to be free of mycoplasma before injection. Human PBMCs (10 × 10⁶) were injected intravenously into the mice. Subcutaneous tumor size measurements were started when the mice had measurable tumors. Tumor size was measured with a slide caliper at least twice a week for 4 weeks, and tumor volume was calculated. When the tumor volume reached 1 cm³, the mice were euthanized, and the tumor 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 either the CD45 engager and luciferase reporter gene, or modified only with the luciferase reporter gene (without modification with the CD45 engager). All cells were tested. It was found that mycoplasma was not present before injection. One day after tumor administration, human PBMCs (10 x 10⁶) were injected intravenously into mice in two consecutive days at a dose of 5 x 10⁶ PBMCs / day. Next, the mice were subcutaneously injected with daratumumab (ADCC-competent anti-CD38 antibody) in the 1-RPMI8226 injection group and trastuzumab (anti-Her2 antibody) in the 2-SKOV3 injection group at a dose of 8 mg / kg for both antibodies, three days after tumor cell injection. Mice under isoflurane conditions were fluorescence-imaged using the In Vivo Imaging System (IVIS) Spectrum (Perkin Elmer, Santa Clara, CA, USA) and analyzed using IVIS imaging software (Perkin Elmer). Imaging was performed on day 0 in all animals, and then twice weekly until the mice were euthanized and the tumors and organs were removed (Figure 32).

[0547] IVIS imaging demonstrated that control mice in the RPMI-8226 group given PBMCs and daratumumab controlled tumor development (Figure 34). However, injection of RPMI-8226 cells expressing the CD45 engager, along with PBMCs and daratumumab, led to tumor development (Figure 33: Photograph from IVIS imaging depicting RPMI-8226 expressing luciferase and the CD45 engager. Mice were treated with PBMCs and daratumumab). In a similar manner, CD45 engager-modified K562 cells led to higher immune evasion, even when accompanied by PBMC administration, 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, but injection of SKOV3 cells expressing the CD45 engager along 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 are incubated on ice with blocking reagents for 10 minutes, then stained on ice for 30–60 minutes with antibody and viability stain. Samples are analyzed using a Fortessa / Symphony flow cytometer (BD Biosciences), and data are analyzed using FlowJo software (TreeStar, Ashland, OR). AriaFusion instrument (BD Biosciences) is used for selection. Selected cells are cultured in antibiotic-containing medium for 2 weeks. Subsequently, cells are cultured without antibiotics.

[0550] Extracellular vesicle (EV)-mediated α-CD45-sc mRNA delivery alleviates 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 via the Nanosight Tracking Analysis System (NTA). EVs are used for mRNA delivery of transgenes used to generate antibodies or nanobodies in vivo. The inventors also tested engagers expressed on extracellular vesicles at different densities.

[0552] Isolation and purification of EVs

[0553] The CM was harvested and pre-clarified by slow 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, any remaining undesirable large vesicles were removed. Next, the CM was filtered using a bottle-top filter (Corning, low protein binding) with a cellulose acetate membrane with a 0.22 μm pore size. The CM medium was then diafiltration by ultrafiltration using 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 4000 × g over a specific time based on the sample concentration. The quality and concentration of the EV were then analyzed using ZetaView (Figure 40).

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

[0555] The collagen-induced arthritis (CIA) mouse model is a well-established and frequently used model that mimics the clinical symptoms and immunopathogenicity of human rheumatoid arthritis (RA). Mice immunized with collagen II (CII) showed increased arthritis scores. The control group showed no significant change. Interestingly, MSC EVs carrying α-CD45-sc mRNA exhibited an inhibitory effect on arthritis severity (Figure 38). In contrast, mRNA-mock MSC EVs had no effect. Additionally, the pathogenesis of RA involves activated immune cells that promote the release of pro-inflammatory cytokines by macrophages. Therefore, serum TNF-α and IL-1β levels were measured by sandwich ELISA. Notably, MSC EVs carrying α-CD45-sc mRNA reduced serum TNF-α and IL-1β levels in CIA mice (Figures 39A and B). These results indicate that α-CD45-sc effectively attenuates inflammation in CIA mice (Figure 38). a-CD45-sc EVs alleviate 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-carrying MSCs Extracellular viable cells (EVs) were injected on days 0, 7, 14, and 21 after induction of arthritis. Eleven EVs (2.5E) were injected into the tail vein. Arthritis scores were assessed every 5 days. Data are expressed 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). MSC EVs carrying a-CD45-sc mRNA or mock mRNA were injected on days 0 and 10 after induction of arthritis. After intravenous injection of 5E11 EVs from a-sc-CD45 or mock mRNA, cytokine (TNF-α and IL-1β) levels were measured on day 40. Data are expressed as mean ± SD.

[0557] Transgene expression system

[0558] Lentiviral and retroviral systems are used for transgene expression in target and / or effector cells. Electroporation or chemical-based methods are used for transient expression. Transgenes are delivered chemically or electrochemically as mRNA, with or without vector-based or nanoparticle-based delivery systems. It is delivered through. 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 clinical applications and cell types, suicide genes may be incorporated into cells. This allows for the destruction of cells typically treated with non-toxic agents, such as ganciclovir. Representative suicide genes are shown in Table 3 below.

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

[0562] Example 9: Results

[0563] According to the chromocyte cytotoxicity assay described above, referring to Figure 7, 51Specific lysis percentage of K562 cells using peripheral blood mononuclear cells (PBMCs) with chromium assay. K562 control cells and K562 cells expressing E3.49K and 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 a Luma plate. The plate was dried overnight and read the following day with a gamma counter. Figure 7 shows reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing ai-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 the 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. The cells were centrifuged, and 20 μL of supernatant was added to a Luma plate. The plate was dried overnight and read the following day with a gamma counter. As shown in Figure 7, the results in Figure 8 clearly show reduced cell lysis for cells expressing UL11 and E3.49K, and complete inhibition of lysis for cells expressing a-CD45-sc.

[0565] Figure 9 shows 51 This shows the specific lysis percentage in K562 cells using a Cr release assay. K562 control cells, K562 expressing E3.49K, UL11, and α-CD45-sc were incubated with NK92 and E:T as shown in Figure 9 for 4 hours. The cells were centrifuged, and 20 μL of supernatant was added to a Luma plate. The plate was dried overnight and read with a gamma counter. As shown 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 α-CD45-sc.

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

[0567] Figure 11 shows 51 This shows the specific lysis percentage of RPMI88226 using a Cr release assay. RPMI88226 control cells and RPMI88226 expressing E3.49K, UL11, or a-CD45-sc were incubated with T cells in E:T for 4 hours. Cells were centrifuged, and 20 μL of supernatant was added to a Luma plate. The plate was dried overnight and read with a gamma counter. As shown 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] To evaluate whether CD45 engager expression affects graft function when graft cells are effector cells (NK cells or T cells), NK-92 and TALL-104 cell lines were transduced with α-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% thermoinactivated fetal bovine serum (Atlanta Biologicals, Norcross, Ga.) and 100 units / ml recombinant human IL-2. Cell lines were repeatedly tested for negative mycoplasma contamination using a commercial polymerase chain reaction kit. Both NK-92 and TALL-104 cells were kept at concentrations of 4–6, in fixed numbers (10 4 (Suspension state in a well)51 4h 51 The cells were tested in a Cr release assay. Unmodified NK-92 and TALL-104 were used as control effector cells. 51 The percentage of Cr release was calculated from the average of three replicas. 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 cytotoxic capacity of NK / T cells against target cells, mediated by the chimeric antigen receptor (a-CD38CAR) for CD38, which is influenced by a-CD45-sc.

[0572] To analyze the influence 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 the CAR, and their cytotoxicity against target cells was evaluated (Figures 24 and 41). This experiment can be readily utilized with single-chain and single-domain approaches to 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 96-well flat-bottom plates in Incucyte. Referring to Figure 44, red cells indicate target cell death, which was counted over a 4-hour period. The data was analyzed using GraphPad Prism.

[0573] Referring to Figure 41, RPMI8226 cells were incubated with NK92 control cells, a-CD38CAR, a-CD38CAR+a-CD45-sc, or NK92 cells expressing a-CD45-sc. After 4 hours of incubation, the cells were centrifuged and 2 0 μL of supernatant was added to the Luma plate.

[0574] B. (CD19) + Evaluation of whether the cytotoxic capacity of NK / T cells against target cells, mediated by the chimeric antigen receptor (CARCD19) for CD19, is affected by α-CD45-sc.

[0575] To analyze the influence 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 the 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 then transduced again with either a-CD45-sc or a control. Jurkat and Raji cells were used as targets. A degranulation assay was performed over 4 hours, and cells were labeled with CD107a along with CD3, CD56, Live / Dead-APC-H7, and CD19h-biotin. After degranulation, cells were flow-cytometerd. Data were analyzed using Flowjo (Figure 43).

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

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

[0578] Example 11: Clinical Application

[0579] The present invention can be used to treat any cells or tissues before introduction into the body. The present invention can also be used to treat autoimmune diseases; hematological cancers including lymphoma and leukemia; bone marrow failure syndromes including anemia and cytopenia; hereditary immune disorders including WAS and SCID; abnormal hemoglobin disorders including sickle cell disease (SCD) and thalassemia; neurological disorders including neuromyelitis optica; cartilage replacement, such as joint replacement, such as knee and hip replacement; and prophylactic management of cytotoxicity.

[0580] Currently, tissue transplantation requires drug-mediated immunosuppression. Immunosuppression may also be required for cell transplantation. Immunosuppressants make patients severely immunocompromised and at high risk of opportunistic infections. Using the constructs and methods taught in Examples 1-13 above, tissue and cell therapies can be designed that require no additional immunosuppression, or only low doses of immunosuppressants.

[0581] The cells and tissues processed may be any mammalian cells or hybrids between humans and other mammals. (Cyranoski D. Japan approved) 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 embodiments, and the present invention is not limited to the following: It is understood that this has the potential to be applied to any cell or tissue therapy involving the introduction of non-autologous cells or tissues of choice, or modified autologous cells or tissues, into living mammals in which the body may recognize and reject the introduced cells or tissues.

[0583] A. Graft

[0584] 1. Solid organ grafts

[0585] This strategy could be utilized by transient or permanent genetic modification of solid organs with vectors encoding CD43, CD45, and / or CD148 engagers, thus potentially safeguarding grafts from T and NK cell-based immune responses due to the lack of synapse formation. This could be hypothetically used in any organ or part of an organ or organoid graft, including but not limited to: the muscular system (including joints, ligaments, muscles, and tendons), the digestive system (including mouth, teeth, tongue, salivary glands, parotid gland, submandibular gland, sublingual gland, pharynx, esophagus, stomach, small intestine, duodenum, jejunum, ileum, large intestine, liver, gallbladder, mesentery, pancreas, and anal canal), the respiratory system (including nasal cavity, pharynx, larynx, trachea, bronchi, lungs, and diaphragm), the urinary system (including kidneys, ureters, bladder, and urethra); the female reproductive system (ovaries, fallopian tubes, uterus, vagina, vulva, clitoris, and placenta); and the male reproductive system (testis, epididymis, vas deferens, seminal vesicles, prostate gland, bulbourethral glands, Penis and scrotum included; endocrine system (including pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland, pancreas); circulatory system (including heart, patent foramen ovale, arteries, veins, capillaries); lymphatic system (including lymphatic vessels, lymph nodes, bone marrow, thymus, spleen, intestinal 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, eardrum, middle ear, scleral ossicles, inner ear, cochlea, vestibule, semicircular canals, olfactory epithelium, tongue, taste buds, dermal system, main article Article: The endothelial system, mammary glands, skin, and subcutaneous tissue. Organs in these parts can be genetically modified using previously defined gene modification strategies. Aravalli RN, Belcher JD, Steer CJ. Liver-targeted gene therapy: Approaches and challenges. Liver Transpl. 2015;21(6):718-37. Essentially, batches of vectors can be used for in vivo or ex vivo gene delivery via hydrodynamic delivery or similar strategies. This potentially enables the use of interspecies tissues.

[0586] 2. Tissue transplantation

[0587] Similar to solid organ grafts, the use of these engagers may allow for the use of tissue or parts of the organ to be transplanted. Complex transplants (hands, limbs, faces) may be 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 infection.

[0588] Immunosuppression typically consists of triple maintenance immunosuppression with induction therapy (anti-thymocyte globulin [ATG] and / or IL-2 receptor blockers), followed by corticosteroids, antiproliferative agents (e.g., basiliximab), and calcineurin inhibitors (see table). In some cases, topical creams containing calcineurin inhibitors or corticosteroids are used. The use of engagers for these tissues through genetic modification would reduce or even suppress the lifelong need for immunosuppression.

[0589] Skin allografts use donor skin (typically from a cadaver). Allografts are used in patients with extensive burns or other conditions that result in large skin loss, such as when the patient does not have enough undamaged skin to provide a graft. Allografts can be used to cover large, exposed compartments, thus reducing fluid and protein loss and preventing invasive infections. Unlike solid organ grafts, 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 the risk of infection. When valves are damaged or diseased and do not function as they should, they may require repair or replacement. Conditions that can cause heart valve dysfunction are stenosis (rigidity) and regurgitation (leakage valve). Diseased valves can be repaired using a ring to support the damaged valve, or the entire valve may 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 a donor). Allografts and xenografts have the challenge of immune rejection. Therefore, patients may need to undergo lifelong immunosuppression. Modification of valve grafts in CD45 engagers can reduce this need for immunosuppression and prolong the time to rejection.

[0590] Nerve grafting and nerve transfer surgery offer new hope for patients with paralyzed limbs or severely injured limbs due to accidents. In most cases, replacement nerves are sourced from cadavers or, in some cases, living donors. In either case, patients must be given immunosuppressants until these nerves regenerate, which can take up to two years. The modification of nerve grafts in CD45 engagers, as envisioned herein, can reduce this need for immunosuppression and extend the time to rejection.

[0591] Cartilage grafts are used for children with congenital nasal or ear defects and for adults with severe injuries or joint destruction (e.g., severe osteoarthritis). Chondrocytes are more resistant to rejection, presumably 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, particularly in older populations. Inclusion of CD45 engagers for these tissues through genetic modification would increase engraftment.

[0592] Bone grafting is used to reconstruct large bone defects (e.g., after extensive resection of bone cancer). While surviving donor osteocytes do not survive in the recipient, the dead matrix from the allogeneic graft stimulates recipient osteoblasts to regenerate the matrix and implant new bone. This matrix acts as a scaffold to bridge and stabilize the defect until new bone is formed. Caddisfly allogeneic grafts are preserved by freezing to reduce bone immunogenicity and by adding glycerol to maintain chondrocyte survival. The use of CD45 engagers for softer bone tissue through genetic modification would reduce the need for this treatment, thus reducing perioperative procedures and lowering postoperative morbidity due to faster bone tissue engraftment.

[0593] Similar strategies may be used for the production of adrenal tissue allogeneic grafts 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 transplanted each year, it is estimated that only about 1 million are actually transplanted.

[0595] 3.Cell graft

[0596] Modified cells in Engager have suicide genes, chemokine receptors, and activated or inhibited receptors. It can be assumed that these can be simultaneously modified with essentially any transgene, including but not limited to CD43, CD45, and / or CD148 engager-modified cells can also be gene-edited using endonucleases or CRISPR / Cas9 or other technologies to remove other genes, including immunological checkpoint receptors, chemokine receptors, hypoxia-responsive receptors, and key differentiation regulators. Use in non-human cells and tissues is envisioned for use in humans.

[0597] a. Stem cell transplantation for cancer treatment and genetically corrected stem cells for single-gene disorders or compound genetic disorders. Allogeneic stem cell transplantation (from umbilical cord blood, peripheral blood, bone marrow, or other sources) is a recognized therapeutic approach for a variety of malignant tumors, including but not limited to acute myeloid leukemia, spinal dysplasia, and multiple myeloma, and has been tested in various solid organ tumors / cancers, including metastases, such as liver cancer, breast cancer, and kidney cancer. Similarly, this approach has shown success in anemia as well as in hereditary disorders affecting the hematopoietic system, such as severe combined immunodeficiency, e.g., SCID-X, Wiscott-Aldrich syndrome. This approach has several drawbacks, and one of the challenges remains the failure of engraftment and the need for high levels of chimerism. Donor-derived hematopoietic stem cells can be genetically modified ex vivo using vectors encoding the CD43, CD45, and / or CD148 engager constructs or derivatives thereof as described in this application, and it may be conceivable that partial or complete chimerism can be achieved in the absence of potentially lymphocyte-depleting regimens. In this way, host immune cells can be deactivated against the graft, and engraftment can be promoted.

[0598] b. Platelet transfusion

[0599] To avoid rapid platelet rejection potentially mediated by NK cells and other effector cells, it may also be conceivable to utilize engagers in platelets by directly modifying platelets or platelet-producing cells. This could be used before the onset of platelet refractory state in patients who receive multiple platelet infusions during their lifetime.

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

[0601] This strategy could also potentially be used to modify erythrocyte progenitor cells and RBCs to avoid cellular rejection of RBC infusion products in patients requiring multiple RBC transfusions, provided the patient has not developed antibodies against the RBC antigen prior to the time of administration.

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

[0603] The methodologies described above can also be used to produce or generate iPSC or hES cell lines and cells derived therefrom. Therefore, in one embodiment of the present invention, compositions and methods are provided for producing target cells having CD43, CD45, and / or CD148 engagers, thereby creating low immunogenic cells. Such low immunogenic cells are expected to be less susceptible to immune rejection by the recipient to whom such cells are transplanted. Upon transplantation, these low immunogenic cells should engraft (not be rejected). In one embodiment, such target cells can engraft and survive with little or no immunosuppression required of the recipient.

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

[0605] e. Donor leukocyte infusion

[0606] Donor leukocyte infusions (DLIs) involving NK cells, T cells, and macrophages, with or without additional genetic modifications encoding transgenes such as T cell receptors, chimeric antigen receptors, dimeric antigen receptors, or any other genes, may be considered safe for graft infusion by utilizing the simultaneous transduction of CD43, CD45, and / or CD148 engagers, thereby avoiding the formation of functional immunological synapses that, without engagers, could lead to recipient cell-mediated rejection of the graft cells. T cells may include any T cell, including but not limited to suppressor T cells, regulatory T cells, gamma delta T cells, and mucosa-associated invariant T cells (MAITs), 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 further genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activating receptors, and cell adhesion receptors. This can be done by additional transduction or simultaneous transduction of CD43, CD45, and / or CD148 engagers, which in the absence of engagers can lead to recipient cell-mediated rejection of the graft cells, thereby making the graft safe for injection and avoiding cell-mediated graft rejection. Currently, CAR-modified T cells are commonly used to treat cancer.

[0609] Representative CAR T cells include those currently being studied for hematological cancers, which include, but are not limited to, the following targets and genes: BCMA (TNFRSF17), CD123 (IL3RA), CD138 (SDCl), CD19 (CD19) (commercial CD19 CARs include axicaputazine silorucel (Yescarta®) and tisagenlecleucel (Kymriah®)), CD20 (MS4Al), CD22 (CD22), CD38 (CD38), CDS (CDS), IgG K chain (IgGK), LeY (FUT3), NKG2D ligand (NKG2D), RORl (RORl), and WTl (WTl).

[0610] CARs for solid tumors include, but are not limited to, the following targets and genes: targets (genes), 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, and hESC-derived NK cells, with or without further genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activating receptors, and cell adhesion receptors. This can be done by additional transduction or simultaneous transduction of CD43, CD45, and / or CD148 engagers, which in the absence of engagers can lead to recipient cell-mediated rejection of the graft cells, thereby making the graft safe for injection and 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, and hESC-derived macrophages, with or without further genetic modification using chimeric antigen receptors, chemokine receptors, T cell receptors, activating receptors, and cell adhesion receptors. This can be done by additional transduction or simultaneous transduction of CD43, CD45, and / or CD148 engagers, which in the absence of engagers can lead to recipient cell-mediated rejection of graft cells, thereby making the graft safe for injection and avoiding cell-mediated graft rejection.

[0615] i. Genetic correction cells for other hereditary disorders

[0616] It can be hypothesized, in a similar manner, that transplanted cells for metabolic disorders may be modified with transgenes encoding engagers to CD43, CD45, and / or CD148 for optimal engraftment, potentially without the need for lymphocyte ablation.

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

[0618] Other cells intended for use / transplantation as described herein include: endoderm-derived cells, e.g.: exocrine epithelial cells (Brunner's gland cells in the duodenum (enzyme and alkaline mucus), shielded goblet cells in the respiratory and gastrointestinal tract (mucus secretion), stomach, pit cells (mucus secretion), chief cells (pepsinogen secretion), parietal cells (hydrochloride secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells in 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, acinar central cells (pancreas), interglandular tubular cells (pancreas), intestinal brush border cells (with microvilli); Hormone-secreting cells: enteroendocrine cells, K cells (secrete gastric inhibitory peptides), L cells (secrete glucagon-like peptide-1, peptide YY3-36, oxytomodulin, and glucagon-like peptide-2), I cells Thyroid cells (secreting cholecystokinin (CCK)), G cells (secreting gastrin), enterochromaffin cells (secreting serotonin), enterochromaffin-like cells (secreting histamine), N cells (secreting neurotensin), S cells (secretin), D cells (secreting somatostatin), Mo cells (or M cells) (secreting motilin), other hormones secreted: vasoactive enteropropyl peptides, substance P, alpha and gamma-endorphins, bombesin; thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells; islets (islets of Langerhans), alpha cells (secreting glucagon), beta cells (secreting insulin and amyrin), delta cells (secreting somatostatin), epsilon cells (secreting ghrelin), PP cells (gamma cells) (secreting pancreatic polypeptides).

[0619] Ectoderm-derived cells, such as exocrine epithelial cells, salivary gland mucinous cells, salivary gland serous cells, and tongue cells. Ebner's gland cells (cleansing taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), earwax gland cells (earwax secretion), dark eccrine sweat gland cells (glycoprotein secretion), clear eccrine sweat gland cells (small molecule secretion), apocrine sweat gland cells (scent secretion, sex hormone sensitive), Moll's gland cells of the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells of the nose (cleansing the olfactory epithelium); hormone-secreting cells, hormone-secreting cells, anterior / intermediate pituitary cells, adrenocorticotropic hormone-producing cells, gonadotropin-producing cells, mammary gland-stimulating hormone-producing cells, melanin Cell-stimulating hormone-producing cells, growth hormone-producing cells, thyroid-stimulating hormone-producing cells, giant cell neurosecretory cells (secreting oxytocin and vasopressin), small cell neurosecretory cells (secreting thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), vasopressin, oxytocin, neurotensin, and prolactin), chromaffin cells (adrenal gland); epithelial cells, e.g., keratinocytes (differentiated epithelial cells); epithelial basal cells (stem cells); melanocytes; trichocytes (producing hair and nail cells), e.g., medullary hair Stem cells, cortical hair stem cells, microdermal hair stem cells, Huxley layer root sheath cells, Henle 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; interglandular duct cells (salivary gland); striatal duct cells (salivary gland); mammary duct cells (mammary gland); ameloblasts (deposits tooth enamel), oral cells, e.g., odontoblasts (dentin formation), cementoblasts (cementum formation); sensory transmission cells, e.g., auditory hair cells of the organ of Corti, auditory cells of the organ of Corti External hair cells, 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; proprioceptor primary sensory neurons; tactile-sensitive primary sensory neurons; chemoreceptor glomus cells of the carotid body (blood pH sensors); outer hair cells of the vestibular system of the ear (acceleration and gravity);Inner hair cells of the ear's vestibular system (acceleration and gravity); taste receptor cells of taste buds; autonomic nerve neurons, e.g.; cholinergic neurons (various types); adrenergic neurons (various types); peptide-glucan neurons (various types); supporting cells of sensory organs and peripheral neurons, 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 cells of the organ of Corti, vestibular supporting cells, taste bud supporting cells, olfactory epithelial supporting cells, olfactory nerve sheath cells, Schwann cells, satellite glial cells, intestinal glial cells, central nervous system Neurons and glial cells, e.g., neuronal cells (interneurons, basket cells, cartwheel cells, astrocytes, Golgi cells, granule cells, Lugaro cells, unipolar brush cells, Multinoti cells, Chandelier cells, Cajal-Retius cells, double bouquet cells, neuroglial cells, retinal horizontal cells, amacrine cells, starburst amacrine cells, spinal interneurons, Renshaw cells); chief cells (spindle neurons, fork neurons, pyramidal cells, place cells, lattice cells, speed cells, head direction cells, Betz cells, astrocytes, border cells, bushy cells, Purkinje cells, medium spiny neurons); astrocytes; oligodendrocytes; ependymal cells, tanocytes; pituitary cells; nervous system cells, e.g., sensory transmission cells, autonomic neuron 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 primarily derived from the mesoderm, for example: metabolic and storage cells (adipocytes: white and brown adipocytes, hepatic adipocytes); secretory cells (adrenal cortical cells, including those of the zona glomerulosa, produce mineralocorticoids; zona fasciculata cells produce glucocorticoids; zona reticularis cells produce androgens); estrogen-secreting follicular linal cells; progesterone-secreting luteal cells of ruptured follicles (granulosa lutein cells, follicular membrane lutein cells); testosterone-secreting Leydig cells of the testis; seminal vesicle cells (secrete seminal fluid components, including fructose for migrating sperm); prostatic cells (secrete seminal fluid components); urethral bulb Glandular cells (mucus secretion); Bartholin's gland cells (vaginal lubricant secretion); Littrey's gland cells (mucus secretion); Endometrial cells (carbohydrate secretion); Paraglideral cells (renin secretion); Renal macula densa cells; Renal peripolar cells; Renal mesangial cells; Urinary tract barrier cells (parietal epithelial cells; podocytes, proximal tubular brush border cells, cells of the narrow part of the loop of Henle) Thin segment cells: Distal tubular cells of the kidney, collecting duct cells of the kidney, chief cells, interstitial cells, transitional epithelium (lining the bladder); Reproductive system: Glandular cells (of the seminal vesicles, prostate, etc.), efferent duct cells, epididymal chief cells, epididymal basal cells; Circulatory system: Endothelial cells; Extracellular matrix cells: Semilunar plane epithelial cells of the vestibular system of the ear (proteoglycan secretion), interdental epithelial cells of the organ of Corti (secreting the tectonic membrane covering hair cells), loose connective tissue fibroblasts, corneal fibroblasts (corneal stromal cells), tendon fibroblasts, bone marrow reticular tissue fibroblasts, other non-epithelial fibroblasts , pericellular cells (hepatic stellate cells (Ito cells)), nucleus pulposus cells of intervertebral discs, hyaline chondrocytes, fibrochondrocytes, elastochondrocytes, osteoblasts / osteocytes, bone progenitor cells (osteoblast stem cells), vitreous cells of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, pancreatic stellate cells; contractile cells, for example: skeletal muscle cells (red skeletal muscle cells (slow contraction), white skeletal muscle cells (rapid contraction), intermediate skeletal muscle cells, nuclear bag cells of the muscle spindle, nuclear chain cells of the muscle spindle, muscle satellite cells (stem cells)), cardiomyocytes (cardiomyocytes, SA nodule cells, Purkinje fibrils) Cells; smooth muscle cells (various types); myoepithelial cells of the iris; myoepithelial cells of exocrine glands; blood and immune system cells, e.g., red blood cells (erythrocytes) and their precursors, erythroblasts, megakaryocytes (platelet precursors), platelets (a subject of debate at this time, but considered as separate cells), monocytes (white blood cells), connective tissue macrophages (various types), epithelial Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils and their precursors (myeloblasts, promyelocytes, myelocytes, metamyelocytes), eosinophilic granulocytes 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 progenitor cells committed to the blood and immune system (various types); germ cells, such as oogonia / oocytes, spermatocytes, spermatocytes, spermatogonia (stem cells for spermatocytes), sperm; nurse cells, granulosa cells (in the ovary), Sertoli cells (in the testes), reticular cells (in the thymus); and stromal cells: interstitial kidney cells.

[0621] 2. Therapeutic particles

[0622] a. Extracellular vesicles

[0623] It may also be conceivable to use the introduction of CD43, CD45, and / or CD148 engagers directly into extracellular vesicles or into parental cells utilized for the production of extracellular vesicles, for the targeted or systemic delivery of therapeutic transgenes or proteins.

[0624] b. Adenovirus gene delivery

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

[0626] c. Oncolytic viruses

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

[0628] This strategy can also be used for cell-drug delivery, where grafted cells deliver drugs to target tissues, in which case these grafted cells are simultaneously modified with CD43, CD45, and / or CD148 engagers.

[0629] C. Specific conditions

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

[0631] mRNA or DNA encoding CD43, CD45, and / or CD148 engagers may also be delivered, either locally or systemically, to patients with chronic inflammatory diseases in which cytotoxicity is part of the disease physiology. This may 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 grafts

[0633] The present invention may be used in conjunction with conventional stem cell therapies to produce cells and tissues for wound treatment without fear 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. Hereditary metabolic disorders

[0635] The present invention may be used to treat hereditary metabolic disorders such as: 17-alpha-hydroxylase deficiency, 17-beta-hydroxysteroid dehydrogenase 3 deficiency, 18-hydroxylase deficiency, 2-hydroxyglutaric aciduria, 2-methyl-3-hydroxybutyrateuria, 2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase 1 deficiency, 3-alpha-hydroxyacyl-CoA dehydrogenase deficiency, 3-hydroxyisobutyrateuria, 3-methylcrotonyl-CoA carboxylase deficiency, 3-methylglutaconyl-CoA hydratase deficiency (AUH deficiency), 5-oxoprolinase deficiency, and 6-pyruvoyl-tetrahydropterincin. Tase deficiency, abdominal obesity metabolic syndrome, abetalipoproteinemia, acatalasemia, aceruloplasminemia, acetyl-CoA acetyltransferase 2 deficiency, acetyl-carnitine deficiency, acrodermatitis enteroplasmosis, acromegaly, acute intermittent porphyria, adenine phosphoribosyltransferase deficiency, adenosine deaminase deficiency, adenosine monophosphate deaminase 1 deficiency, adenirosuccinase deficiency, adrenal spinal neuropathy, adult polyglucosan body disease, adult-onset type II citrullinemia, albinism deafness syndrome, ocular albinism delayed 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, Argininosuccinateuria, aromatic L-amino acid decarboxylase deficiency, joint contractures, renal dysfunction, cholestatic syndrome, Arts syndrome, aspartylglycosamiuria, ataxia type 1 with oculomotor apraxia, ataxia with vitamin E deficiency, atransferrinemia, atypical Gaucher disease due to saposin C deficiency, 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 iron deposition disorder, Birth 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 disorder, biotinidase deficiency, Bjornstad syndrome, Blue Diaper syndrome, carbamoyl phosphate synthetase 1 deficiency, carnitine palmitoyltransferase 1A Deficiency, carnitine-acylcarnitine translocase deficiency, carnosinemia, central diabetes insipidus, cerebral folate deficiency, cerebral tendon xanthomatous cerebrospinal fluid 1, shanalin-dolfmann syndrome, chediak-Higashi syndrome, CHILD syndrome, childhood hypophosphatasia, childhood-onset cerebral X-linked adrenoleukodystrophy, cholesteryl ester storage disorder, chondrocalcinosis 1, chondrocalcinosis 2, chondrocalcinosis due to apatite crystal deposition, chondrodysplasia punctate 1, X-linked recessive, chronic progressive extraocular muscle palsy, 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 deficiency16, congenital bile acid synthesis deficiency type 1, congenital bile acid synthesis deficiency type 2, congenital glycosylation disorder type I / IIX, congenital erythrodysplasia type 2, congenital erythropoiesis porphyria, congenital lactase deficiency, congenital muscular dystrophy with or without intellectual disability - dystroglycanopathy (type B), copper deficiency, familial benign, CoQ-responsive OXPHOS deficiency, Crigler-Nadjar syndrome type 1, Crigler-Nadjar syndrome type 2, cystinosis, ocular nonnephropathy, cytochrome c oxidase deficiency, D-2-hydroxyglutaric aciduria, D-bifunctional protein deficiency, D-glycerate phosphateemia, Danon disease, DCMA syndrome, DDOST-CDG (CDG-Ir), hearing loss, dystonia, and cerebral white matter dysplasia, dentatorubral-pallidoluysian atrophy, desmosterolosis, diamond-Blackfan anemia, dicarboxylic acid amino aciduria, dihydrolipidemia Amide dehydrogenase deficiency, dihydropteridine reductase deficiency, dihydropyrimidinase deficiency, dihydropyrimidine dehydrogenase deficiency, thirst-induced diabetes insipidus, DOLK-CDG (CDG-Im), Dopa-responsive dystonia, dopamine beta-hydroxylase deficiency, Dowling-Degos disease, DPAGT1-CDG (CDG-Ij), DPM1-CDG (CDG-Ie), DPM2-CDG, DPM3-CDG (CDG-Io), Dubin-Johnson syndrome, encephalopathy due to prosaposin deficiency (sphingolipidosis), erythropoiesis Adult protoporphyria, erythropogenic uroporphyria associated with myeloma, ethylmalonate encephalopathy, Fabry disease, familial HDL deficiency, familial hypocalciuric hypercalcemia type 1, familial hypocalciuric hypercalcemia type 2, familial hypocalciuric hypercalcemia type 3, familial LCAT deficiency, familial partial lipodystrophy type 2, Fanconi-Bickel syndrome, Faber disease, fatal infantile encephalomyopathy, fatty acid hydroxylase-associated neurodegeneration, fish-eye disease, fructose-1,6-bisphosphatase deficiency, fucosidosis,Fukuyama muscular dystrophy, fumarase deficiency, Galactokinase deficiency, galactosialidosis, gamma-aminobutyrate transaminase deficiency, gamma-cystathionase deficiency, Gaucher disease, Gaucher disease-ophthalmoplegia-cardiovascular calcification (Gaucher disease), perinatal fatal Gaucher disease, Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, diabetes insipidus of pregnancy, Gilbert's syndrome, Guittelmann's syndrome, glucose transporter type 1 deficiency syndrome, glucose-galactose malabsorption, glutamate hormone Muiminotransferase deficiency, glutamine deficiency, congenital, glutaric acidemia type 1, glutaric acidemia type 2, glutaric acidemia type 3, glutathione synthetase deficiency, glutathionuria, 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, Glyco Glycogen storage disease type 5, glycogen storage disease type 6, glycogen storage disease type 7, glycoprotein storage disorder, 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 gynocerebral atrophy, Heim-Munk syndrome, Hartnapp disease, Hawkinsinuria, 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 hyperfrightening disorder, Hereditary multiple osteochondroma, Hereditary sensory autonomic neuropathy type 1E, Hereditary sensory neuropathy type 1, Hermanski-Padlak syndrome type 2, Histidineemia, HMG CoA lyase deficiency, homocarnosinosis, homocysteinemia, homocystinuria due to CBS deficiency, homocystinuria due to MTHFR deficiency, Hurler syndrome, Hurler-Scheyet syndrome, hydroxykynurenuria, hyper-IgD syndrome, hyperbeta-alanininemia, hypercoagulation syndrome due to glycosylphosphatidylinositol deficiency, hyperglycemia, hyperinsulinemia due to glucokinase deficiency,Hyperinsulinemia-hyperammonemia syndrome, hyperlipidemia type 3, hyperlipoproteinemia type 5, hyperlysinemia, hyperphenylalaninemia due to dehydratase deficiency, hyperprolinemia, hyperprolinemia type 2, hypertryptophanemia, hypolipoproteinemia, hypophosphatasia, I-cell disease, Imerslund-Grasbeck syndrome, iminoglycinuria, inclusion body myopathy 2, inclusion body myopathy 3, infantile free sialic acid storage (free sialic acid storage), infantile neuroaxonal dystrophy, infant-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, latosterosis, LCHAD deficiency, Leber hereditary optic neuropathy, Leigh syndrome, French-Canadian type, Lesch-Nyhan syndrome, infantile leucine-sensitive hypoglycemia, leukoencephalopathy-dystonia-disorder Dynamic 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 20, limb-girdle muscular dystrophy type 2T (limb-girdle muscular dystrophy), type 2C, complex lipase deficiency, lipoic acid synthetase deficiency, Urbach-Wiethe lipoid proteinosis, low-eye encephalorenal syndrome, lysinuric protein intolerance, malonyl-CoA decarboxylase deficiency, MAN1B1-CDG, Mannor Streptococcal 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, maternal genetic diabetes 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 with homocystinuria cbl C,Homosis, Methylmalonic acidemia with tinuria (cblD type), methylmalonic acidemia with homocystinuria (cblF type), methylmalonic acidemia with homocystinuria (cblJ type), methylmalonic aciduria (cblA type), methylmalonic aciduria (cblB type), mevalonic aciduria, MGAT2-CDG (CDG-IIa), mild phenylketonuria, mitochondrial complex I deficiency, mitochondrial complex II deficiency, mitochondrial complex III deficiency, mitochondrial DNA depletion syndrome, morphology of encephalomyopathy with methylmalonic aciduria, mitochondrial DNA related 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-related hepatencephaloencephalopathy 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 anomalies-hypotension-seizure syndrome, multiple congenital anomalies-hypotension-seizure syndrome type 2, multiple endocrine neoplasia type 2B, multiple sulfatase deficiency, multiple symmetrical lipomatosis, myoophthalmoencephalopathy, muscular dystrophy, congenital, giant Large cone type, muscle phosphorylase kinase deficiency, myoconstrictive Ehlers-Danlos syndrome, myoclonus epilepsy with red rag 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, New Laxova (Neu Laxova syndrome, neuroferritinosis, 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, triglyceride storage disorder with myopathy, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Northern epilepsy, other unspecified 3-MGAuria type, Occipital-Horn syndrome, ocular albinism type 1, oculocutaneous albinism type 1, oculocutaneous albinism type 1B, oculocutaneous albinism type 2, oculocutaneous albinism type 3, OPA3 deficiency, optic nerve atrophy type 1, ornithine transcarbamylase deficiency, ornithine translocase deficiency syndrome, O Lotinic aciduria type 1, Papillon-Lefevre syndrome, Parkinson's disease type 9, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, pentoseuria, permanent neonatal diabetes, peroxisome formation disorder, peroxisome disorder, Perot syndrome, Peters-Plass syndrome, PGM1-CDG, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphoribosyl pyrophosphate synthetase deficiency, PMM2-CDG (CDG-Ia), pontocerebellar dysplasia type 6, late-onset cutaneous plaque Rufirinosis, primary carnitine deficiency, primary hyperoxaluria type 1, primary hyperoxaluria type 2, primary hyperoxaluria type 3, primary hypomagnesemia with secondary hypocalcemia, progressive extraocular palsy, autosomal recessive type 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 phosphorylation This condition is caused by ze deficiency, dysostosis, 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 pipecholic acidemia, Refsum disease, infantile form, renal glycosuria, renal hypomagnesemia², renal hypomagnesemia-⁶, renal tubular disease, diabetes mellitus, and mitochondrial DNA replication. Cerebellar ataxia, RFT1-CDG (CDG-In), chondrodysplasia rhizoides type 3 (chondrodysplasia rhizoides), Rotor syndrome, saccharopinuria, Salla disease (free sialic acid storage), sarcosinemia, Schayet syndrome, Schimke immune dysplasia, Schindler's disease type 1, cochlear pelvic dysplasia, SCOT deficiency, Seablue histiocytosis, Sengels syndrome, ataxic neuropathy, dysarthria, and ophthalmoplegia, sepiapterin reductase deficiency, severe combined immunodeficiency, short-chain acyl-CoA dehydrogenase deficiency, sialidosis type 1, sial Dosis type II, sialic aciduria, French type, sideroblastic anemia and mitochondrial myopathy, sitosterolemia, Sjögren-Larsson syndrome, SLC35A1-CDG (CDG-IIf), SLC35A2-CDG, SLC35C1-CDG (CDG-IIc), Smith-Lemley-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, spinal dysplasia Ehlers-Danlos syndrome, Spine epiphyseal dysplasia, arthrolacia, SRD5A3-CDG (CDG-Iq), SSR4-CDG, succinate semialdehyde dehydrogenase deficiency, Tangier disease, Tay-Sachs disease, thiamine-responsive megaloblastic anemia syndrome, thiopurine S-methyltransferase deficiency, tiglinemia, TMEM165-CDG (CDG-IIk), transaldolase deficiency, transcobalamin 1 deficiency, transient neonatal diabetes mellitus, trehalase deficiency, trimethylaminuria, triose phosphate isomerase deficiency, tyrosine hydroxylase deficiency X-linked adrenoleukodystrophy, X-linked Charcot-Marie-Tooth disease type 5, X-linked creatine deficiency, X-linked chondrodysplasia 2, X-linked sideroblastic anemia, xanthinuria type 1, xanthinuria type 2, and Zellweger syndrome.

Claims

1. It is a therapeutic agent, It contains cells or extracellular vesicles expressing one or more CD45-binding molecules configured to inhibit functional immunological synapse formation with cytotoxic cells, thereby preventing cytotoxicity. The one or more CD45-binding molecules comprise a recombinant protein containing, in this order: (i) a signal peptide, (ii) a heavy chain of an anti-CD45 antibody, (iii) a first linker, (iv) a light chain of an anti-CD45 antibody, (vi) a stalk, and (vii) a transmembrane region. The first linker contains an SGGGG motif and / or its length varies over 5 to 60 amino acids. The aforementioned stalk has a length of 8 to 200 amino acids. A therapeutic agent wherein the transmembrane region is derived from CD34, CD45, CD28, and / or CD8a.

2. The therapeutic agent according to claim 1, wherein the recombinant protein comprises in this order: (i) a signal peptide, (ii) a heavy chain of an anti-CD45 antibody, (iii) a first linker, (iv) a light chain of an anti-CD45 antibody, (v) a second linker whose length varies by 5 to 60 amino acids, (vi) a stalk, (vii) a transmembrane region, and (viiii) an intracellular region.

3. The therapeutic agent according to claim 1, wherein one or more CD45-binding molecules contain a single-chain antibody.

4. The therapeutic agent according to claim 1, comprising extracellular vesicles.

5. The therapeutic agent according to claim 1, comprising a protein having the sequence described in Sequence ID No. 5, 54, 56, 58, or 60, or a protein having at least 80% identity with Sequence ID No. 5, 54, 56, 58, or 60.

6. The therapeutic agent according to claim 1, comprising cells having one or more CD45-binding molecules expressed on the surface of the cells.

7. The therapeutic agent according to claim 6, wherein the cells include graft cells.

8. The therapeutic agent according to claim 6, wherein one or more CD45-binding molecules can retain CD45 in developing immunological synapses on the surface of the cytotoxic cells, thereby interfering with the formation of functional immunological synapses.

9. A therapeutic agent according to any one of claims 1 to 8, for use as a pharmaceutical.

10. A therapeutic agent according to any one of claims 1 to 8, for use in the prevention or treatment of one or more conditions of autoimmune diseases, hematological cancers, bone marrow failure syndromes, hereditary immune disorders, abnormal hemoglobin disorders, neurological disorders, and graft-versus-host diseases.

11. A therapeutic agent according to any one of claims 1 to 8, for use in the prevention or treatment of one or more of psoriasis and vitiligo, for promoting escape from T cell-mediated lysis.

12. The therapeutic agent according to any one of claims 1 to 8, wherein graft-versus-host disease is prevented when xenograft cells for transplantation are exposed to the therapeutic agent.

13. The therapeutic agent according to any one of claims 1 to 8, wherein the cell or extracellular vesicle is bound to the surface of a different cell.

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