MXPA06003128A

MXPA06003128A - Therapeutic binding molecules

Info

Publication number: MXPA06003128A
Application number: MXPA/A/2006/003128A
Authority: MX
Inventors: Kolbinger Frank; Carballido Herrera Josem; Aszodi Andras; Saldanha Josew; M Hall Bruce; Gregori Silvia; Grazia Roncarolo Maria; Loux Veronique; Aversa Gregorio; Jeschke Margit
Original assignee: Aszodi Andras; Aversa Gregorio; Carballido Herrera Jose M; Gregori Silvia; M Hall Bruce; Jeschke Margit; Kolbinger Frank; Loux Veronique; Novartis Ag; Novartis Pharma Gmbh; Grazia Roncarolo Maria; Saldanha Jose W
Priority date: 2003-09-18
Filing date: 2006-03-17
Publication date: 2007-04-10

Abstract

A molecule comprising at least one antigen binding site, comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-Ile-IIe-His (NYIIH), said CDR2 having the amino acid sequence Tyr-Phe-Asn-Pro -Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe -Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT);e.g. further comprising in sequence the hypervariable regions CDR1', CDR2'and CDR3', CDR1'having the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-GIy-Thr -Ser-IIe-Gln (RASQNIGTSIQ), CDR2'having the amino acid sequence Ser-Ser-Ser-Glu-Ser -Ile-Ser (SSSESIS) and CDR3'having the amino acid sequence Gln-GIn-Ser-Asn-Thr-Trp - Pro-Phe-Thr (QQSNTWPFT), e.g. a chimeric or humanised antibody, useful as a pharmaceutical.

Description

THERAPEUTICAL BINDING MOLECULES FIELD OF THE INVENTION The present invention relates to organic compounds, such as the binding molecules against CD45 antigen isoforms, such as for example monoclonal antibodies (mAbs), and their use.

BACKGROUND OF THE INVENTION One aspect in the treatment of a variety of diseases is to achieve the elimination or inactivation of pathogenic leukocytes and the potential for the induction of tolerance to inactive immune-pathogenic responses. It is believed that the rejection of organ, cell and tissue transplantation and the various autoimmune diseases are the main result of immune responses mediated by T cells that are capable of recognizing specific antigens that are captured, processed and presented to the T helper cells through of the antigen presenting the cell (APC) such as macrophages and dendritic cells, in the form of an antigen-MCH complex, ie the helper T cell when recognizing specific antigens is stimulated to produce cytokines such as IL-2 and to express or upregulate some cytokine receptors and other activation molecules and to proliferate. Some of these helper T cells can act directly or indirectly, i.e., by helping the T cells or cytotoxic activating B cells, to destroy the cells or tissues expressing the selected antigen. After the termination of the immune response some of the clonally selected mature cells remain as the memory aid and memory cytotoxic T cells, which circulate in the body and quickly recognize the antigen if it appears again. If the antigen that triggers this response is an innocuous environmental antigen the result is an allergy, this may result, if the antigen is not a foreign antigen, but it is a homogenous antigen, it can result in an autoimmune disease; If the antigen is an antigen from a transplanted organ, the result may be rejection of the graft. The immune system has been developed to recognize homogeneous from non-homogeneous. This property allows an organism to survive in an environment exposed to daily pathogen challenges. This specificity for nonhomogeneous and tolerance towards homogeneous originates during the development of the T cell repertoire towards the thymus through positive and negative selection processes, which also includes the recognition and elimination of auto reactive T cells. This type of tolerance is referred to as a central tolerance. However, some of the self-reactive cells escape this selective mechanism and have a potential risk for the development of autoimmune diseases. To control self-reactive T cells that have escaped from the periphery, the immune system has peripheral regulatory mechanisms that provide protection against autoimmunity. These mechanisms are a basis for peripheral tolerance. The cell surface antigens recognized by specific mAbs are generally designated by a CD number (Differentiation Group) assigned by International Leukocyte Typing Workshops and the term CD45 applied herein refers to the common antigen of the cell surface leukocyte CD45; and a mAb to that antigen which is referred to herein as "anti-CD45". Antibodies against common leukocyte antigen (LCA) or CD45 with a major component of anti-lymphocyte globulin (ALG). CD45 belongs to the family of transmembrane tyrosine phosphatases and is both a positive and negative regulator of cell activation, depending on the interaction of the receptor. The activity of CD45 phosphatases appears to be required for the activation of cinases of the Src family associated with the B and T cell antigen receptor (Trowbridge IS et al., Annu Rev Immunol., 1994; 12: 85-116). Thus, in T cell activation, CD45 is essential for signal 1 and CD45 deficient cells have profound defects in activation events measured by TCR. The CD45 antigen exists in different isoforms that comprise a family of transmembrane glycoproteins. Different isoforms of CD45 differ in their structure of the extracellular domain that originates from the alternative division of three variable exons that encode part of the extracellular region of CD45 (Streuli MF., Et al., J. Exp. Med. 1987; 166: 1548-1566). The various isoforms of CD45 have different extracellular domains, but have the same transmembrane and cytoplasmic segments that have two highly conserved phosphatase domains, homologues of approximately 300 residues. Different combinations of isoforms are differentially expressed in sub-populations of T and B lymphocytes (Thomas ML, et al., Immunol., Today 1988; 9: 320-325). Some monoclonal antibodies recognize a common epitope for all the different isoforms, while other mAbs have a restricted specificity (CD45R), dependent on which alternatively divided exons they recognize. For example, monoclonal antibodies recognizing the product of exon A are consecutively designated CD45RA, these recognize the various isoforms containing exon B which has been designated CD45RB (Beverley PCL et al., Immunoi. Supp. 1988; 1: 3-5 ). Antibodies such as UCHL1 selectively bind to the 180 kDa isoform CD45RO (without any of the variable exons, A, B or C) that appear to be restricted to a subset of activated T cells, memory cells and cortical thymocytes and not they are detected in B cells (Terry LA et al., Immunol., 1988; 64: 331-336).

DESCRIPTION OF THE FIGURES Figure 1 shows that the inhibition of the primary MLR through the "candidate mAb" depends on the dose in the range of 0.001 and 10 μg / ml. "Concentration" is the concentration of the "candidate mAb". Figure 2 shows the plasmid map of HCMV-G1 vector expression HuA6-VHQ comprising the heavy chain having the nucleotide sequence SEQ ID NO: 12 (3921-4274) in the nucleotide sequence of the full expression vector ID NO: 15. Figure 3 shows the plasmid map of the expression vector HCMV-G1 HuA6-VHE comprising the heavy chain having the nucleotide sequence SEQ ID NO: 11 (3921-4274) in the nucleotide sequence of the complete expression vector SEQ ID NO: 16. Figure 4 shows the plasmid map of the HCMV-K expression vector HuAb-VL1 humV1 comprising the light chain having the nucleotide sequence SEQ ID NO: 14 (3964-4284) in the nucleotide sequence of the complete expression vector SEQ ID NO: 17. Figure 5 shows the plasmid map of the HCMV-K expression vector HuAb-VL1 humV2 comprising the light chain having the nucleotide sequence SEQ ID NO: 13 (3926-4246) in the nucleotide sequence of The complete expression vector SEQ ID NO: 18. Figure 6 shows the plasmid map of the expression vector LCVL1SP20 comprising the light chain having the nucleotide sequence SEQ ID NO: 33 in the nucleotide sequence of the complete expression vector SEQ ID NO: 36. Figure 7 shows the plasmid map of the expression vector LCVL2SP20 comprising the light chain having the nucleotide sequence SEQ ID NO: 13 in the nucleotide sequence of the complete expression vector SEQ ID NO: 39 Figure 8 shows the plasmid map of the expression vector HCVHEN73D Sp20 comprising the heavy chain having the nucleotide sequence SEQ ID NO: 34 in the nucleotide sequence of the complete expression vector SEQ ID NO: 37. Figure 9 shows the plasmid map of the expression vector HCVHQN73D Sp20 comprising the heavy chain having the nucleotide sequence SEQ ID NO: 35 in the nucleotide sequence of the expression vector Complete SEQ ID NO: 38. Figure 10 shows the plasmid map of the HCVHESp20 expression vector comprising the heavy chain having the nucleotide sequence SEQ ID NO: 11 in the nucleotide sequence of the complete expression vector SEQ ID NO: 40. Figure 11 shows the plasmid map of the HCVHQSp20 expression vector comprising the heavy chain having the nucleotide sequence SEQ ID NO: 12 in the nucleotide sequence of the complete expression vector SEQ ID NO: 41. Figure 12 shows the Analysis of Size Exclusion Chromatography of VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2, and of VHE / humV1 together with VHE-N73D / humV1. Figure 13 shows the Ion Exchange Chromatography of VHE / humV2, VHE / humV1, VHQ / humV2, VHQ / humV1 and VHE / humV2 together with VHE-N73D / humV1. Figure 14 shows the Reverse Phase Chromatography of VHE / humV2 and VHE-N73D / humV1.

DESCRIPTION OF THE INVENTION A linker molecule comprising a polypeptide sequence that binds CD45RO and CD45RB has been found, hereinafter also referred to as a "linker molecule" CD45RO / RB. "These linker molecules according to the invention can induce immunosuppression, inhibit T cell responses and induce T cell tolerance. In addition, the binding molecules of the invention inhibit primary mixed lymphocyte (MLR) responses. CD45RO / RB bond preferably also have weakened proliferative responses in secondary MLR even in the absence of the CD45RO / RB binding molecules in the secondary MLR.These weakened proliferative responses in secondary MLR are an indication of the ability of the binding molecules of the invention In addition, it is found that the CD45RO / RB binding molecules of the invention can induce Circulation of cell death through apoptosis in human T lymphocytes, can support the differentiation of T cells with regulatory T cell phenotype characteristics (Treg); and / or can induce T-regulatory cells capable of suppressing simple T cell activation. In addition, it is found that in vivo administration of the binding molecule, CD45RO / RB to mice with severe combined immunodeficiency (SCID) that undergo xeno-GVHD after injection with human PBMC can prolong the survival of the mice, compared to the treated mice with control, even though human T cells in circulation can still be detected in the CD45RO / RB binding molecule. The CD45RB / RO binding molecule can also suppress the inflammatory process mediating skin rejection of the human allograft. Furthermore, it is found that the CD45RO / RB binding molecule can suppress the process that mediates rejection of human allograft skin, in particular, it can suppress the inflammatory process mediating rejection of human allograft skin in vivo in SCID mice transplanted with human skin and grafted with mononuclear splenocytes. And further, it is found that the CD45RB / RO binding molecule can lead to prolonged human islet allograft survival through the prevention of graft infiltration and through the inhibition of leukocyte-mediated graft rejection reaction in vivo. . By "CD45RO / RB binding molecule" is meant any molecule capable of binding specifically to the CD45RB and CD45RO sofmas of the CD45 antigen, either alone or in association with other molecules. The binding reaction can be shown by standard methods (qualitative assay) including for example, any kind of binding assay such as direct or indirect immunofluorescence together with fluorescence or cytofluorimetric microscopy (FACS) analysis, the immunosorbent assay. enzyme linked (ELISA) or radioimmunoassay where the link of the molecule can be visualized to the cells that express a particular CD45 isoform. For example, the inhibition of the primary or secondary mixed lymphocyte response (MLR) can be determined, such as an in vitro assay or a bioassay to determine the inhibition of the primary or secondary MLR in the presence and absence of a CD45RO binding molecule. / RB and the determination of the differences in the inhibition of primary MLR. Alternatively, in vitro functional modulatory effects can also be determined by measuring PBMC or proliferation of T cells or CD4 + cells, the production of cytokines, the change in the expression of cell surface molecules, for example, after cell activation in MLR, or after stimulation with a specific antigen such as tetanus toxoid or other antigens, or with polyclonal stimulators such as phytohemagglutinin (PHA) or anti-CD3 and anti-CD29 antibodies or phorbol esters and ionophores Ca2 +. The cultures are established in a similar manner as described with respect to MLR, except that allogeneic cells such as soluble stimulator antigen, or polyclonal stimulators are used instead of such as those mentioned above. T cell proliferation is preferably measured as described above through the incorporation of 3H-thymidine. Cytokine production is preferably measured through the sandwich ELISA where an antibody capture cytokine covers the surface of a 96-well plate, culture supernatants are added and incubated for 1 hour at room temperature and then add an antibody for specific detection for the particular cytokine, after a second pass antibody conjugated with an enzyme such as horseradish peroxidase followed by the corresponding substrate and the absorbance is measured in a plate reader. The change in molecules at the cell surface can preferably be measured through direct or indirect immunofluorescence after staining the target cells with antibodies specific for a particular cell surface molecule. The antibody can be either directly labeled with fluorochrome or a fluorescently labeled second step antibody specific for the first antibody can be used., and the cells are analyzed with a cytofluorimeter. The binding molecule of the invention has a binding specificity for both CD45RO and CD45RB ("the CD45RB / RO binding molecule"). Preferably the binding molecule binds the CD45RO isoforms with a dissociation constant (Kd) <20nM, preferably with a Kd < 15 nM or < 10 nM, more preferably with a Kd < 5 nM. Preferably linker molecule binds to the CD45RB isoforms with a Kd < 50 nM, preferably with a Kd < 15 nM or < 10 nM, more preferably with a Kd < 5 nM. In a preferred additional embodiment the binding molecule of the invention binds those CD45 isoforms which 1) includes the A and B epitopes but not the C epitope of the CD45 molecule; and / or 2) includes the B epitope but not A and not the C epitope of the CD45 molecule; and / or 3) does not include any of the A, B or C epitopes or the CD45 molecule. In a further preferred embodiment the binding molecule of the invention does not bind CD45 isoforms including 1) all epitopes A, B and C of the CD45 molecule; and / or 2) both B and C epitopes but not the A epitope of the CD45 molecule. In further preferred embodiments, the linker molecule of the invention further 1) recognizes memory T cells and alloactivates in vivo; and / or 2) binds to its target in human T cells, such as for example PEER cells; wherein said link preferably is with a Kd < 15 nM, more preferably with a K < 10 nM, preferably with a Kd < 5nM; and / or 3) inhibits the function of the alloreactive T cell in vitro, preferably with an IC50 of less than 100 nM, preferably less than 50 nM or 30 nM, preferably with an IC50 of about 10 or 5 nM, preferably with an IC50 of approximately 0.5 nM or even 0.1 nM; and / or 4) induces cell death through apoptosis in human lymphocytes; and / or 5) induces the tolerance of the alloantigen specific T cell in vitro; and / or 6) prevents xenogeneic graft-versus-host-host disease (GvHD) induced in SCID mice through injection of human PBMC when administered in an effective amount; I 7) binds to T lymphocytes, monocytes, stem cells, natural killer cells, and / or granulocytes, but to platelets or B lymphocytes; and / or 8) supports the differentiation of T cells with a characteristic T-regulatory cell phenotype (Treg); and / or 9) induces regulatory T cells capable of suppressing simple T cell activation; and / or 10) suppresses the inflammatory process mediating the rejection of human allograft skin, in particular, suppresses the inflammatory process mediating rejection of human allograft skin in vivo in SCID mice transplanted with human skin and grafted with mononuclear splenocytes; and / or 11) prolong the survival of the human islet allograft in a model of hu-PBL-NOD / SCID mice. In a further preferred embodiment the binding molecule of the invention binds to the same epitope as the monoclonal antibody "A6" as described by Aversa et al., Cellular Immunoiogy 158, 314-328 (1994). Due to the binding properties described above and the biological activities, said binding molecules of the invention are particularly useful in medicine, for example therapy and / or prophylaxis. Diseases wherein the binding molecules of the invention are particularly useful include autoimmune diseases, rejection to transplantation, dermatitis, inflammatory spleen disease and / or allergies, as will be set forth below. It has been found that a molecule comprising a polypeptide of SEQ ID NO: 1 and a polypeptide of SEQ ID NO: 2 is a CD45RO / RB binding molecule. The hypervariable regions CDR1 ', CDR2' and CDR3 'have also been found in a CD45RO / RB binding molecule of SEQ ID NO: 1, CDR1' has the amino acid sequence Arg-Ala-Ser-GIn-Asn-ile-Gly -Thr-Ser-lle-Gin (RASQNIGTSIQ) (SEQ ID NO: 19), CDR2 'has the amino acid sequence Ser-Ser-Ser-Glu-Ser-lle-Ser (SSSESIS) (SEQ ID NO: 20) and CDR3 'has the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT) (SEQ ID NO: 21). The hypervariable regions CDR1, CDR2 and CDR3 have also been found in a CD45RO / RB binding molecule of SEQ ID NO: 2, CDR1 has the amino acid sequence Asn-Tyr-lie-lie-His (NYIIH) (SEQ ID NO: 22), CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) (SEQ ID NO: 23) and CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT) (SEQ ID NO: 24). The CDRs are 3 specific complementary determination regions that are also called hypervariable regions, which essentially determine the characteristics of the antigen binding. These CDRs are part of the variable region, for example, of SEQ ID NO: 1 or SEQ ID NO: 2, respectively, where these CDRs alternate with structure regions (FR's), for example the constant regions. An A SEQ ID NO: 1 is part of a light chain, for example of SEQ ID NO: 3, and a SEQ ID NO: 2 is part of a heavy chain, for example of SEQ ID NO: 4, in a chimeric antibody according to the present invention. The CDRs of a heavy chain together with the CDRs of an associated light chain essentially constitute the antigen binding site of a molecule of the present invention. It is known that the contribution made by a variable chain region to the binding energy is very low compared to that made by the associated heavy chain variable region and that of the isolated heavy chain variable regions have a binding activity to its own antigen . Said molecules are commonly referred to as individual domain antibodies.

In one aspect the present invention provides a binding molecule comprising at least one antigen binding site, for example a CD45RO / RB binding molecule, which sequentially comprises the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 has the amino acid sequence Asn-Tyr-lle-lle-His (NYIIH) (SEQ ID NO: 22), said CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys- Tyr-Asn-GIu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) (SEQ ID NO: 23) and said CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT ) (SEQ ID NO: 24); for example and direct equivalents thereof. In another aspect the present invention provides a molecule comprising at least one antigen binding site, for example a CD45RO / RB binding molecule, comprising a) a first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 has the amino acid sequence Asn-Tyr-lle-lle-His (NYIIH) (SEQ ID NO: 22), said CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His- Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) (SEQ ID NO: 23) and said CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe -Asp-Thr (SGPYAWFDT) (SEQ ID NO: 24); and b) a second domain comprising in sequence the hypervariable regions CDR1 ', CDR2' and CDR3 ', CDR1' has the amino acid sequence Arg-AIa-Ser-GIn-Asn-lle-Gly-Thr-Ser-lle-Gln ( RASQNIGTSIQ) (SEQ ID NO: 19), CDR2 'has the amino acid sequence Ser-Ser-Ser-GIu-Ser-ile-Ser (SSSESIS) (SEQ ID NO: 20) and CDR3' has the amino acid sequence Gln- Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT) (SEQ ID NO: 21), for example and direct equivalents thereof. In a preferred embodiment the first domain sequentially comprises the hypervariable regions CDR1, CDR2 and CDR3 is an immunoglobulin heavy chain, and the second domain sequentially comprises the hypervariable regions CDR1 ', CDR2' and CDR3 'in an immunoglobulin light chain. In a further aspect the present invention provides a molecule for example to a CD45RO / RB binding molecule, comprising a polypeptide of SEQ ID NO: 1 and / or a polypeptide of SEQ ID NO: 2, preferably comprising in domain a polypeptide of SEQ ID NO: 1 and in another domain a polypeptide of SEQ. ID NO: 2, for example a chimeric monoclonal antibody and in another aspect, a molecule, for example a CD45RO / RB binding molecule, comprising a polypeptide of SEQ ID NO: 3 and / or a polypeptide of SEQ ID NO: 4 , preferably comprising in one domain a polypeptide of SEQ ID NO: 3 and in another domain a polypeptide of SEQ ID NO: 4, for example a chimeric monoclonal antibody. When the antigen binding site comprises both, the first and second domains or a polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3, respectively, and a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 , respectively, these can be located in the same polypeptide, or, preferably each domain can be in a different chain, for example the first domain being part of a heavy chain, for example the immunoglobulin heavy chain, or a fragment thereof and the second domain being part of a light chain, for example an immunoglobulin light chain or fragment thereof. It has been found that a CD45RO / RB binding molecule according to the present invention is a CD45RO / RB binding molecule in a mammal, for example human body environment. A CD45RO / RB binding molecule according to the present invention can thus be designated as a monoclonal antibody (mAb), wherein the binding activity is mainly determined through the CDR regions as described above, for example said regions CDRs are associated with other molecules without a binding specificity, such as a structure, for example constant regions, which are substantially of human origin. In another aspect the present invention provides a CD45RO / RB binding molecule that is not the monoclonal antibody "A6" as described by Aversa et al., Cellular Immunology 158, 314-328 (1994), which are incorporated herein by reference to the passages that characterize A6. In another aspect the present invention provides a CD45RO / RB binding molecule according to the present invention which is a chimeric, humanized or fully human monoclonal antibody. Examples of a CD45RO / RB binding molecule include chimeric or humanized antibodies for example antibody derivatives as produced by B cells or hybridomas or any fragment thereof, for example F (ab ') 2 and Fab fragments, as well as individual chain or single domain antibodies. An individual chain antibody consists of variable regions of light and heavy chains of the antibody covalently linked through a peptide linker, usually consisting of 10 to 30 amino acids, preferably 15 to 25 amino acids. Accordingly, said structure does not include the constant part of the heavy and light chains, and it is believed that the small peptide spacing should be less antigenic than a complete constant part. By "chimeric antibody" is meant an antibody wherein the constant regions of the heavy and light chains or both are of human origin while the variable domains of the heavy and light chains are not of human origin (eg, murine). By "humanized antibody" is meant an antibody in which the hypervariable regions (CDRs) are not of human origin (eg murine) while all or substantially all other parts, e.g., constant regions and highly conserved parts of the Variable regions are of human origin. A humanized antibody can, however, retain a few amino acids of the murine sequence in the regions of the variable regions adjacent to the hypervariable regions. The hypervariable regions, i.e., CDRs according to the present invention can be associated with any kind of structure regions, for example constant parts of light and heavy chains, of human origin. Suitable framework regions are for example described in "Sequences of proteins of immunological interest", Kabat, E.A. and others, US Department of Health and Human Services. Preferably the constant part of a human heavy chain can be of the IgG 1 type, including subtypes, preferably the constant part of the human light chain can be of the K or? Type, more preferably of the K type. Preferably, said heavy chain comprises no more than one glycosylation site, more preferably the glycosylation site is an N-glycosylation site, and more preferably the glycosylation site is located in the constant part of the heavy chain. More preferably no glycosylation site is present in the variable region, preferably no glycosylation site in the region of the structure. A preferred constant part of a heavy chain is a polypeptide of SEQ ID NO: 4 (without the portions of the sequence of CDR1 ', CDR2' and CDR3 'that were specified above) and a preferred constant part of a light chain is a polypeptide of SEQ ID NO: 3 (without the parts of the sequence of CDR1, CDR2 and CDR3 that were specified above). A humanized antibody comprising a light chain variable region of amino acid SEQ ID NO: 7 or amino acid SEQ ID NO: 8, comprising CDR1 ', CDR2' and CDR3 'according to the present invention and / or a heavy chain variable region of SEQ ID NO: 9 or SEQ ID NO: 10, comprising CDR1, CDR2 and CDR3 according to the present invention. Another additional humanized antibody comprising a light chain variable region of the amino acid SEQ ID NO: 7 or of the amino acid SEQ ID NO: 8, comprising CDR1 ', CDR2' and CDR3 'according to the present invention and / or a heavy chain variable region of SEQ ID NO: 31 or SEQ ID NO: 32, comprising CDR1, CDR2 and CDR3 according to the present invention. In another aspect the present invention provides a humanized antibody comprising a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10 and polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8. Still a further aspect of the present invention provides a linker molecule which is a humanized antibody comprising a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 32 and to polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8. In another aspect, the present invention provides a humanized antibody comprising - a polypeptide of SEQ ID NO: 9 and a polypeptide of SEQ ID NO: 7 (such as VHE / humV2), - a polypeptide of SEQ ID NO: 9 and a polypeptide of SEQ ID NO: 8 (such as VHE / humV1), - a polypeptide of SEQ ID NO: 10 and a polypeptide of SEQ ID NO: 7 (such as VHQ / humV2), - a polypeptide of SEQ ID NO: 10 and a polypeptide of SEC ID NO: 8 (such as VHQ / humV1), - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 7 (such as VHEN73D / humV2), - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 8 (such as VHEN73D / humV1), - a polypeptide of SEQ ID NO: 32 and a polypeptide of SEQ ID NO: 7 (such as VHQN73D / humV2), or - a polypeptide of SEQ ID NO: 32 and to a polypeptide of SEQ ID NO: 8 (such as VHQN73D / humV1). A polypeptide according to the present invention, for example of a specified sequence, for example of CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), CDR3 (SEQ ID NO: 24), CDR1 '(SEQ. ID NO: 19), CDR2 '(SEQ ID NO: 20), CDR3' (SEQ ID NO: 21), or of a SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32 include direct equivalents of said (poly) peptide (sequence); for example including a functional derivative of said polypeptide. Said functional derivative may include covalent modifications of a specified sequence, and / or said covalent modifications of a specified sequence, and / or functional derivative may include variants of the amino acid sequence of a specified sequence. "Polypeptide", unless otherwise specified herein, includes any peptide or protein comprising amino acids linked to one another via peptide bonds, which have an amino acid sequence starting at the N-terminus and ending at the C-terminus -terminal. Preferably the polypeptide of the present invention is a monoclonal antibody, more preferred is a chimeric (V-grafted) or humanized (CDR-grafted) monoclonal antibody. The humanized monoclonal antibody (CDR-grafted) may or may not include additional mutations introduced into the sequences of the structure (FR) of the acceptor antibody. Preferably the humanized or chimeric antibody comprises no more than one glycosylation site. More preferably said a glycosylation site is an N-glycosylation site. More preferably said glycosylation site is an N-glycosylation site. More preferably no glycosylation site is present in the variable region, and even more preferably no glycosylation site is present in the variable region of the heavy chain, more preferably no glycosylation site is present in the framework regions (FR's). A functional derivative of a polypeptide as used herein, includes a molecule having a qualitative biological activity in common with a polypeptide of the present invention, i.e., has the ability to bind to CD45RO and CD45RB. A functional derivative includes peptide fragments and analogs of a polypeptide according to the present invention. The fragments comprise regions within the sequence of a polypeptide according to the present invention, for example of a specified sequence. The term "derivative" is used to define variants of the amino acid sequence, and covalent modifications of a polypeptide according to the present invention. For example of a specified sequence. Functional derivatives of a polypeptide according to the present invention, for example of a specified sequence, preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, preferably at least about 95% homology of the overall sequence with the polypeptide amino acid sequence according to the present invention, for example of a specified sequence, and substantially retain the ability to bind to CD45RO and CD45RB. Preferably, the functional derivative has at least the binding affinity of a binding molecule comprising a polypeptide of SEQ ID NO: 1 and / or a polypeptide of SEQ ID NO: 2, of a humanized antibody comprising a polypeptide of SEC ID NO: 9 or SEQ ID NO: 10 and / or a polypeptide of SEQ ID NO: 7 or SEC. ID NO: 8; or a humanized antibody comprising a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8. The term "covalent modification" includes modifications of a polypeptide according to the present invention, for example of a specified sequence; or a fragment thereof with an organic or non-proteinaceous protein derivatization agent, fusions to heterologous polypeptide sequences, and post-translational modifications. Polypeptides covalently modified, for example from a specified sequence, still have the ability to bind to CD45RO and CD45RB through entanglement. Covalent modifications are traditionally introduced through the reaction of activated amino acid residues with an organic derivatization agent that is capable of reacting on selected sides or terminal residues, or through mechanisms of utilization of post-translational modifications that work in the recombinant host cells. Certain post-translational modifications are the result of the action of the recombinant host cells on the expressed polypeptide. The asparaginyl and glutaminyl residues are frequently discharged post-translationally to the corresponding glutamyl and aspartyl residues. Alternatively, these residues deaminate under mild acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues, mutilation of the a-amino groups of side chains of lysine, arginine and histidine, see for example, TE Creighton , Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983). Covalent modifications for example include fusion proteins comprising a polypeptide according to the present invention, for example of a specified sequence and its variants of the amino acid sequence, such as immunoadhesins, N-terminal fusions to heterologous signal sequences. "Homology" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of the amino acid residues in the candidate sequence that are identical to the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if it is necessary, to achieve the maximum percentage of homology, and not considering any conservative substitution as part of the identity of the sequence. Neither N- or C-terminal extensions should be constructed as reducing identity or homology. The methods and computer programs for alignment are well known.

"Amino acid (s)" refers to all naturally occurring L-a-amino acids, for example and including D-amino acids. Amino acids are identified through either single-letter or three-letter well-known designations. The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a polypeptide according to the present invention, for example of a specified sequence. Variants of the amino acid sequence of a polypeptide according to the present invention, for example of a specified sequence, still have the ability to bind to CD45RO and CD45RB. Substitution variants are those that have at least one amino acid residue removed and a different amino acid inserted in its place in the same position in a polypeptide according to the present invention, for example of a specified sequence. These substitutions can be individual, when only one amino acid in the molecule has been replaced, or they can be multiple, when two or more amino acids have been substituted in the same molecule. The insertion variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a polypeptide according to the present invention, for example of a specified sequence. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. Deletion variants are those with one or more amino acids in a polypeptide according to the present invention, for example of a specified sequence, removed. Generally, elimination variants will have one or two amino acids removed in a particular region of the molecule. It was also found that the polynucleotide sequences of - GGCCAGTCAGAACATTGGCACAAGCATACAGTG (SEQ ID NO: 25), which encodes the amino acid sequence of CDR1; - TTCTTCTGAGTCTATCTCTGG (SEQ ID NO: 26), which encodes the amino acid sequence of CDR 2; - ACAAAGTAATACCTGGCCATTCACGTT (SEQ ID NO: 27), which encodes the amino acid sequence of CDR 3; - TTATATTATCCACTG (SEQ ID NO: 28), which encodes the amino acid sequence of CDR1 ', TTTTATTCCTTACAATCATGGTACTAAGTACAATGAGAAGTT CAAAGGCAG (SEQ ID NO: 29), which encodes the amino acid sequence of CDR2'; AGGACCCTATGCCTGGTTTGACACCTG (SEQ ID NO: 30), which encodes the amino acid sequence of CDR3 '; - SEQ ID NO: 5 encoding a polypeptide of SEQ ID NO: 1, ie the variable region of a light chain of a mAb according to the present invention; - SEQ ID NO: 6 encoding a polypeptide of SEQ ID NO: 2, ie the variable region of a heavy chain of a mAb according to the present invention; - SEQ ID NO: 11 encoding a polypeptide of SEQ ID NO: 9, ie, a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention; - SEQ ID NO: 12 encoding a polypeptide of SEQ ID NO: , i.e. a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention; - SEQ ID NO: 13 encoding a polypeptide of SEQ ID NO: 7, ie, a light chain variable region including CDR1 ', CDR2' and CDR3 'according to the present invention; - SEQ ID NO: 14 encoding a polypeptide of SEQ ID NO: 8, ie a light chain variable region including CDR1 ', CDR2' and CDR3 'according to the present invention; - SEQ ID NO: 33 encoding a polypeptide of SEQ ID NO: 8, ie a variable region of light chain including CDR1 ', CDR2 'and CDR3' according to the present invention; - SEQ ID NO: 34 encoding a polypeptide of SEQ ID NO: 31, i.e. a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention; and - SEQ ID NO: 35 encoding a polypeptide of SEQ ID NO: 32, i.e. a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention. In another aspect the present invention provides isolated polynucleotides comprising polynucleotides encoding the linker CD45RO / RB, eg, encoding the amino acid sequence of CDR1, CDR2 and CDR3 according to the present invention and / or, preferably and, polynucleotides encoding the amino acid sequence of CDR1 ', CDR2' and CDR3 'according to the present invention; and The polynucleotides comprising a SEC polynucleotide ID NO: 5 and / or, preferably, and, a polynucleotide of SEQ ID NO: 6; and polynucleotides comprising polynucleotides that encode a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8 and / or, and preferably, a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10; for example, which encodes - a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 9, - a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 10, - a polypeptide of SEQ ID NO: 8 and a polypeptide of SEQ ID NO: 9, or - a polypeptide of SEQ ID NO: 8 and a polypeptide of SEQ ID NO: 10; and polynucleotides comprising a polynucleotide of SEQ ID NO.

NO: 11 or SEQ ID NO: 12 and / or, preferably and, to polynucleotide of SEQ ID NO: 13 or a polynucleotide of SEQ ID NO: 14, preferably comprising - a polynucleotide of SEQ ID NO: 11 and a polynucleotide SEQ ID NO: 13, - a polynucleotide of SEQ ID NO: 11 and a polynucleotide of SEQ ID-NO: 14, - a polynucleotide of SEQ ID NO: 12 and a polynucleotide of SEQ ID NO: 13, or - a polynucleotide of SEQ ID NO: 12 and a polynucleotide of SEQ ID NO: 14; and polynucleotides comprising polynucleotides encoding a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or, and preferably, to a peptide of SEQ ID NO: 7 or SEQ ID NO: 8; for example, which encodes - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 7, - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 8, - a polypeptide of SEQ ID NO: 32 and a SEC polypeptide ID NO: 7, or - a polypeptide of SEQ ID NO: 32 and a polypeptide of SEQ ID NO: 8; and polynucleotides comprising a polynucleotide of SEQ ID NO: 34 or SEQ ID NO: 35 and / or, preferably and, a polynucleotide of SEQ ID NO: 33; SEQ ID NO: 14 or 13. - a polypeptide of SEQ ID NO: 34 and a polypeptide of SEQ ID NO: 33, - a polypeptide of SEQ ID NO: 34 and a polypeptide of SEQ ID NO: 14, - a polypeptide of SEQ ID NO: 34 and a polypeptide of SEQ ID NO: 13, a polypeptide of SEQ ID NO: 35 and a polypeptide of SEQ ID NO: 33, a polypeptide of SEQ ID NO: 35 and a polypeptide of SEC ID NO: 14, or - a polypeptide of SEQ ID NO: 35 and a polypeptide of SEQ ID NO: 13. "Polynucleotide", unless otherwise specified herein, includes any polyribonucleotide or polidesoxiribunucleótido, which may be RNA or DNA unmodified , or modified RNA or DNA, which includes, without limitation, single and double chain structure RNA, and RNA which is a mixture of single and double chain structure regions. A polynucleotide according to the present invention, for example to polynucleotide encoding the amino acid sequence CDR1, CDR2, CDR3, CDR1 ', CDR2', CDR3 ', or of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32 respectively, such as a polynucleotide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35 respectively, includes the allelic variations thereof and / or their respective complements; for example it includes a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, respectively; for example, which encodes the polypeptide having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEC ID NO: 32, respectively, for example, which includes a functional derivative of said polypeptide, for example said functional derivative having at least 65% homology with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32, respectively, for example said derivative functional which includes covalent modifications of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEC ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32, respectively, for example said functional derivative including variants of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32, respectively; for example, a SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, respectively includes a sequence, as a result of the redundancy (degeneracy) of the genetic code, it also encodes a polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQ ID NO: 32, respectively, or encode a polypeptide with a sequence of amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31 or SEQID NO: 32, respectively. Preferably, the allelic variants or functional derivatives have at least the binding affinity to a binding molecule comprising a polypeptide of SEQ ID NO: 1 and / or a polypeptide of SEQ ID NO: 2, of a humanized antibody comprising a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8; or a humanized antibody comprising a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8. A binding molecule CD45RO / RB, by example which is a chimeric or humanized antibody, can be produced through recombinant DNA techniques. In this way, one or more DNA molecules encoding the CD45RO / RB can be constructed, placed under appropriate control sequences and transferred to a suitable host (organism) for the expression of an appropriate vector. In another aspect the present invention provides a polynucleotide encoding a light, heavy and / or light chain of a CD45RO / RB binding molecule according to the present invention; and the use of a polynucleotide according to the present invention for the production of a CD45RO / RB binding molecule according to the present invention through recombinant means. A CD45RO / RB binding molecule can be obtained according, for example, analogically, to a conventional method together with the information provided herein, for example the knowledge of the amino acid sequence of the hypervariable or variable regions and the polynucleotide sequences that they encode these regions. A method for constructing a variable domain gene for example is described in EP 239400 and can be briefly summarized as follows: A gene encoding a variable region of a mAb of any specificity can be cloned. The DNA segments encoding the structure and the hypervariable regions are determined and the DNA segments encoding the hypervariable regions are removed. The synthetic CDR cassettes of double chain structure are prepared through DNA synthesis according to the CDR and the CDR sequences as specified herein. These cassettes are provided with sticky ends so that they can be ligated at the intersections of a desired structure of human origin. The polynucleotides encoding the individual chain antibodies can also be prepared according to, for example, analogically, with a conventional method. A polynucleotide according to the present invention in this prepared form can be conveniently transferred into a suitable expression vector. The appropriate cell lines can therefore be found, for example, analogically with a conventional method. Expression vectors, for example comprising the suitable promoter (s) and genes encoding heavy and light chain constant parts are known for example and are commercially available. Suitable hosts are known or can be found accordingly, for example, analogically, with a conventional method and include cell culture or transgenic animals. In another aspect the present invention provides an expression vector comprising a polynucleotide encoding the CD45RO / RB binding molecule according to the present invention, for example of the sequence SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41. In another aspect the present invention provides an expression system comprising a polynucleotide according to the present invention wherein said expression system is capable of producing an CD45RO / RB binding molecule according to the present invention, wherein said expression system or part thereof is present in a host cell . compatible; and - An isolated host cell comprising an expression system as defined above. In addition it has been found that a CD45RO / RB binding molecule according to the present invention inhibits primary autoimmune responses in a dose-dependent manner as determined by MLR in vitro. The results indicate that cells that have been alloactivated in the presence of a CD45RO / RB binding molecule according to the present invention are weakened in their responses to alloantigen. This 0 confirms the indication that a CD45RO / RB binding molecule according to the present invention can act directly on the alloreactive T cells of the producer and modulates its function. In addition, the functional properties of primary MRL T cells were further studied in re-stimulation experiments in secondary MLR, using specific stimulator cells or third-party stimulators to evaluate the specificity of the functional effects observed. It was found that cells derived from primary MLRs wherein a CD45RO / RB binding molecule according to the present invention is present, were weakened in their ability to respond to subsequent optimal stimulation with specific stimulator cells, although no antibody was added to secondary crops. The specificity of the inhibition was demonstrated through the ability of cells treated with a CD45RO / RB binding molecule according to the present invention to respond normally to stimulator cells from unrelated third-party donors. The re-stimulation experiments using T cells derived from primary MLR cultures in this manner indicate that the cells that have alloactivated a CD45RO / RB binding molecule according to the present invention are hyporesponsive, ie tolerant, to the original alloantigen. In addition, the biological activities are described in Examples 7, and 9 to 13. In addition, it was found that the proliferation of cells in cells pretreated with a CD45RO / RB binding molecule according to the present invention could be rescued through exogenous IL-2. . This indicates that the treatment of alloreactive T cells with a CD45RO / RB binding molecule according to the present invention induces a state of tolerance. Indeed, the reduced proliferative responses observed in the cells treated with a CD45RO / RB binding molecule according to the present invention, was due to the weakening of the T cell function, and these cells were able to respond to exogenous IL-2. , indicating that these cells are in an anergic state, truly irresponsive. The specificity of this response was shown through the ability of cells treated with a CD45RO / RB binding molecule according to the present invention to normally proliferate donor cells unrelated to the level of cells treated with control. In addition, the experiments indicate that the binding of a CD45RO / RB binding molecule according to the present invention to CD45RO and CD45RB can inhibit memory responses of peripheral blood mononuclear cells (PBMC) from donors immunized for the specific recovery antigen. The binding of a CD45RO / RB binding molecule according to the present invention to CD45RO and CD45RB in this way is also effective in inhibiting memory responses for soluble Ag. The ability of a CD45RO / RB binding molecule according to the present invention to inhibit tetanus recovery responses in PBMC from humanized donors indicates that a CD45RO / RB binding molecule according to the present invention is capable of activating and modulating the activation of memory T cells. For example, these data indicate that a CD45RO / RB binding molecule according to the present invention in addition to recognizing the activated and activated alloreactive T cells is capable of modulating its function, resulting in an induction of the T cell's lack of energy. This property may be important in the treatment of ongoing immune responses to autoantigens and allergens and possibly to alloantigens as seen in autoimmune diseases, allergy and chronic rejection, and diseases such as psoriasis, inflammatory spleen disease, where the responses play a role in maintaining the state of the disease. It is believed to be an important feature where memory responses that self-antigens play a major role in maintaining the disease. It has also been found that a CD45RO / RB binding molecule according to the present invention can modulate the T cell proliferative responses in a mixed lymphocyte (MLR) response in vivo, i.e., a CD45RO binding molecule was found. / RB according to the present invention has the corresponding inhibitory properties in in vivo tests, such as for example, the prevention of lethal xanthogenic graft-versus-host disease (GvHD) or suppression of the inflammatory process mediating allograft skin rejection in human SCID mouse models, or prolong the survival of other human islet allografts in a model of hu-PBL-NOD / SCID mice. A CD45RO / RB binding molecule according to the present invention in this way can have immunosuppressive and tolerogenic properties and can be useful for induction of tolerance in vivo and ex vivo for alloantigens, autoantigens, and antigens of intestinal flora, by example to CD45RO / RB binding molecule according to the present invention may be useful in the treatment and prophylaxis of diseases, for example including autoimmune diseases, such as, but not limited to, rheumatoid arthritis, psoriatic arthritis, autoimmune thyroiditis, Graves disease, type I and type II diabetes, multiple sclerosis, Crohn's disease (CD), ulcerative colitis (UC), systemic lupus erythematosus, Sjogren's syndrome, scleroderma, autoimmune gastritis, glomerulonephritis, transplant rejection, such as , but not limited to rejection of allograft and organ and tissue xenograft, for example for the treatment of recipients of e.g. example heart, lung, heart-lung combined, liver, kidney, pancreatic, skin or corneal transplants, graft-versus-host disease (GVHD), such as after spinal cord transplantation, and / or rejection of islet cell transplantation pancreatic, and / or also psoriasis, dermatitis such as atopic and contact dermatitis that includes allergic contact dermatitis, inflammatory bowel disease and / or allergies, which include allergic asthma. In another aspect, the present invention provides the use of a CD45RO / RB binding molecule according to the present invention as a pharmaceutical, for example in the treatment of prophylaxis of autoimmune diseases, rejection to transplantation, for example rejection to cell transplantation. pancreatic islet, or graft-versus-host disease (GVHD), psoriasis, dermatitis, inflammatory bowel disease and / or allergies. In another aspect, the present invention provides a CD45RO / RB binding molecule according to the present invention for the production of a medicament in the treatment and prophylaxis of diseases associated with autoimmune diseases, rejection to transplantation, for example rejection to cell transplantation. pancreatic islet, or graft-versus-host disease (GVHD), psoriasis, dermatitis, inflammatory bowel disease and / or allergies. In another aspect the present invention a method for the treatment and / or prophylaxis of diseases associated with autoimmune diseases, rejection to transplantation, psoriasis, dermatitis, inflammatory bowel disease and / or allergies comprising administering to a subject in need of said treatment and / or prophylaxis an effective amount of a CD45RO / RB binding molecule according to the present invention, for example in the form of a pharmaceutical composition according to the present invention. One embodiment of the present invention provides a method for the treatment and / or prophylaxis of a disease associated with the rejection of islet cell transplantation, for example rejection of islet cell transplantation, which comprises administering to a subject in need of said treatment and / or prophylaxis an effective amount of a molecule or a humanized antibody according to the present invention. In preferred embodiments said CD45RO / RB binding molecule for use as a pharmaceutical, for the production of a medically or in a method for the treatment and / or prophylaxis of a disease associated with autoimmune diseases, rejection to transplantation, psoriasis, dermatitis, disease of the inflammatory bowel and / or allergies comprises a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8. Preferably the binding molecule CD45RO / RB comprises a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 8. An "effective amount" of a CD45RO / RB binding molecule is an amount sufficient to achieve beneficial or desired results that include clinical results such as the decrease of one or more symptoms that result from the autoimmune disease, rejection of the transplant, psoriasis, dermatitis, inflammatory bowel disease and / or allergy, decrease in the quality of life of those who that suffer from, decrease in the dose of other medications required to treat such diseases, improving the effect of another medication, delaying the progression of the disease, and / or prolonging the survival of patients, either directly or indirectly. An effective amount may be administered in one or more administrations and may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. In this way an "effective amount" can be considered in the context of an administration of one or more therapeutic agents, and a single agent can be considered as given in an effective amount if, in conjunction with one or more other agents, it can give or achieve a desired result. It is further provided that a "CD45RO / RB binding" molecule of the invention can be administered as the active ingredient alone or together with other drugs in immunomodulatory regimens, or other anti-inflammatory agents for example for the treatment or prevention of diseases associated with diseases autoimmune, rejection to transplantation, psoriasis, dermatitis, inflammatory bowel disease and / or allergies For example, the CD45RO / RB binding molecule of the invention can be used in combination with a calcineurin inhibitor, for example cyclosporin A, cyclosporin G, FK-506, ABT-281, ASM 981; an mTOR inhibitor, for example rapamycin, 40-0- (2-hydroxy) ethyl-rapamycin, CC1779, ABT578, AP23573, AP23464, AP23675, AP23841, TAFA-93, biolimus-7 or bioimus-9; a corticosteroid; cyclophosphamide; azathioprine; methotrexate; an S1P receptor agonist, for example FTY 720 or an analogue thereof; leflunomide or analogs thereof; mizoribin; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or analogues thereof; immunosuppressive monoclonal antibodies, for example monoclonal antibodies to leukocyte receptors, for example, MHC, CD2, CDS ', CD4, CD11a / CD18, CD7, CD25, CD27, CD7, CD45, CD45, CD137, ICOS, CD150 (SLAM) ), OX40, 4-1BB or its ligands, for example CD154; or other immunomodulatory compounds, for example a recombinant binding molecule having a portion of the extracellular domain of CTLA4 or a mutant thereof, for example in at least an extracellular portion of CTLA4 or a mutant thereof linked to a non-protein sequence. -CTLA4, for example CTLA41g (for example the one designated ATCC 68629) or a mutant thereof, for example LEA29Y, or other inhibitors of the adhesion molecule, for example mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists. An effective amount of a CD45RO / RB binding molecule of the invention, alone or in conjunction with another drug, compound, or pharmaceutical composition can be administered through any conventional route, which includes Injection and through gradual infusion over time. . The administration can, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, topical or transdermal. By "co-administration" is meant administration of the components of the compositions of the invention together with or substantially at the same time, either in the same vehicle or in separate vehicles, so by oral administration, for example, both compounds are present simultaneously in the gastrointestinal tract. Preferably, the compounds are administered as a fixed combination.

In another aspect the present invention provides a pharmaceutical composition comprising a CD45RO / RB binding molecule according to the present invention in association with at least one pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable carrier or diluent" as used herein means one or more compatible solid or liquid fillers, diluents or substances for encapsulation which are suitable for administration to a mammal including humans. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations can routinely contain pharmaceutically acceptable concentrations of salts, pH regulating agents, preservatives, compatible carriers, complementary immune enhancing agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable but pharmaceutically acceptable salts can not be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. The pharmaceutical compositions may contain suitable pH regulating agents, including: acetic acid in a salt; citric acid in a salt, boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal. The doses of the polypeptide or nucleic acid encoding said polypeptide administered to a subject can be selected according to different parameters, in particular according to the mode of administration used and the condition of the subject. Other factors include the desired treatment period. In the case where a response in a subject is insufficient to the initial doses applied, higher doses (or indeed higher doses via a different, more localized delivery route) may be employed to encompass the tolerance that the patient allow. The pharmaceutical compositions can conveniently be presented in a unit dosage form and can be prepared by any of the methods known in the pharmacy art. All methods include the step of bringing the active ingredient in association with a carrier that constitutes one or more secondary ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound in association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for oral administration can be presented as distinct units, such as capsules, tablets, dragees, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous or non-aqueous liquids such as a syrup, elixir, or an emulsion. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of a polypeptide or nucleic acid encoding the polypeptide, which is preferably isotonic with the blood of the recipient. This preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic, parenterally-acceptable diluent or solvent, for example, as a solution of 1,3-butan diol. Among the vehicles and acceptable solvents that can be used are water, Ringer's solution, isotonic sodium chloride solution. In addition, fixed, sterile oils are conveniently employed as a solvent or suspension medium. For this purpose any soft fixed oil may be employed, which includes mono or synthetic diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. The carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration. can be found at Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. A pharmaceutical composition may further comprise, for example, active ingredients, for example other immunomodulatory antibodies such as, but not confined to anti-lCOS, anti-CD154, anti-CD134L or recombinant proteins such as, but not limited to rCTLA-4 ( CD152), rOX40 (CD134), or anti-inflammatory agents or immunomodulatory compounds such as, but not limited to cyclosporin A, FTY720, RAD, rapamocin, FK506, 15-deoxyspergualin, steroids; as described above. Said pharmaceutical composition may comprise a CD45RO / RB binding molecule according to the invention and immunomodulatory drugs and / or anti-inflammatory agents in separate unit dosage forms, preferably wherein the unit dosage forms are suitable for the administration of the component compounds in synergistically effective amounts, together with instructions for their use, optionally with additional means to facilitate compliance with the administration of the component compounds, for example, a label or drawings. The compositions of the invention can be administered as a fixed combination, or they can be formulated in a fixed combination. Absolute dosages of the compounds will vary depending on a number of factors, for example, the rate of release of the active ingredient and the nature and severity of the condition to be treated.

Diseases as described above that are to be treated with a CD45RO / RB binding molecule of the present invention alone or in combination with other drugs include, but are not limited to, autoimmune diseases, including rheumatoid arthritis, psoriatic arthritis, autoimmune thyroiditis , Graves disease, type I and type II diabetes, multiple sclerosis, Crohn's disease (CD), ulcerative colitis (UC), systemic lupus erythematosus, Sjögren's syndrome, scleroderma, autoimmune gastritis and glomerulonephritis; rejection of transplantation, including, but not limited to rejection of allograft and organ and tissue xenograft, for example for the treatment of recipients of for example heart, lung, heart-lung combined, liver, kidney, pancreatic, skin or corneal transplants graft-versus-host disease (GVHD), such as after spinal cord transplantation, and / or rejection of pancreatic islet cell transplantation; psoriasis; dermatitis such as atopic and contact dermatitis that includes allergic contact dermatitis; Inflammatory bowel disease and / or allergies, which include allergic asthma.

EXAMPLES The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. In the following examples all temperatures are in degrees Celsius.

The "candidate mAb" or "chimeric antibody" is a CD45RO / RB binding molecule according to the present invention comprising the light chain of SEQ ID NO: 3 and the heavy chain of SEQ ID NO: 4. The "humanized antibody" is a linker molecule CD45RO / RB according to the present invention comprising a polypeptide of SEQ ID NO: 8 and a polypeptide of SEQ ID NO: 9 (VHE / humVI, VHENL1 or VHENLh), a polypeptide of SEQ ID NO: 8 and a polypeptide of SEQ ID NO: 10 (VHQ / humV1, VHQNL1 or VHQNLh); a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 9 (VHE / humV2, VHENL2 or VHENLm); a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 10 (VHQ / humV2, VHQNL2 or VHQNLm); a polypeptide of SEQ ID NO: 8 and a SEC polypeptide ID NO: 31 (VHEN73D / humV1, VHEN73DNL1 or VHEN73DNLh); a polypeptide of SEQ ID NO: 8 and a polypeptide of SEQ ID NO: 32 (VHQN73D / humV1, VHQN73DNL1 or VHQN73DNLh); a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 31 (VHEN73D / humV2, VHEN73DNL2 or VHEN73DNLm); or a polypeptide of SEQ ID NO: 7 and a polypeptide of SEQ ID NO: 32 (VHQN73D / humV2, VHQN73DNL2 or VHEN73D NLm). The following abbreviations were used: APC antigen presentation cell CEX cation exchange chromatography c. p. m. counts per minute dhfr dihydrofolate reductase EDTA acid ethylene dinitrile tetra acid ELISA enzyme-linked immunosorbent assay ESI-Q-TOF flight time, quadruple ionization by electrospray FACS fluorescence activated cell sorting Fe fragment crystallizable F (ab ') 2 link of the antigen fragment; bivalent FITC fluorescein isothiocyanate FBS fetal bovine serum GVHD graft-versus-host disease HCMV human cytomegalovirus promoter HPLC High Performance Liquid Chromatography IFN-? interferon gamma igE isotope E immunoglobulin IgG isotope G immunoglobulin IL-2 interIeucin-2 IU international units MALDI-TOF flight time ionization assisted laser-assisted matrix MLR mixed lymphocyte reaction MLC mixed lymphocyte culture MP1 protein matrix 1 of hemophilic influenza MTX methotrexate saline PBS regulated in its pH of PBL phosphate peripheral blood leukocytes PBMC peripheral blood mononuclear cells PCR polymerase chain reaction RP reverse phase chromatography SEC size exclusion chromatography SCID severe combined immunodeficiency 'reg regulatory T cells xGVHD xenograft versus host disease EXAMPLE 1 Response of the primary mixed lymphocyte (MLR) Blood samples were obtained from healthy human donor cells. Peripheral blood mononuclear cells (PBMC) were isolated through centrifugation on Ficoll-Hypaque (Pharmacia LKB) of whole peripheral blood leukocytes, leukapheresis or leukocyte layer with known blood type, but unknown HLA type. In some MLR experiments, PBMC is used directly as the stimulator cells after irradiation at 40 Gy. In other experiments, T cells are consumed from PBMC by using Dynabeads CD2 or CD3 (Dynal, Oslo, Norway). The pearls and polluting cells are removed through the magnetic field. PBMC consumed from T cells, CD3 + T cells or CD4 + T cells are used as the responding cells in MLR. The cells are prepared from different donors for the stimulator cells. T cells CD3 + are purified through negative selection using the anti-CD16 mAb (Zymed, CA), Dynabead goat anti-mouse IgG, Dynabeads anti-CD14, Dynabeads CD19. In addition Dynabeads anti-CD8 are used to purify CD4 + T cells. The cells obtained are analyzed by FACScan or FACSCalibur (Becton Dickinson & Co., CA) and the purity of the cells obtained was > 75% The cells were suspended in RPMI1640 medium, supplemented with 10% heat-inactivated FBS, penicillin, streptomycin and L-glutamine.

Reagents The "candidate mAb" chimeric anti-CD45RO / RB mAb and an isotope of the chimeric control antibody compared was also generated. The mouse control IgG antibody (human) specific for KLH (keyhole limpet hemocyanin) or recombinant human IL-10 was purchased from BD Pharmingen (San Diego, CA).

The anti-human mAb 5c8 CD154 is according to Lederman and others 1992.

Primary mixed lymphocyte (MLR) response Aliquots of 1 x 10 5 PBMC or 5 x 10 4 cells were mixed CD3 + or CD4 + with 1 x 105 irradiated PBMC or PBMC irradiated consumed T cells of 5 x 104 (50 Gy) in each well of 96-well plates (Costar, Cambridge, MA) in the presence of the indicated mAb or absence of Ab . In some experiments, the goat anti-mouse Ig F (ab ') 2 fragment or goat anti-human Ig specific for the Fe moiety (Jackson ImmunoResearch, West Grove, PA) was added at 10 μg / ml in addition to the candidate mAb to ensure optimal in vitro entanglement of target CD45 molecules. The mixed cells were cultured for 4-5 days at 37 ° C in 5% C02 and proliferation was determined by pulsing the cells with thymidine during the last 16-20 hours of culture. Other experiments are similar to those described above, but with the following exceptions: 1) The medium used is EX-VIVO (Bio-Whittaker) containing 10% FBS and 1% human plasma; 2) Anti-mouse IgG (5 μg / L) was used as the secondary entanglement step; 3) The irradiation of the stimulator cells is 60 Gy. The primary MLR was carried out in the presence of the "candidate mAb" or control chimeric IgG (10 μg / ml), both with a second step of the reagent, the F (ab ') 2 fragment of goat anti-human Ig specific for the Fe portion (10 μg / ml). Percent inhibition through the "candidate mAb" was calculated in comparison to cell proliferation in the presence of control IgG. The results are shown in the following TABLE 1: TABLE 1 Inhibition of primary MLR by 10 μg / ml of candidate mAb according to the present invention Significantly different from the control value (P <0.001) A candidate mAb according to the present invention inhibits the primary MLR as can be seen in TABLE 1. The average inhibitory effect is 60.83 ± 6.83% in four different CD4 + T cells derived from donors and statistically significant. Inhibition of primary MLR through "candidate mAb" is shown as being dose dependent in the range of 0.001 and 10 μg / ml of the "candidate mAb" as shown in Figure 1. IC50 for inhibition of MLR Primary through a "candidate mAb" was determined from the results of three separate MLR experiments using a donor PBMC as responding cells. Thus, the CD4 + T cells that respond as stimulators are mixed in the presence of a "candidate mAb" or control chimeric Ab with 10 μg / ml of the goat anti-human Ig F (ab ') 2 fragment. The experiments were repeated 3 times and the percentage of proliferation in the presence of a "candidate mAb" was calculated in comparison to the proliferation of the T cell in the presence of control Ab. The IC50 value was determined using Origin (V. 6.0®). The IC50 value of the cellular activity was calculated as being 0.87 + 0.35 nM (0.13 ± 0.0524 μg / ml).

EXAMPLE 2 Secondary MLR In order to assess whether a "candidate mAb" induces a lack of response of CD4 + T cells to specific alloantigens, the secondary MLR is carried out in the absence of any antibody after the primary MLC. The CD4 + T cells are cultured with irradiated allogeneic stimulator cells (PBMC consumed T cells) in the presence of the indicated antibody in 96-well culture dishes for 10 days (primary MLC). The cells are then harvested, stratified on a Ficoll-Hypaque gradient to remove the dead cells, washed twice with RPMI, and restimulated with the same stimulator, the third stimulator cells or IL-2 (50 U / ml). The cells are cultured for 3 days and the proliferative response is determined by pulsing the cells with 3H-thymidine for the last 16-20 hours of the culture. Specifically, CD4 + T cells are cultured with irradiated allogeneic stimulator cells (PBMC consumed from T cells taken from other donors) in the presence of 10 μg / ml of the "candidate mAb", the control IgG 1 chimeric Ab and the F fragment (ab ') 2 of goat anti-human Ig. Proliferation of primary MLR was determined on day 5. For the secondary MLR, responding and stimulatory cells are cultured for 10 days in the presence of the "candidate mAb", then the cells are harvested, washed twice in RPM11640 and they are stimulated again with the specific stimulator, third stimulators or IL-2 (50 U / ml) in the absence of any Ab. The proliferation of cells is determined on day 3. The results are established in TABLE 2: TABLE 2 * Significantly different from the control value (p = &0.001 determined through the test t, SigmaStat V. 2.03). # p = < 0.046 In order to test whether weakened proliferation is due to lack of response as a consequence of treatment with a "candidate mAb", cells derived from the primary MLR were cultured in the presence of IL-2 (50 U / ml). In addition to the results of IL-2 in the rescue of proliferative responses of T cells that have been treated with a "candidate mAb" in primary MLR, levels similar to those observed in the presence of IgG, Ab control. These data indicate that the weakened secondary response in T cells treated with a "candidate mAb" is due to the functional alteration of the responding T cells, which become non-sensitive to the specific stimulator cells. The inhibition percentage is calculated according to the following formula: c.p.m. with control Ab - c.p.m with "candidate mAb" x 100 c.p.m. with control Ab Statistical analysis was carried out using SigmaStat (Vers 2.03). Data were analyzed through two-way ANOVA followed by the Dunnett method. At all probabilities of the < 0.05 were considered significant. In some tests t of the experiments was used (SigmaStat V. 2.03).

EXAMPLE 3 In vivo survival studies in SCID mice Hu-PBL grafts in SCID mice Human peripheral blood mononuclear cells (PBMC) were injected intraperitoneally in SCID CB 17 mice / Lystbg GbmsTac-Pr / cc / csc c 'mice (Taconic, Germantown, NY) in an amount sufficient to induce a xenogeneic graft versus host disease (xGvHD) in > 90% of the mice within four weeks after the cell transfer. Said SCID mice treated below are designated as hu-PBL-SCID mice.

Mab treatment of hu-PBL-SCID mice Hu-PBL-SCID mice were treated with a "candidate mAb" or controlled mAb compared to mouse or chimeric isotope on day 0, immediately after the PBMC injection, on the day 3, on the 7th and weekly intervals thereafter. The mabs were delivered subcutaneously in 100 μl of PBS to a final concentration of 5 mg / kg of body weight. The treatment was stopped when all the control mice died.

Evaluation of treatment outcomes The main criterion for evaluating the efficacy of a "candidate mAb" in this study was the survival of hu-PBL-SCID mice. The importance of the results was evaluated through the statistical method of survival analysis using the Log-rank test (Mantel method) with the help of software Systat v9.01. The method of survival analysis is a test or parametric, which only considers if a particular mouse is still alive but also if it was sacrificed for irrelevant reasons for treatment / disease such as the requirement to carry out in vitro analyzes with its organs. cells Liver, lung, kidney and bladder biopsies were obtained from dead mice for further evaluation. In addition, the PBL-SCID mice were weighed at the beginning (before the cell transfer) and throughout (every two days) of the experiment as an indirect estimate of their health status. Linear regression lines were generated using the body weight against the values of the transfer days after PBMC obtained from each mouse and subsequently, their deviations (control mice against mice treated with anti-CD45) were compared using the Mann test -Whitney non-parametric.

Results All hu-PBL-SCID mice treated with mAb controls had human leukocytes infiltrated into the lung, liver, and spleen and died (4/4) within approximately 2 to 3 weeks after cell transfer. Death is probably a consequence of xGvHD. Mice treated with mAb further lost weight in a linear fashion, approximately 10% and more within 3 weeks. All mice treated with hu-PBL-SCID with a "candidate mAb" survived (4/4) with no apparent signs of disease more than four weeks, although treatment with the "candidate mAb" was stopped after 3 weeks. Mice treated with "candidate mAb" gained weight linearly, up to approximately} 5% within 4 weeks.

EXAMPLE 4 Expression of antibodies of the invention Expression of the humanized antibody comprising SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10 SEQ ID NO: 31 or SEQ ID NO: 32 The expression vectors according to the maps of the plasmid shown in Figures 2 to 11 were constructed, the nucleotides comprising the coding of the amino acid sequence of the humVi of the humanized light chain variable region (SEQ ID NO: 8, Figures 4 and 6), the humV2 of the humanized light chain variable region (SEQ ID NO: 7, Figures 5 and 7), the humanized heavy chain variable region HEV (SEQ ID NO: 9, Figure 3 and 10), or VHQ of the variable region of humanized heavy chain (SEQ ID NO: 10, Figures 2 and 11), VHE-N73D of the humanized heavy chain variable region (SEQ ID NO: 31, Figure 8) or humanized heavy chain variable region VHQ-N73D ( SEQ ID NO: 32, Figure 9), respectively. These expression vectors have the DNA (nucleotide) sequences SEQ ID NO: 15 and SEQ ID NO: 41 (VHQ), SEQ ID NO: 16 and SEQ ID NO: 40 (VHE), SEQ ID NO: 17 and SEQ ID NO 36 (humV1), SEQ ID NO: 18 and SEQ ID NO: 39 (humV2), or SEQ ID NO 37 (VHE-N73D) and SEQ ID NO 38 (VHQ-N73D).

Construction of light and heavy chain expression vectors of the humanized antibody for expression in COS cells Human kappa light chain expression vectors for VLh and VLm versions In order to construct the final expression vector encoding the complete humanized light chain of the human human kappa isotope, DNA fragments encoding the complete light chain variable regions (VLh and VLm) of VLh and VLm containing the cloning vectors of the PCR Instruction Group (Stratagene) (VLm region) were excised using Hindlll and BglII. The gel purified fragments were then subcloned into the Hindlll and Bamlll sites of the kappa expression vector C21-HCMV which was created during the construction of the humanized anti-IgE TESC-21 antibody (Kolbinger et al. 1993) and which was originally received from M. Bendig (MRC Collaborative Center, London, UK) (Maeda et al., 1991). The ligation products were purified through phenol / chloroform extraction and electrophoresed within the XLI-Blue Epicurian Coli® competent electroporation chain structure (Cat. No. # 200228, Stratagene). After plating on LB / amp agar plates overnight at 37 ° C, every 12 colonies were selected to prepare the plasmid DNA from a 3 ml culture using the BioRobot 9600 (Qiagen). This produced the light chain expression vectors for the VLh and VLm versions of the humanized antibody, respectively, as described further in the Figures.

Human gamma-1 heavy chain expression vectors for HCV For the construction of the VHQ expression vector, a method was taken in steps. First, the complete variable region of VHQ was assembled via PCR according to the ethology as described in Kolbinger et al. 1993 (Protein Eng. 1993 Nov; 6 (8): 971-80) and subcloned into the expression of C21-HCMV-gama-1 from which the C21 insert has been removed using the same enzymes. A HindlIl / BamHI fragment of HCV was then subcloned from the clone of the PCR Instructions Group containing the entire variable region within the expression vector C21-HCMV-gamma-1 divided with the same enzymes. This produced the final expression vector for the VHQ version of the humanized antibody.

Human 1-range heavy chain expression vectors for HEV The construction of the final VHE expression vector encoding the complete humanized heavy chain of the human gamma-1 isotope was achieved by directly ligating a restricted PCR fragment Hindlll and BamHI encoding the variable region within of the Hindlll and BamHI sites of the C21-HCMV gamma-1 expression vector, which was created during the construction of the humanized anti-IgE TESC-21 antibody (Kolbinger et al., 1993) which was also originally received from M. Bendlg (MRC Collaborative Center, London, UK) (Maeda et al., 1991).

Temporal expression in COS cells The following transfection protocol was adapted for adherent COS cells in 150 mm dishes of cell culture, using the SuperFect Transfection Reagent (Cat. No. 301305, Qiagen). The four different expression vectors described above were used for the temporary transfection of the cells. For the expression of a humanized antibody, each of the two clones containing the heavy chain inserts (VHE or VHQ, respectively) were co-transfected into cells with each of the two clones encoding the light chains (humV1 or humV2, respectively ), in total 4 different combinations of heavy and light chain expression vectors (VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2). Prior to transfection, the plasmids were made linear with restriction endonuclease Pvul that is divided into the region encoding the resistance gene for ampicillin. The day before transfection, 4 x 10 6 COS cells were seeded in 30 ml of fresh culture medium in 150 mm cell culture dishes. Sowing at this cell density generally produced 80% confluence after 24 hours. On the day of transfection, four different combinations of light and heavy chain DNA expression vectors were diluted in linear form (15 μg each) in a total volume of 900 μl of fresh medium without serum and antibiotics. Then 180 μl of the SuperFect Transfection Reagent was mixed vigorously with the DNA solution. The DNA mixture was incubated for 10 minutes at room temperature to allow complex formation. While complex formation took place, the growth medium was removed from the COS cell cultures, and the cells were washed once with PBS. Then 9 ml of fresh culture medium (containing 10% FBS and antibiotics) was added to each reaction tube containing the transfection complexes-and mixed well. The final preparation is immediately transferred to each of the four cultures to be transfected and mixed lightly. The cell cultures were then incubated with the DNA complexes for 3 hours at 37 ° C and 5% C02. After incubation, the medium containing the transfection complexes was removed and replaced with 30 ml of fresh culture medium. The supernatants were harvested 24 hours after transfection.

Concentration of culture supernatants For ELISA and FACS analysis, culture supernatants harvested from COS cells transfected with heavy and light chain plasmids were concentrated as follows. 10 ml of each supernatant was added to Centriprep Centrifugal Filter Devices YM-50 (Cat. No. 4310, Millipore) as described by the manufacturer. The Centrlprep filters were centrifuged for 10 minutes at 3000 rpm at room temperature. The centrifugation step was then repeated again with the remaining 20 ml of the supernatant using only 5 minutes of centrifugation and monitoring the evolution of the concentration. 500 μl of intermediate was recovered from the concentrated supernatant, transferred to the new Micrcon Centrifugal Filter Devices (Cat. No. 42412, Microcon) and further concentrated according to the manufacturer's protocol. The concentrated supernatants were centrifuged four times for 24 minutes at 3000 rpm at room temperature, once for 10 minutes at 6000 rpm and then, three times for 5 minutes, always monitoring the evolution of the concentration. The final volume of the concentrated conditioned medium achieved is 100-120 μl corresponding to a concentration of 250 to 300 times the original culture medium and stored at 4 ° C until use. For comparison and control, the culture medium of non-transfected cells is similarly concentrated, using the same centrifugation protocol described above.

Generation of stable Sp2 / 0 myeloma transfectants that secrete anti-CD45RQ / RB antibodies The Sp2 / 0 mouse myeloma cell line (ATCC, CRL-1581) was electrophoresed with the above-described CHO expression vectors encoding the heavy chain (VHE or VHQ) and light (humV1 or humV2) of the humanized antibodies binding CD45RO / RB. Four different combinations of heavy and light chain expression vectors (VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2) were used for the transfection according to the following protocol: 20 μg of super-linked DNA was mixed. each plasmid in an electroporation tube (0.4 cm egg) with 8 x 106 of Sp2 / 0 cells suspended in DMEM culture medium / 10% FCS. The configuration of the electroporation is 1500 V, 25 μF using a GenePulser instrument from BioRad. After electroporation, the cells were cultured for 20 hours in culture medium (DMEM supplemented with 10% penicillin FCS, streptomycin and L-glutamine). On day two the drug of selection G418 (Cat. No. 10131-019, Gibco) was added to a final concentration of 1 mg of active drug / ml and the cells were distributed in a 96-well plate, 200 μl each cavity with approximately 105 cells per cavity. Fifteen days later, survival clones G418 were expanded in medium containing G418. The secretion of the humanized mAbs from these transfectants was evaluated through ELISA, using a goat anti-human IgG / Fcy antibody layer (Cat. No. 109-005-098, Jackson Labs) and a peroxidase-coupled antibody against the human kappa light chain (Cat. No. A-7164, Sigma). The transfectants, which recorded positive points in this assay, were selected for a comparison of productivity on bases per cell per day, again using ELISA (see below). The best clone of each transfectant was selected for immediate subcloning through limiting dilution, using a seeding density of 1 cell per cavity. The productivity of the surviving subclones G418 is again determined as described above. The subclones were expanded in selection medium containing G418, until the volume of the culture reached 150 ml, at which stage the cultivation continued without G418 in flasks intended to feed roller bottles. After this first transfection and selection, the stable transfectants grew out of 96-well plates at a frequency of 20.8% for VHE / humV1, 11.5% for HCV / humV1, 18.8% for HEV / humV2 and 7.3% for HCV / humV2 . After two rounds of subcloning, the two best producers are clone 1.33.25 (3.87 pg / cell / day) and clone 1.33.26 (3.43 pg / cell / day) for HEV / humV1 and clone 12.1.4 ( 1.19 pg / cell / day) and clone 12.1.20 (1:05 pg / cell / day) for HCV / humV1. The stable Sp2 / 0 transfectants for HEV / humV1 and VHQ / humV1 subsequently expanded for the production and purification of the antibody. The antibodies were purified from the supernatants of stably transfected SP2 / 0 myeloma cell lines containing 10% FCS through a combination of affinity chromatography using an immobilized anti-human IgGFc matrix and size exclusion chromatography. If required, the endotoxin is removed using an Acticlean Etox (Sterogene Bioseparations) column.

Construction of heavy and light chain expression vectors of the humanized antibody for expression in Sp2 / 0 cells. Human kappa light chain expression vectors for VLh and VLm versions: The cAMP humV1 (= VL1) or humV2 (= VL2) was amplified through PCR of the expression plasmid CHO SEQ ID NO: 17 or SEQ ID NO: 18 respectively, using the primers HuCD45LC-Mlu (5'-AAAACGCGTTGTGACATTCTGCTGACCCAGTCT-3 '; SEQ ID NO: 42) and HuCD45LC-Hind (5' -AAAAAAGCTTGGTCCCCTGGCCGAACGTGAA-3 '; SEQ ID NO: 43). Each 321 bp PCR fragment was digested with Mlul and Hindlll and ligated directly into the light chain expression vector chA6HCk.dhfr which was digested with the same enzymes. The resulting plasmids were named LCVL1 Sp20 (SEQ ID NO: 36; Figure 6) and LCVL2Sp20 (SEQ ID NO: 39; Figure 7) respectively. LCVL1Sp20 is then used for the expression in Sp2 / 0 cells of the binding molecules CD45RO / RB VHE / humV1, VHQ / humV1 or VHE-N73D / humV1; LCVL2Sp20 can then be used for expression in Sp2 / 0 cells in the binding molecules CD45RO / RB VHE / humV2, VHQ / humV2 or VHE-N73D / humV2.

Human gamma-1 heavy chain expression vectors for HCV and for HEV The two humanized VH cDNA regions were amplified by PCR of the recombinant plasmids HCMV-G1 HuA6-VHE (SEQ ID NO: 16; Figure 3) and HCMV -G1 HuA6-VHQ (SEQ ID NO: 15 Figure 2) respectively, using the PCR primers HuCD45HCEup (5'- CAGGCAGAGGTGCAGCTGGTGGAGTCA-3 '; SEQ ID NO: 44) or HuCD45HCQup (5'- CAGGCACAGGTGCAGCTGGTGGAGTCA-3'; NO: 45) and HuCD45HCIo (5'-AAATCCTTCTAGAACTCACCTGAGGAGAC-3 '; SEQ ID NO: 46). PCR fragments of 3 'ends were digested with BstEII. Each PCR fragment was then cloned into the vector cut-off of the heavy chain cassette HCcassREAL with BstEll and the blunt-ended cutter Hindll. The resulting plasmids are intermediates for the final expression constructs and are named HCcassHVESP20 and HCcassHVQSP20, respectively. This subcloning also leads to a change in the leader sequence that is associated with both VH regions in the original vectors. While the amino acid sequence of the leader sequence is MDWTWRVFCLLAWAPGAHS (SEQ ID NO: 47), it has been replaced during subcloning with MAWVWTLPFLMAAAQSVQA (SEQ ID NO: 48). Intermediates HCcassVHESP20 or HCcassVHQSP20 are digested with Seal and subsequently, the plasmids are digested with BamHI and EcoRl. The 2.9 kb fragments containing the Ig heavy chain promoter and the VH region are purified and ligated with the 8.7 kb BamHI- and EcoRI fragment digested from a heavy chain expression construct. The resulting plasmids are named HCVHESp20 (SEQ ID NO: 40; Figure 10) and HCVHQSp20 (SEQ ID NO: 41; 11), respectively; and are used for the expression in Sp2 / 0 cells of the humanized CD45RO / RB binding molecules VHE / humVI, VHQ / humV1.

Human gamma-1 heavy chain expression vector for VHEN73D The HCVHESp20 expression vectors (SEQ ID NO: 40, Figure 10) and HCVHQSp20 (SEQ ID NO: 41, Figure 11), were used as templates for site-directed mutagenesis to achieve a N73D mutation (exchange of an asparagine to aspartate at position 73 of the amino acid of the heavy chain of the amino acid molecule CD45RO / RB). This mutation eliminates a putative N-site glycosylation. Mutagenesis with the QuikChange® Multi-site Targeted Mutagenesis team and the aCD45H-N73D primer (5'-phosphate GCCACACTAACTGCAGACAAATCCATCAGCACAGC-3 '; SEQ ID NO: 49) according to the equipment manual. The sequence of the resulting constructs HCVHEN73DSp20 and HCVHQN73DSp20 are confirmed and described as SEC NO: 37 and SEC NO: 38, and Figures 8 and 9, respectively. HCVHEN73DSp20 (SEQ NO: 37) together with the LCVL1 light chain expression construct SP20 (SEQ NO: 36) are used for expression in Sp2 / 0 cells of the VHE-N73D / humV1a CD45RO / RB binding molecule.

Generation of stable Sp2 / 0 myeloma transfectants that secrete 'humanized anti-CD45RO / RB antibody VHE-N73D / humV1 The Sp14 / 10 mouse myeloma cell line Sp2 / 0 Ag14.10 is electroporated with vectors encoding the heavy chain (HCVHEN73DSp20 SEQ ID NO: 37) and light (LCVL1SP20; SEQ ID NO: 36) of the linker molecule VHEN73D / humV1 CD45RO / RB humanized. They are used for the transfection of cells in the exponential growth phase with a viability greater than 95%. The cells were washed twice with cold pH-regulator TF (272 mM sucrose, 1 mM MgCl2, 7 mM pH 7.4 phosphate buffer) and the cell concentration was adjusted to 2 x 10 7 cells / ml in buffer of pH TF. 0.8 ml of the cell suspension was mixed with 15 μg of each of the heavy and light chain constructs and placed on ice for 10 minutes. 5 transfections were made through electroporation using the Biorad Gene Push Button (280V and 25 μF). After electroporation the cells were placed on ice for 15 minutes followed by transfer into 50 ml of cold culture medium (RPMI-based medium without FCS) and incubated for 2 days at 37 ° C and 5% C02. For the selection of transfectants, cells were cultured in the presence of 1.1 mg / ml of G418 (Geneticin, Gibco lot 3069464) for approximately 2 to 3 weeks. The amplification marker of dhfr (dihydrofolate reductases) located in the construction of the light chain allowed the amplification of the dhfr gene as well as the transgene through methotrexate of the folic acid analogue (MTX). For gene amplification, G418 resistant cells were cultured in the presence of 200 nM MTX for a period of 2-3 weeks followed by a further increase in MTX concentration to 1 μM resulting in a heterogeneous cell group amplified. The concentration of the antibody was determined by HPLC of analytical Protein A. The best producing groups were selected for cloning through limiting dilution, using a seeded density of 0.3 cells per cavity to isolate the most productive clones.

EXAMPLE 5 Determination of the expression of recombinant human IgG through ELISA Characterization of VHE / humV1. VHE / humV2. VHQ / humV1 v VHQ / humV2 To determine the IgG concentrations of the recombinant human antibody expressed in the culture supernatants, a sandwich ELISA protocol was developed and optimized using IgG as standard. Flat bottom 96-well microtitre plates (Cat. No. 4-39454, Nunc Immunoplate Maxisorp) were coated overnight at 4 ° C with 100 μl goat anti-human IgG (complete molecule, Cat. No. 11011). , SIGMA) at the final concentration of 0.5 μg / ml in PBS. The cavities were then washed 3 times with wash buffer (PBS containing 0.05% Tween 20) and blocked for 1.5 hours at 37 ° C block pH buffer (0.5% BSA in PBS). After 3 wash cycles, samples of the antibody and standard human IgG (Cat. No. 14506, SIGMA) were prepared through a 1.5 fold serial dilution in blocking pH buffer. 100 μl of diluted or standard samples were transfected in duplicate for the cover plate and incubated for 1 hour at room temperature. After incubation, the plates were washed three times with wash buffer and subsequently incubated for 1 hour with 100 μl with peroxidase-conjugated light chain kappa goat anti-human IgG (Cat. No. A-7164, SIGMA) diluted to 1/4000 in blocking pH regulator. Control cavities received 100 μl of blocking pH regulator or concentrated normal culture medium. After washing, the colorimetric quantification of the peroxidase bond in the sample and the standard cavities was performed, using an EIA TMB Peroxidase Substrate Kit (Cat. No. 172-1067, Bio-Rad) according to the manufacturer's instructions . The peroxidase mixture was added to 10 μl per well and incubated for 30 minutes at room temperature in the dark. The colorimetric reaction was stopped by the addition of 100 μl of 1M sulfuric acid and the absorbance in each well was read at 450 nm, using an ELISA plate reader (Model 3350-UV, BioRad). With a correlation coefficient of 0.998 for the standard IgG curve, the following concentrations were determined for the four different culture concentrates (approximately 250-300 times concentrated) obtained from transfected COS cells: VHE / humV1 supernatant = 8.26, μg / ml VHE / humV2 supernatant = 6.27, μg / ml VHQ / humV1 supernatant = 5.3, μg / ml VHQ / humV2 supernatant = 5.56, μg / ml Size Exclusion Chromatography (SEC) Analysis Purified affinity antibodies VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2 were analyzed through Size Exclusion Chromatography (SEC) on a TSKgel Super SW3000SWXL with the In order to determine the protein content, the percentage of small aggregates (oligomeric antibodies) as well as as possible through, and degradation products. The chromatograms show that for HEV / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2 the main peak is divided and that the separation is more pronounced for groups with heavy chain E (Figure 12). The results suggest that there are at least two molecules with a typical retention time for an IgG antibody present in each sample.

SDS-PAGE under reducing conditions Analysis of binding molecules CD45RO / RB VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2 through SDS-PAGE under reducing conditions (gradient gel, 4 to 20% of Glycine Tris-gel, Novex) shows the presence of an unexpected additional band, migrating slightly above the band corresponding to the heavy chain band of each sample. The difference in mass is estimated as being in the range of 2 to 4 kDa. Western staining analysis reveals that the upper band is recognized by anti-human antibodies (H + L) suggesting that the additional band is a heavy chain variant.

Cation Exchange Chromatography (CEX) Charge heterogeneity was assessed through cation exchange chromatography (CEX) using a PolyCatA column (PolyLC Inc.). Since the chimeric mAb is essentially free of charge heterogeneity different from the C-terminal lysine variants, all humanized CD45RO / RB binding molecules, ie, VHE / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2 , they were found to be very heterogeneous in charge. A common reason for the charge heterogeneity in antibodies is the presence / absence of Lys at the C-terminal end of the heavy chain of the antibody resulting in three peaks in CEX. The highest number of peaks visible in each chromatogram (< 10; Figure 13) indicates that at least one additional modification is present in the four CD45RO / RB link molecules.

SDS-PAGE In order to evaluate the molecular differences between the two antibody species found in the HEV / humV1, VHE / humV2, VHQ / humV1 and VHQ / humV2 protein groups, VHE / humV2 is selected to be further analyzed. The two SEC peaks of VHE / humV2 were collected in a semi-preparative mode and retested through SEC to confirm their purity. The fractions collected by SEC (Fraction 1 and Fraction 2) were then analyzed by SDS-PAGE under reducing conditions. The ratio of the unexpected additional band to about 49 kDa is different between the two fractions. Fraction 2 is almost free of this additional higher band.

Mass Spectrometry ESI-Q-TOF The SEC fractions F1, F2 and and I group of the HEV protein / humV2 were then further analyzed through mass spectrometry ESI-Q-TOF. In the spectrograms of the protein group and Fraction 2, the same signal groups were observed. The first group of signals at approximately 148O00 Da was found in the group of proteins and Fraction 2 was not detected in Fraction 1. In Fraction 1, a second group of signals was detected (at approximately 150'300 Da) in addition to another group of signals at around 152'500 Da. The second group of signals at around 150'320 Da can not be correlated with any form of antibody usually observed. These findings suggest that the two peaks in SEC and the two upper bands in reduced SDS-PAGE correspond to each of the variants of an unexpected protein.

Reverse Phase Chromatography To obtain a less complex pattern in mass spectrometry, the antibody chains are also analyzed separately. The fractions are reduced and rented. In order to confirm the degree of completion of the reactions, the reduced and alkylated samples are analyzed through reverse phase chromatography (RP) (SORBAX, Poroshell 300SB-C8). After reduction and alkylation, the shape of the peak is almost the same as only after reduction. A change in retention time was observed. Similar patterns were obtained for the reduced and alkylated samples of Fraction 1 and Fraction 2 SEC. The sample of the protein group VHE / humV2, Fraction 1 SEC and Fraction 2 SEC (reduced and alkylated) was analyzed with mass spectrometry ESI-Q-TOF. Several peaks of mass can be assigned to the expected antibody forms. The same main peaks are found in all three samples (group of proteins, F1 and F2) but their intensities in Fraction 2 are very low (Figure 14).

Carbohydrate analysis of heavy chain variants Reduced and alkylated antibody was injected into a reverse phase chromatography (RP) column and the two peaks corresponding to the heavy chain variants were collected. The characteristics of the oligosaccharide of these fractionated RP antibodies are then determined. Among the anticipated GO and G1 oligosaccharide residues found in all the samples, several other peaks were detected in the chromatogram of Fraction 1 (traces of those peaks are also detectable in the Fraction 2 chromatograms and the protein group). The results taken together suggest that a larger antibody species contains additional glycosylation, not typically found in monoclonal antibodies expressed SP2 / 0, counting for the difference in mass. Since the large complex glycosylation found through carbohydrate analysis and mass spectrometry would be very unlikely for the conserved glycosylation site in the constant region of the antibody, a search is made for other possible sites in the amino acid sequence of the antibodies humanized A potenial site (N73) for N-glycosylation (N-X-S) was identified in the variable domain of the two heavy chain variants.

Preparative fractionation through CEX Additional analyzes were carried out using purified material from the VHE / humV1 group. To reduce the heterogeneity of the material, the N-terminal antibody was fractionated through preparative cation exchange chromatography using SP Sepharose (Pharmacia). The column was equilibrated with 25 mM sodium phosphate pH regulator, pH 6.0 (= regulated pH A) and the protein binding was eluted with 250 mM NaCl in buffer pH A (= buffer pH B; gradient of 0-65% B). The purity of the fractions was analyzed using CEX. The collected fractions were reinjected in the SEC column to find a correlation between the results obtained with the two techniques. The chromatogram indicates that the first group of peaks in CEX correlates with the pre-peak in the SEC analysis of the antibody, that second group of peaks in CEX leads along with the first peak in the SEC analysis of the protein group (or Fraction 1 ), and that last peak in CEX is eluted as the last peak in SEC (Fraction 2). In conclusion, the results described above (oligosaccharide profile for fractions 1 and 2 SEC, the CEX pattern obtained after decarboxylation, the SEC pattern obtained for the fractions collected in CEX, mass spectrometry strongly suggest the presence of a species of antibody with complex oligosaccharides causing a) mass heterogeneity, leading to a double peak in SEC and b) heavy chain double band with reduced SDS-PAGE charge heterogeneity, leading to the CEX pattern.

Analytical characterization of CEX fractions Since large antibody species are eluted first in CEX, showing that they are less positively charged, the presence of sialic acid (a component of complex glycosylation and negatively charged) is measured in the different fractions obtained through of CEX preparation. It was found that sialic acid is present in all four samples to vary the degrees. It was found that sialic acid is most likely the N-glycolyl-neuraminic acid form (based on the shorter retention time as compared to standard N-acetyl-neuraminic acid). The very low presence of sialic acid in a CEX fraction in contrast to its very high abundance in another CEX fraction strongly suggests that the negatively charged sugar component contributes to the mass / charge heterogeneity. The CEX fractions were analyzed through mass spectrometry ESI-Q-TOF.

SDS-PAGE The same fractions were also analyzed by SDS-PAGE under reducing conditions. It can be concluded that the upper heavy chain band corresponds to the portion of an antibody with a complex oligosaccharide profile containing sialic acid. The lower heavy chain band corresponds to the expected antibody that displays a conventional oligosaccharide profile.

Separation in a Mono-S column of CEX fractions digested in papain In order to confirm the use of the potential glycosylation site in the variable domain of the heavy chain, about 1 mg of each CEX fraction was treated with papain to separate the part Fab and antibody Fe. Preparative chromatography was carried out on a Mono-S column. Each of the sub-fractions collected was injected back into the RP column to identify its content in terms of Fab and / or Fe domains. Each of the sub-fractions collected afterwards was analyzed with mass spectrometry ESI-Q-TOF . The results obtained show that a) the conserved glycosylation site in the Fe part of the heavy chain is occupied by the bi-antenna oligosaccharide forms without any (GO), one (G1) or two (G2) terminal galactose units; b) that in contrast, the irregular glycosylation site (N73) carries complex oligosaccharides containing N-glycolyl neuraminic acid. In conclusion, the asparagine residue N73 in the variable domain of the heavy chain was found to be partially N-glycosylated. These sugar species cause mass heterogeneity as well as charge heterogeneity which is detected by SDS-PAGE, size exclusion chromatography and cation exchange chromatography.

Characterization of VHE-N73D / humV1 In order to eliminate the heterogeneity of humanized VHE / humVI, VHE / humV2, VHQ / humV1 and VHQ / humV2 antibodies, the asparagine residue at position N73 in the heavy chain variable domain is replaced by a residue of aspartic acid (see example 4). The VHE-N73D / humV1 was then further analyzed: Size Exclusion Chromatography Analysis (SEC) SEC was carried out as described above. A clear distance was observed between VHE / humV1 and VHE- N73D / humV1 in SEC. In contrast to the double peak obtained for VHE / humV1, only one peak was obtained for VHE-N73D / humV1 (Figure 12). Approximately 0.2% of aggregates will be quantified through SEC.

SDS-PAGE under reducing conditions VHE / humV2 and VHE-N73D / humV1 were further analyzed via SDS-PAGE under reducing conditions as described above. Only one band for VHE-N73D / humV1 is visible at the expected heavy chain (HC) position (around 50 kDa). The HC band observed for VHE-N73D / humV1 corresponds to the lower band of the doublet observed for VHE / humV2. The position of the light chain band is the same for the proteins analyzed.

Cation Exchange Chromatography (CEX) Chromatography (CEX) was carried out as described above. The results obtained for the CEX analysis show that the charge heterogeneity of VHE-N73D / humV1 is reduced compared to the high charge heterogeneity of HEV / humV2 (> 10; Figure 13) MALDI TOF analysis (mass spectrometry) The mass detected for the heavy chain and the light chain obtained through a MALDI TOF analysis (mass spectrometry) are in accordance with the expected mass, deduced from the amino acid sequence of VHE-N73D / humV1.

Reverse Phase Chromatography (RP) After reduction with DDT the two humanized antibodies VHE / humV2 and VHE-N73D / humV1 were analyzed through reverse phase chromatography (RP). Due to partial glycosylation on asparagine N73, two "heavy chains" were observed for VHE / humV2 (double peak at about 18.5 minutes) between the peak corresponding to the light chain (about 17.3 minutes). For VHE-N73D / humV1, only one peak was observed for the heavy chain (Figure 14).

EXAMPLE 6 FACS Competency Analysis (link affinity) The PEER T cell line was chosen as the target cell for FACS analysis because it expresses the CD45 antigen on its cell surface. To analyze the binding affinity of the supernatants of a humanized antibody, competition experiments using the chimeric antibody labeled FITC as a reference were carried out and compared with the inhibition of the purified mouse antibody and the chimeric antibody. The PEER cell cultures were centrifuged for 10 seconds at 3000 rpm and the medium was removed. Cells were resuspended in pH buffer FACS buffer (PBS containing 1% FBS and 0.1% sodium azide) and seeded in a 96-well round bottom microtiter plate at a cell density of 1 x 105 per cavity. The plate was centrifuged and the supernatant discarded. For the block studies, first 25 μl of concentrated non-transfected medium or control antibody compared to the isotope (negative controls), unlabeled mouse antibody or chimeric antibody (positive controls) were added, as well as the concentrated supernatant containing the various combinations of a humanized antibody (samples) in each cavity at the concentrations indicated in the text. After 1 hour of incubation at 4 ° C, the PEER cells were washed with 200 μl of pH regulator FACS through centrifugation. The cells were subsequently incubated for 1 hour at 4 ° C with the chimeric antibody conjugated with FITC in 25 μl of pH regulator FACS at the final concentration of 20 μg / ml. Cells were washed and resuspended in 300 μl pH regulator FACS containing 2 μg / ml propidium iodide, which enabled viable cells to be activated. The cell preparations were analyzed on a flow cytometer (FACSCalibur, Becton Dickinson). FACS analyzes indicate a dose-dependent blockade of the chimeric antibody labeled with fluorochrome through culture supernatants of the concentrated humanized antibody. No dose-dependent blockade of the chimeric antibody binding was seen with the control antibody compared to the isotope, indicating that the block effect across the different humanized antibody combinations are epitope-specific and the epitope specificity It seems that it is retained after the process of humanization. The undiluted supernatant of the aforementioned SP2 / 0 isotope transfectants or chimeric antibody (negative controls) or control antibody compared to the 2 μg / ml in culture medium were incubated with 1.5 x 10 5 PEER cells in 100 μl for 30 minutes at 4 ° C. Then, 100 μl of PBS containing the chimeric antibody labeled FITC was added to each sample and incubation at 4 ° C continued for another 30 minutes. After washing, the cells were resuspended in FACS-PBS containing 1 μg / ml of 7-Amino-Actinomycin D and analyzed by flow cytometry using a Becton Dickinson FACSCalibur instrument and the CelIQuest Pro Software. was on living cells, ie negative events of 7-Amino-Actinomycin D The 'FACS analyzes show that the unlabeled humanized CD45RB / RO binding molecules, for example VHE / humV1 and VHQ / humV1 but not the control antibody compared with isotope compete with the FITC-labeled chimeric antibody for binding to the CD45-positive PEER T cell line.

Specificity of VHE-N73D / humV9 To evaluate whether modification of the humanized VHE / humV1 CD45R0 / RB binding molecule, ie the exchange of asparagine by aspartate at amino acid position 73 of the heavy chain of the CD45RO binding molecule / RB, modified the reactivity with the cognate epitope, the reactivity of VHE-N73D / humV1 with the human T cell line PEER expressing CR45 was analyzed in a competition binding experiment. The PEER cells were incubated with VHE-N73D / humV1, with their chimeric predecessors or with a non-binding isotope control IgG1 antibody. The unbound antibody was washed and the cells were incubated with an FITC-labeled isotope IgG 1 control antibody or fluorochrome fluorescein isothiocyanate (FITC) chimeric anti-CD45R0 / B mAB mAB. After washing, the cells were subjected to flow cytometric analysis to quantify the amount of FITC-labeled antibody bound to the pre-incubated PEER cell: PEER cell cultures were centrifuged for 10 minutes at 400 times the standard gravity force (g), and the medium was removed. Cells were resuspended in pH regulator FACS (PBS containing 1% v / v of FBS, 0.1 p / v of EDTA and 0.1% w / v of sodium azide) and seeded in V-bottom microtiter plates. of 96 cavities at a cell density of 1 x 105 cells per well. The plate was centrifuged and the supernatant discarded. Each cell sample was incubated for 30 minutes at 4 ° C in 50 μl of pH regulator FACS containing 20 μg / ml of either chimeric anti-CD45RO / RB mAb, VHE-N73D / humV1 or IgG 1 control Ab. Then the PEER cells were washed twice with 150 μl of pH-regulator FACS through centrifugation. The cells were subsequently incubated for 30 minutes at 4 ° C in 50 μl of pH regulator FACS containing 20 μg / ml of either chimeric mAB conjugated with FITC or control Ab 3G5 conjugated with FITC. Finally, the cells were washed and resuspended in 200 μl of pH regulator FACS containing 7-amino actinomycin D (7-AAD) at 1 μg / ml, which allowed the identification of viable cells during the analysis. The cell preparations were analyzed in a FACSCalibur flow cytometer (Becton Dickinson). It was observed that unlabeled VHE-N73D / humV1 and non-labeled chimeric anti-CD45RO / RB mAB but not antibody gg1 prevent the FITC labeled chimeric anti-CD45RO / RB mAb from binding to PEER cells expressing CD45 as determined by a decrease in the fluorescent signal. In conclusion, it is found that modification of the VHE / humV1 antibody to VHE-N73D / humV1 does not change the epitope specificity of the humanized anti-CD45R0 / RB binding molecule.

Affinity of VHE-N73D / humV1 for cynomolgus or human CD45 To determine the affinity of the VHE-N73D / humV1 antibody for its epitope, the reactivity of VHE-N73D / humV1 with a PEER T cell line expressing human CD45 or the line of HSC-F T cells expressing CD45 from cynomolgus monkey can be quantitated in a competitive binding experiment, similar to the procedure described in Daley et al., 1995; J. Mol. Biol. 253: 243. Briefly, the intensity of staining of the PEER or HSC-F cells with the FITC-labeled antibody in the presence of various concentrations of the unlabeled competitor antibody was used to calculate the affinities of the unlabeled antibodies. More precisely, in a first step, the concentration and the fluorochrome labeling stoichiometry of the mouse derivative CD45RB / RO A6 (mA6) molecule derived from FITC-labeled mouse were measured. Next, the concentration of the target molecule, CD45, on the surface of the PEER or HSC-F cell line is determined with the aid of the mA6 yes antibody conjugated with FITC (anti-huCDer derived from mouse) and using fluorescent beads with a known molecular labeling degree. With the concentration of cellular CD45 receptors and their known FITC conjugated ligands, the affinity of mAb conjugated with FITC for cellular CD45 receptors is determined through the equilibrium titration procedure based on FACS. Finally, the affinity of the unlabeled antibody VHE-N73D / humV1 is determined from the competition staining reactions with mA6 conjugated with FITC or PEER or HSC-F cells by measuring the increase in FITC fluorescence as a function of the total concentration of VHE-N73D / humV1. If the cubic equation representing the algebraically explicit description of the link equilibrium in a mixture of two competing ligands that bind to a receiver is implemented in the Origin 7.5 program, the software program was used to calculate the value of the constant of dissociation (Kd) for VHE-N73D / humV1 on the repetitive curve fit analysis.

In one experiment the dissociation constant of VHE-N73D / humV1 for the PEER cellular target was calculated as 2.44 ± 0.07 nM, for the target of the HSC-F cell cynomolgus was calculated as 1.67 ± 0.00 nM, assuming the binding of the divalent antibody to the target molecules CD45RO / RB.

EXAMPLE 7 Biological Activities of the CD45EB / RO binding molecules In this study, we conducted if the CD45RB / RO chimeric antibody, when present in the cultures of polyclonally activated primary human T cells (i) support the differentiation of T cells with a characteristic Treg phenotype, (ii) prevents or ameliorates apoptosis after activation of the T cell, and (iii) affects the expression of subgroup-specific antigens and receptors after re-stimulation.

The CD45RB / RO binding antibody improves cell death in primary T cells of polyclonally activated T cells Primary T cells (mixture of CD4 + and CD8 + T subgroups) were subjected to activation via anti-CD3 mAb plus anti-CD28 (200 μg / ml each) in the presence or absence (= control) of the CD45RB / RO binding chimeric antibody. The excess antibodies were removed by washing on day 2. 7-amino-actinomycin D (7-AAD) was used as a staining dye of DNA absorbed by the apoptotic and necrotic cells to measure the death of the cell after of the activation. The results show that the activation of T cells in the presence of the CD45RB / RO binding chimeric antibody increased the fraction of positive 7-AAD cells to double on day 2 after activation. On day 7, the portion of the 7-AAD positive cells was again similar in the control cultures and treated with the CD45RB / RO binding chimeric antibody.

The chimeric CD45RB / RO binding antibody but not the T cells treated with control mAb display a T-regulatory cell phenotype (Treg) The increased expression of CD25 and the negative regulatory protein CTLA-4 (CD152) is a Treg cell marker . Functional suppression of primary and secondary T cells through the CD45RB / RO chimeric binding antibody may be due to the induction of Treg cells. To drive this aspect, the T cells when activated through anti-CD3 + mAbs mAbs in the presence of the CD45RB / RO chimeric binding antibody or anti-LPS control mAb. The time course of CTLA-4 and CD25 expression reveals marked differences between controls and treated T cells with the chimeric CD45RB / RO binding antibody on days 1 and 3 after secondary stimulation, indicating a Treg phenotype.

The expression intracellular CTLA-4 is sustained in the presence of the chimeric binding antibody CD45RB / RO It has been reported that substantial amounts of CTLA-4 can also be found intracellularly. Therefore, in parallel to CTLA-4 surface staining, extracellular CTLA-4 expression was analyzed. Moderate differences were seen between the T cell cultures on day 4 after stimulation. After prolonged culture, however, high levels of intracellular CTLA-4 were sustained only in T cells treated with the CD45RB / RO binding chimeric antibody but not the control T cells.

T cells treated with the CD45RB / RO binding chimeric antibody become double positive for CD4 and CD8 After stimulation, T cells induce and upregulate the expression of several surface receptors, such as CD25, CD152 (CTLA -4), CD154 (Ligand CD40) and others. In contrast, the level of CD4 or CD8 expression is believed to remain relatively constant. A strong increase of both CD4 and CD8 antigens in T cells treated with the CD45RB / RO binding chimeric antibody but not in T cells treated with Ab after activation was reproducibly observed. The emergence of a CD4 / CD8 double positive T cell population seems to be due to the upregulation of the CD4 subgroup in the CD8 + subgroup and the CD8 + subgroup in the CD4 + subgroup. This contrasts with a moderately low percentage of double positive t cells in control cultures.

High expression of the alpha chain of the IL-2 receptor, but very low expression of the beta chain through T cells treated with the chimeric antibCD45RB / RO It is known that Treg cells are constitutively positive for CD25, the alpha chain of the IL-2 receptor. The regulation of other subgroups of the trimeric IL-2 receptor in Treg cells is not known. Recently, the expression of the beta chain of the IL-2 receptor, for example CD122, was compared in activated T cells and propagates in the presence or absence of the CD45RB / RO binding chimeric antib The results show that T cells treated with the CD45RB / RO binding chimeric antibhave about 10 times lower CD122 expression compared to T cells in control cultures. This difference may indicate that Treg cells require factors other than IL-2 to proliferate.

EXAMPLE 8 Sequences of the invention (the CDR sequences of the invention are underlined) SEQ ID NO: 1 Part of the chimeric light chain amino acid sequence DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGSGTKLEIK SEQ ID NO: 2 Part of the chimeric heavy chain amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTNYHHWVKQEPGQGLEWIGYFNPYNHGTKY NEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSS SEQ ID NO: 3 Amino acid sequence of chimeric light chain DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSES1SGIPSRFSG SGSGTDFTLS1NSVESEDIADYYCQQSNTWPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID N0: 4 Amino acid sequence of chimeric heavy chain EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEW1GYFNPYNHGTKY NEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5 Nucleotide sequence encoding a polypeptide of SEQ ID NO: 1 GACATTCTGCTGACCCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCA GTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAGA ACAAATGGTTCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCCT TCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGA GTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGG CTCGGGGACCAAGCTTGAAATCAAA SEQ ID NO: 6 Nucleotide sequence encoding a polypeptide of SEQ ID NO: 6 NO: 2 GAGGTGCAGCTGCAGCAGTCAGGACCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAG ATGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAGACAAATCCTCCAACACA GCCTACATGGACCTCAGCAGCCTGACCTCTGAGGACTCTGCGATCTACTACTGTGCAA GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA SEQ ID NO: 7 Amino acid sequence part of humanized light chain designated humV2 (humV2 = VLm) DILLTQSPAT LSLSPGERAT FSCRASQNIG TSIQWYQQKT NGAPRLLIRS SSES1SGIPS RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK SEQ ID NO: 8 Humanized light chain amino acid sequence part designated humV1 (humV1 = VLh) DILLTQSPAT LSLSPGERAT LSCRASQNIG TSIQWYQQKP GQAPRLÜRS SSESISGIPS RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK SEQ ID NO: 9 Humanized heavy chain amino acid sequence part designated VHE EVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS SEQ ID NO: 10 Part of amino acid sequence of humanized heavy chain designated VHQ QVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS SEQ ID NO: 11 Nucleotide sequence encoding amino acid sequence SEQ ID NO: 9 GAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA SEQ ID NO: 12 Nucleotide sequence encoding amino acid sequence SEQ ID NO: 10 CAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATI'iTAATCCTTACAATCATGGTACTA AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA SEQ ID N0: 13 Nucleotide sequence encoding amino acid sequence SEQ ID NO: 7 GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA CTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA ACAAATGGTGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 14 Nucleotide sequence encoding amino acid sequence SEQ ID NO: 8 GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA CTCTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA CCAGGTCAGGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG AGCCTGAAGATTTTGCAGTGTATrACTGTCAACAAAGTAATACCTGGCCATTCACGTTC GGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 15 Nucleotide sequence of the expression vector HCMV-G1 HuA6-VHQ (Complete DNA sequence of a heavy chain expression vector comprising SEQ ID NO: 12 (VHQ) of 3921-4274) 1 AGCTTTTTGC AA? AGCCTAG GCCTCCA? AA AAGCCTCCTC ACTACTTCTG 51 GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA 101 TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC 151 GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT AATTGAGATG 201 CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC TGACTAATTG 251 AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG GGGACTTTCC 301 ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT TTCGGTGATG 351 ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT 401 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG 451 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG 501 GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG 551 TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC 601 CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 651 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 701 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 751 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA 801 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA 851 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA 901 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 951 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 1001 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 1051 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG 1101 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA 1151 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC 1201 TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG TAT TTGGTAT 1251 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 1301 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 1351 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT 1401 TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 1451 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA 1501 AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 1551 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 1601 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 1651 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC 1701 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG 1751 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT 1801 AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 1851 CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC TCGTCGTTTG 1901 GTATGGCTTC GATCAAGGCG AGTTACATGA ATTCAGCTCC GGTTCCCAAC 1951 TCCCCCATGT TGTGCAAAAA CTCCGATCGT 2001 TGTCAGAAGT AGCGGTTAGC TCCTTCGGTC ACTCATGGTT ATGGCAGCAC AAGTTGGCCG CAGTGTTATC TGCATAATTC TCTTACTGTC 2051 TAAGATGCTT TTCTGTGACT 2101 GGTGAGTACT ATGCCATCCG ATTCTGAGAA TAGTGTATGC GGCGACCGAG 2151 CAACCAAGTC TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA CATAGCAGAA 2201 CTTTAAAAGT GCTCATCATT GGAAAACGTT AAAACTCTCA CTTCGGGGCG 2251 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC 2301 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA 2351 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA 2401 TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA 2451 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 2501 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC 2551 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC 2601 GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG AATAATAAAA 2651 TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG CCGACTAAAT 2701 TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG GCGATATTGG 2751 AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG CCGATGTGAG 2801 TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT TTTGGGCATA 2851 CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA TGGCGATAGA 2901 CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT CGCAGTTTCG 2951 ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA 3001 AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG 3051 CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 3101 TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG 3151 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT 3201 TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT 3251 CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG 3301 ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 3351 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 3401 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG 3451 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC 3501 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT 3551 TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT 3601 TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 3651 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC 3701 CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 3751 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA 3801 CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCAA 3851 GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC CTGCTGGCCG 3901 TGGCCCCCGG CGCCCACAGC CAGGTGCAGC TGGTGGAGTC AGGAGCCGAA 3951 GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG CCTCTGGATA 4001 CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG CCTGGTCAGG 4051 GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG TACTAAGTAC 4101 AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA AATCCATCAG 4151 CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC ACTGCGGTCT 4201 ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC CTGGGGCCAA 4251 GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG ATCCCAAGCT 4301 AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG 4351 GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA ATGCCCATGA 4401 GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA CCCAGGGGCC 4451 TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC ATGGCACCAC 4501 CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT 4551 CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG 4601 GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC 4651 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT 4701 CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC 4751 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA 4801 AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG 4851 CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC CAGTCCAGGG 4901 CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT CTGCCCGCCC 4951 CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA GGCTCTGGGC 5001 AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA AAGGGGCAGG 5051 TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC CCTGCCCCTG 5101 ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA GCTCGGACAC 5151 CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT CTGCAGAGCC 5201 CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GGTAAGCCAG 5251 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC TAGAGTAGCC 5301 TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT 5351 CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC 5401 CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG 5451 CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT 5501 ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG 5551 CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA 5601 GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC 5651 TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG TGGGACCCGT 5701 GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA CCCTCTGCCC 5751 TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA GCCCCGAGAA 5801 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA 5851 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG 5901 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT 5951 CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT 6001 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC 6051 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 6101 GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG GCTCTCGCGG 6151 TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT CCCGGGCGCC 6201 CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT GCGAGACTGT 6251 GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA GTGGCATGAG 6301 ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA TCGATCCAGA 6351 CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT 6401 GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA 6451 ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT GCATTCATTT 6501 TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA AGCAAGTAAA 6551 ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC AAGGCACTAT 6601 ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA GCTAAAGGTA 6651 CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT GCCTGTGTGG 6701 AGTAAGAAAA AACAGTATGT TATGATTATA ACTGTTATGC CTACTTATAA 6751 AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG TGCAGCTTTT 6801 TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT TACTAAACAC 6851 AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC TTGGGGTCTT 6901 CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA GTCAGCAGTA 6951 GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA GGTTTTCCTC 7001 ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA TAGGTTGGAA 7051 TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA TTATACACTT 7101 AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG GTAGTTTGTC 7151 CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG ATCCGGGACC 7201 AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AG TTCGGGGG CATGGATGCG 7251 CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG CACTGCCGGT 7301 AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA CCAACTCGCG 7351 AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG 7401 CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC 7451 TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC 7501 GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA GAAGAACTCG 7551 TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG CGGCGATACC 7601 GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC TCTTCAGCAA 7651 TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC 7701 CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT 7751 CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA 7801 TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC 7851 TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA TCCGAGTACG 7901 TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG CAGGTAGCCG 7951 GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC TCGGCAGGAG 8001 CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC 8051 CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG 8101 CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC 8151 TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA AAAGAACCGG 8201 GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG CAGCCGATTG 8251 TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA 8301 GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC 8351 TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA 8401 AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC 8451 GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA CCGCCCAGTC 8501 TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT CTCTTTGCGC 8551 TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT 8601 CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA 8651 GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT SEQ ID NO: 16 Nucleotide sequence of the expression vector HCMV-G1 HuA6-VHE (Complete DNA sequence of a humanized heavy chain expression vector comprising SEQ ID NO: 11 (VHE) of 3921-4274) 1 AGCTTTTTGC AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC ACTACTTCTG 51 GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA 101 TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC 151 GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT AATTGAGATG 201 CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC TGACTAATTG 251 AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG GGGACTTTCC 301 ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT TTCGGTGATG 351 ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT 401 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG 451 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG 501 GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG 551 TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC 601 CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 651 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 701 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 751 CAGCAAAAGG CCAGGAACCG- TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA 801 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA 851 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA 901 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 951 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 1001 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 1051 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG 1101 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA 1151 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC 1201 TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG TATTTGGTAT 1251 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 1301 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 1351 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT 1401 TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 1451 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA 1501 AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 1551 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 1601 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 1651 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC 1701 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG 1751 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT 1801 AATTGTTGCC GGGAAGCTAG AGTÁAGTAGT TCGCCAGTTA ATAGTTTGCG 1851 CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC TCGTCGTTTG 1901 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA 1951 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT 2001 TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC 2051 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT 2101 GGTGAGTACT CAACCAAGTC ATTCTGAGAA T AGTGTATGC GGCGACCGAG 2151 TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA CATAGCAGAA 2201 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 2251 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC 2301 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA 2351 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA 2401 TGTTGAATAC TCATACTCTT CCTTTTTTCAA TATTATTGAA GCATTTATCA 2451 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC 2501 2551 TAAGAAACCA TTATTATCAT GACATTAACC ACCTGACGTC TATAAAAATA GGCGTATCAC 2601 GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG AATAATAAAA 2651 TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG CCGACTAAAT 2701 TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG GCGATATTGG 2751 7AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG CCGATGTGAG 2801 TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT TTTGGGCATA 2851 CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA TGGCGATAGA 2901 CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT CGCAGTTTCG 2951 ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA 3001 AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG 3051 CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 3101 TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG 3151 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT 3201 TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT 3251 CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG 3301 ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 3351 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 3401 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG 3451 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC 3501 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT 3551 TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT 3601 TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 3651 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC 3701 CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 3751 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA 3801 CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCAA 3851 GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC CTGCTGGCCG TGGCCCCCGG 3901 CGCCCACAGC GAGGTGCAGC TGGTGGAGTC AGGAGCCGAA 3951 GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG CCTCTGGATA 4001 CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG CCTGGTCAGG 4051 GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG TACTAAGTAC 4101 AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA AATCCATCAG 4151 CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC ACTGCGGTCT 4201 ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC CTGGGGCCAA 4251 GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG ATCCCAAGCT 4301 AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG 4351 GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA ATGCCCATGA 4401 GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA CCCAGGGGCC 4451 TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC ATGGCACCAC 4501 CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT 4551 CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG 4601 GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC 4651 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT 4701 CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC 4751 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA 4801 AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG 4851 CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC CAGTCCAGGG 4901 CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT CTGCCCGCCC 4951 CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA GGCTCTGGGC 5001 AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA AAGGGGCAGG 5051 TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC CCTGCCCCTG 5101 ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA GCTCGGACAC 5151 CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT CTGCAGAGCC 5201 CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GGTAAGCCAG 5251 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC TAGAGTAGCC 5301 TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT 5351 CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC 5401 CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG 5451 CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT 5501 ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG 5551 CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA 5601 GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC 5651 TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG TGGGACCCGT 5701 GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA CCCTCTGCCC 5751 TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA GCCCCGAGAA 5801 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA 5851 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CT ATCCCAGC GACATCGCCG 5901 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT 5951 CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT 6001 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC 6051 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 6101 GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG GCTCTCGCGG 6151 TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT CCCGGGCGCC 6201 CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT GCGAGACTGT 6251 GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA GTGGCATGAG 6301 ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA TCGATCCAGA 6351 CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT 6401 GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA 6451 ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT GCATTCATTT 6501 TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA AGCAAGTAAA 6551 ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC AAGGCACTAT 6601 ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA GCTAAAGGTA 6651 CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT GCCTGTGTGG 6701 AGTAAGA? AA AACAGTATGT TATGATTATA ACTGTTATGC CTACTTATAA 6751 AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG TGCAGCTTTT 6801 TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT TACTAAACAC 6851 AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC TTGGGGTCTT 6901 CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA GTCAGCAGTA 6951 GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA GGTTTTCCTC 7001 ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA TAGGTTGGAA 7051 TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA TTATACACTT 7101 AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG GTAGTTTGTC 7151 CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG ATCCGGGACC 7201 AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AGTTCGGGGG CATGGATGCG 7251 CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG CACTGCCGGT 7301 AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA CCAACTCGCG 7351 AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG 7401 CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC 7451 TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC 7501 GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA GAAGAACTCG 7551 TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG CGGCGATACC 7601 GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC TCTTCAGCAA 7651 TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC 7701 CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT 7751 CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA 7801 TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC 7851 TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA TCCGAGTACGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG CAGGTAGCCG 7951 GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC 8001 TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC 8051 CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG 8101 CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC 8151 TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA AAAGAACCGG 8201 GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG CAGCCGATTG 8251 TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA 8301 GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC 8351 TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA 8401 AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC 8451 GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA CCGCCCAGTC 8501 TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT CTCTTTGCGC 8551 TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT 8601 CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA 8651 GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT SEQ ID N0: 17 Secuenca nucleotide expression vector HCMV-K Huab-VL1 hum V1 (Complete DNA sequence of a humanized light chain expression vector comprising SEQ ID NO: 14 (humV1 = VLh) of 3964-4284) 1 CTAGCTTTTT GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC TCACTACTTC 51 TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT AAATAAAAAA 101 AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC GGAGTTAGGG 151 GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA CTAATTGAGA 201 TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT GCTGACTAAT 251 TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT 301 CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC GTTTCGGTGA 351 TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT 401 GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG 451 GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG_501_CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG 551 AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAAAT 601 ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG 651 GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG 701 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG 751 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC 801 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA 851 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG 901 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC 951 CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG 1001 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG 1051 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT 1101 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC 1151 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT 1201 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT 1251 ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC 1301 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA 1351 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC 1401 TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT 1451 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT 1501 AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT 1551 GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT 1601 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA 1651 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC 1701 CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG 1751 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA 1801 TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG 1851 CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GCTCGTCGTT 1901 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT 1951 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC 2001 GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC 2051 ACTGCATAAT TCTCTTACTG TCATGCCATC CG TAAGATGC TTTTCTGTGA 2101 CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG 2151 AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CACATAGCAG 2201 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT 2251 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA 2301 CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC 2351 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGAGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT 2451 CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA 2501 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG 2551 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC 2601 ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG TGAATAATAA 2651 AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT CGCCGACTAA 2701 ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA TGGCGATATT 2751 GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT CGCCGATGTG 2801 AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA TTTTTGGGCA 2851 TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG GATGGCGATA 2901 GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT ATCGCAGTTT 2951 CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA GAGGCGACAT 3001 CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG AATCAATATT 3051 GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT CAATATTGGC 3101 TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT ACATTTATAT 3151 TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA TTGACTAGTT 3201 ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG 3251 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA 3301 CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC 3351 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT 3401 GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT 3451 TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA 3501 CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT 3551 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG 3601 GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA 3651 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC 3701 TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA 3751 TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC 3801 CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGC 3851 AAGCTTGATA TCGAATTCCT GCAGCCCGGG GGATCCGCCC GCTTGCCGCC 3901 ACCATGGAGA CCCCCGCCCA GCTGCTGTTC CTGCTGCTGC TGTGGCTGCC 3951 CGACACCACC GGCGACATTC TGCTGACCCA GTCTCCAGCC ACCCTGTCTC 4001 TGAGTCCAGG AGAAAGAGCC ACTCTCTCCT GCAGGGCCAG TCAGAACATT 4051 GGCACAAGCA TACAGTGGTA TCAACAAAAA CCAGGTCAGG CTCCAAGGCT 4101 TCTCATAAGG TCTTCTTCTG AGTCTATCTC TGGGATCCCT TCCAGGTTTA 4151 GTGGCAGTGG ATCAGGGACA GATTTTACTC TTACCATCAG CAGTCTGGAG 4201 CCTGAAGATT TTGCAGTGTA TTACTGTCAA CAAAGTAATA CCTGGCCATT 4251 CACGTTCGGC CAGGGGACCA AGCTGGAGAT CAAACGTGAG TATTCTAGAA 4301 AGATCCTAGA ATTCTAAACT CTGAGGGGGT CG GATGACGT GGCCATTCTT 4351 TGCCTAAAGC ATTGAGTTTA CTGCAAGGTC AGAAAAGCAT GCAAAGCCCT 4401 CAGAATGGCT GCAAAGAGCT CCAACAAAAC AATTTAGAAC TTTATTAAGG 4451 AATAGGGGGA AGCTAGGAAG AAACTCAAAA CATCAAGATT TTAAATACGC 4501 TTCTTGGTCT CCTTGCTATA ATTATCTGGG ATAAGCATGC TGTTTTCTGT 4551 CTGTCCCTAA CATGCCCTGT GATTATCCGC AAACAACACA CCCAAGGGCA 4601 GAACTTTGTT ACTTAAACAC CATCCTGTTT GCTTCTTTCC TCAGGAACTG 4651 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC. CATCTGATGA GCAGTTGAAA 4701 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 4751 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 4801 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 4851 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 4901 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 4951 ACAGGGGAGA GTGTTAGAGG GAGAAGTGCC CCCACCTGCT CCTCAGTTCC 5001 AGCCTGACCC CCTCCCATCC TTTGGCCTCT GACCCTTTTT CCACAGGGGA 5051 CCTACCCCTA TTGCGGTCCT CCAGCTCATC TTTCACCTCA CCCCCCTCCT 5101 CCTCCTTGGC TTTAATTATG CTAATGTTGG AGGAGAATGA ATAAATAAAG 5151 TGAATCTTTG CACCTGTGGT TTCTCTCTTT CCTCATTTAA TAATTATTAT 5201 CTGTTGTTTA CCAACTACTC AATTTCTCTT ATAAGGGACT AAATATGTAG_5251_TCATCCTAAG GCGCATAACC ATTTATAAAA ATCATCCTTC ATTCTATTTT 5301 ACCCTATCAT CCTCTGCAAG ACAGTCCTCC CTCAAACCCA CAAGCCTTCT 5351 GTCCTCACAG TCCCCTGGGC CATGGTAGGA GAGACTTGCT TCCTTGTTTT 5401 CCCCTCCTCA GCAAGCCCTC ATAGTCCTTT TTAAGGGTGA CAGGTCTTAC 5451 AGTCATATAT CCTTTGATTC AATTCCCTGA GAATCAACCA AAGCAAATTT 5501 TTCAAAAGAA GAAACCTGCT ATAAAGAGAA TCATTCATTG CAACATGATA 5551 TAAAATAACA ACACAATAAA AGCAATTAAA TAAACAAACA ATAGGGAAAT 5601 GTTTAAGTTC ATCATGGTAC TTAGACTTAA TGGAATGTCA TGCCTTATTT 5651 ACATTTTTAA ACAGGTACTG AGGGACTCCT GTCTGCCAAG GGCCGTATTG 5701 AGTACTTTCC ACAACCTAAT TTAATCCACA CTATACTGTG AGATTAAAAA 5751 CATTCATTAA AATGTTGCAA AGGTTCTATA AAGCTGAGAG ACAAATATAT 5801 TCTATAACTC AGCAATCCCA CTTCTAGATG ACTGAGTGTC CCCACCCACC 5851 AAAAAACTAT GCAAGAATGT TCAAAGCAGC TTTATTTACA AAAGCCAAAA 5901 ATTGGAAATA GCCCGATTGT CCAACAATAG AATGAGTTAT TAAACTGTGG 5951 TATGTTTATA CATTAGAATA CCCAATGAGG AGAATTAACA AGCTACAACT 6001 ATACCTACTC ACACAGATGA ATCTCATAAA AATAATGTTA CATAAGAGAA 6051 ACTCAATGCA AAAGATATGT TCTGTATGTT TTCATCCATA TAAAGTTCAA 6101 AACCAGGTAA AAATAAAGTT AGAAATTTGG ATGGAAATTA CTCTTAGCTG 6151 GGGGTGGGCG AGTTAGTGCC TGGGAGAAGA CAAGAAGGGG CTTCTGGGGT 6201 CTTGGTAATG TTCTGTTCCT CGTGTGGGGT TGTGCAGTTA TGATCTGTGC 6251 ACTGTTCTGT ATACACATTA TGCTTCAAAA TAACTTCACA TAAAGAACAT 6301 CTTATACCCA GTTAATAGAT AGAAGAGGAA TAAGTAATAG GTCAAGACCA 6351 CGCAGCTGGT AAGTGGGGGG GCCTGGGATC AAATAGCTAC CTGCCTAATC 6401 CTGCCCTCTT GAGCCCTGAA TGAGTCTGCC TTCCAGGGCT CAAGGTGCTC 6451 AACAAAACAA CAGGCCTGCT ATTTTTCCTGG CATCTGTGCC CTGTTTGGCT 6501 AGCTAGGAGC ACACATACAT AGAAATTAAA TGAAACAGAC CTTCAGCAAG 6551 GGGACAGAGG ACAGAATTAA CCTTGCCCAG ACACTGGAAA CCCATGTATG 6601 AACACTCACA TGTTTGGGAA GGGGGAAGGG CACATGTAAA TGAGGACTCT 6651 TCCTCATTCT ATGGGGCACT CTGGCCCTGC CCCTCTCAGC TACTCATCCA 6701 TCCAACACAC CTTTCTAAGT ACCTCTCTCT GCCTACACTC TGAAGGGGTT 6751 CAGGAGTAAC TAACACAGCA TCCCTTCCCT CAAATGACTG ACAATCCCTT 6801 TGTCCTGCTT TGTTTTTCTT TCCAGTCAGT ACTGGGAAAG TGGGGAAGGA 6851 CAGTCATGGA GAAACTACAT AAGGAAGCAC CTTGCCCTTC TGCCTCTTGA 6901 GAATGTTGAT GAGTATCAAA TCTTTCAAAC TTTGGAGGTT TGAGTAGGGG 6951 TGAGACTCAG TAATGTCCCT TCCAATGACA TGAACTTGCT CACTCATCCC 7001 TGGGGGCCAA ATTGAACAAT CAAAGGCAGG CATAATCCAG CTATGAATTC 7051 TAGGATCGAT CCAGACATGA TAAGATACAT TGATGAGTTT GGACAAACCA 7101 CAACTAGAAT GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT 7151 ATTGCTTTAT TTGTAACCAT TATAAGCTGC AATAAACAAG TTAACAACAA 7201 CAATTGCATT CATTTTATGT TTCAGGTTCA GGGGGAGGTG TGGGAGGTTT 7251 TTTAAAGCAA GTAAAACCTC TACAAATGTG GTATGGCTGA TTATGATCTC 7301 TAGTCAAGGC ACTATACATC AAATATTCCT TA TTAACCCC TTTACAAATT 7351 AAAAAGCTAA AGGTACACAA TTTTTGAGCA TAGTTATTAA TAGCAGACAC 7401 TCTATGCCTG TGTGGAGTAA GAAAAAACAG TATGTTATGA TTATAACTGT 7451 TATGCCTACT TATAAAGGTT ACAGAATATT TTTCCATAAT TTTCTTGTAT 7501 AGCAGTGCAG CTTTTTTCCTT TGTGGTGTAA ATAGCAAAGC AAGCAAGAGT 7551 TCTATTACTA AACACAGCAT GACTCAAAAA ACTTAGCAAT TCTGAAGGAA 7601 AGTCCTTGGG GTCTTCTACC TTTCTCTTCT TTTTTGGAGG AGTAGAATGT 7651 TGAGAGTCAG CAGTAGCCTC ATCATCACTA GATGGCATTT CTTCTGAGCA 7701 AAACAGGTTT TCCTCATTAA AGGCATTCCA CCACTGCTCC CATTCATCAG 7751 TTCCATAGGT TGGAATCTAA AATACACAAA CAATTAGAAT CAGTAGTTTA 7801 ACACATTATA CACTTAAAAA TTTTATATTT ACCTTAGAGC TTTAAATCTC 7851 TGTAGGTAGT TTGTCCAATT ATGTCACACC ACAGAAGTAA GGTTCCTTCA 7901 CAAAGATCCG GGACCAAAGC GGCCATCGTG CCTCCCCACT CCTGCAGTTC 7951 GGGGGCATGG ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG ATGCCGACGG 8001 ATTTGCACTG CCGGTAGAAC TCCGCGAGGT CGTCCAGCCT CAGGCAGCAG 8051 CTGAACCAAC TCGCGAGGGG ATCGAGCCCG GGGTGGGCGA AGAACTCCAG 8101 CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC CGGAAAACGA 8151 TTCCGAAGCC CAACCTTTCA TAGAAGGCGG CGGTGGAATC GAAATCTCGT 8201 GATGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTCGAACC CCAGAGTCCC 8251 GCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC GCTGCGAATC 8301 GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC CATTCGCCGC 8351 CAAGCTCTTC AGCAATATCA CGGGTAGCCA ACGCTATGTC CTGATAGCGG 8401 TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA AGCGGCCATT 8451 TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC ACGACGAGAT 8501 CCTCGCCGTC GGGCATGCGC GCCTTGAGCC TGGCGAACAG TTCGGCTGGC 8551 GCGAGCCCCT GATGCTCTTC GTCCAGATCA TCCTGATCGA CAAGACCGGC 8601 TTCCATCCGA GTACGTGCTC GCTCGATGCG ATGTTTCGCT TGGTGGTCGA 8651 ATGGGCAGGT AGCCGGATCA AGCGTATGCA GCCGCCGCAT TGCATCAGCC 8701 ATGATGGATA CTTTCTCGGC AGGAGCAAGG TGAGATGACA GGAGATCCTG 8751 CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT TCAGTGACAA 8801 CGTCGAGCAC AGCTGCGCAA GGAACGCCCG TCGTGGCCAG CCACGATAGC 8851 CGCGCTGCCT CGTCCTGCAG TTCATTCAGG GCACCGGACA GGTCGGTCTT 8901 GACAAAAAGA ACCGGGCGCC CCTGCGCTGA CAGCCGGAAC ACGGCGGCAT 8951 CAGAGCAGCC GATTGTCTGT TGTGCCCAGT CATAGCCGAA TAGCCTCTCC 9001 ACCCAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT CAATCATGCG 9051 AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC CTGCGCCATC 9101 AGATCCTTGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA GGGCTTCCCA 9151 ACCTTACCAG AGGGCGCCCC AGCTGGCAAT TCCGGTTCGC TTGCTGTCCA 9201 TAAAACCGCC CAGTCTAGCT ATCGCCATGT AA GCCCACTG CAAGCTACCT 9251 GCTTTCTCTT TGCGCTTGCG TTTTCCCTTG TCCAGATAGC CCAGTAGCTG 9301 ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT TCTACGTGTT 9351 CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG CAGCGTGAAG SEQ ID NO: 18 Nucleotide sequence of HCMV-K expression vector HuAb-VL1 hum V2 (Complete DNA sequence of a humanized light chain expression vector comprising SEQ ID NO: 13 (humV2 = VLm) of 3926-4246 )T GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC TCACTACTTC 51 TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT AAATAAAAAA 101 AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC GGAGTTAGGG 151 GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA CTAATTGAGA 201 TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT GCTGACTAAT 251 TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT 301 CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC GTTTCGGTGA 351 TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT 401 GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG 451 GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG_501_CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG 551 AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAAAT 601 ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG 651 GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG 701 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG 751 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC 801 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA 851 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG 901 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC 951 CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG 1001 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG 1051 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT 1101 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC 1151 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT 1201 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT 1251 ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC 1301 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA 1351 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC 1401 TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT 1451 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 1501 TTTTAAATCA 1551 ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT 1601 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA 1651 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC 1701 CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG 1751 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA 1801 TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG 1851 CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GCTCGTCGTT 1901 TGGTATGGCT 1951 TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC 2001 GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC 2051 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA 2101 CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG 2151 AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CACATAGCAG 2201 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT 2251 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA 2301 CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC 2351 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA 2401 AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT 2451 CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA 2501 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG 2551 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC 2601 ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG TGAATAATAA 2651 AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT CGCCGACTAA 2701 ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA TGGCGATATT 2751 GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT CGCCGATGTG 2801 AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA TTTTTGGGCA 2851 TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG GATGGCGATA 2901 GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT ATCGCAGTTT 2951 CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA GAGGCGACAT 3001 CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG AATCAATATT 3051 GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT CAATATTGGC 3101 TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT ACATTTATAT 3151 TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA TTGACTAGTT 3201 ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG 3251 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA 3301 CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC 3351 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT 3401 GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT 3451 TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA 3501 CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT 3551 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG 3601 GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA 3651 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC 3701 TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA 3751 TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC 3801 CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGC 3851 AAGCTTGCCG CCACCATGGA GACCCCCGCC CAGCTGCTGT TCCTGCTGCT 3901 GCTGTGGCTG CCCGACACCA CCGGCGACAT TCTGCTGACC CAGTCTCCAG 3951 CCACCCTGTC TCTGAGTCCA GGAGAAAGAG CCACTTTCTC CTGCAGGGCC 4001 AGTCAGAACA TTGGCACAAG CATACAGTGG TATCAACAAA AAACAAATGG 4051 TGCTCCAAGG CTTCTCATAA GGTCTTCTTC TGAGTCTATC TCTGGGATCC 4101 CTTCCAGGTT TAGTGGCAGT GGATCAGGGA CAGATTTTAC TCTTACCATC 4151 AGCAGTCTGG AGCCTGAAGA TTTTGCAGTG TATTACTGTC AACAAAGTAA 4201 TACCTGGCCA TTCACGTTCG GCCAGGGGAC CAAGCTGGAG ATCAAACGTG 4251 AGTATTCTAG AAAGATCCTA GAATTCTAAA CTCTGAGGGG GTCGGATGAC 4301 GTGGCCATTC TTTGCCTAAA GCATTGAGTT TACTGCAAGG TCAGAAAAGC 4351 ATGCAAAGCC CTCAGAATGG CTGCAAAGAG CTCCAACAAA ACAATTTAGA 4401 ACTTTATTAA GGAATAGGGG GAAGCTAGGA AGAAACTCAA AACATCAAGA 4451 TTTTAAATAC GCTTCTTGGT CTCCTTGCTA TAATTATCTG GGATAAGCAT 4501 GCTGTTTTCT GTCTGTCCCT AACATGCCCT GTGATTATCC GCAAACAACA 4551 CACCCAAGGG CAGAACTTTG TTACTTAAAC ACCATCCTGT TTGCTTCTTT 4601 CCTCAGGAAC TGTGGCTGCA CCATCTGTCT TCATCTTCCC GCCATCTGAT 4651 GAGCAGTTGA AATCTGGAAC TGCCTCTGTT GTGTGCCTGC TGAATAACTT 4701 CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC GCCCTCCAAT 4751 CGGGTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA GGACAGCACC 4801 TACAGCCTCA GCAGCACCCT GACGCTGAGC AAAGCAGACT ACGAGAAACA 4851 CAAAGTCTAC GCCTGCGAAG TCACCCATCA GGGCCTGAGC TCGCCCGTCA 4901 CAAAGAGCTT CAACAGGGGA GAGTGTTAGA GG GAGAAGTG CCCCCACCTG 4951 CTCCTCAGTT CCAGCCTGAC CCCCTCCCAT CCTTTGGCCT CTGACCCTTT 5001 TTCCACAGGG GACCTACCCC TATTGCGGTC CTCCAGCTCA TCTTTCACCT 5051 CACCCCCCTC CTCCTCCTTG GCTTTAATTA TGCTAATGTT GGAGGAGAAT 5101 GAATAAATAA AGTGAATCTT TGCACCTGTG GTTTCTCTCT TTCCTCATTT 5151 AATAATTATT ATCTGTTGTT TACCAACTAC TCAATTTCTC TTATAAGGGA 5201 CTAAATATGT AGTCATCCTA AGGCGCATAA CCATTTATAA AAATCATCCT 5251 TCATTCTATT TTACCCTATC ATCCTCTGCA AGACAGTCCT CCCTCAAACC 5301 CACAAGCCTT CTGTCCTCAC AGTCCCCTGG GCCATGGTAG GAGAGACTTG 5351 CTTCCTTGTT TTCCCCTCCT CAGCAAGCCC TCATAGTCCT TTTTAAGGGT 5401 GACAGGTCTT ACAGTCATAT ATCCTTTGAT TCAATTCCCT GAGAATCAAC 5451 CAAAGCAAAT TTTTCAAAAG AAGAAACCTG CTATAAAGAG AATCATTCAT 5501 TGCAACATGA TATAAAATAA CAACACAATA AAAGCAATTA AATAAACAAA 5551 CAATAGGGAA ATGTTTAAGT TCATCATGGT ACTTAGACTT AATGGAATGT 5601 CATGCCTTAT TTACATTTTT AAACAGGTAC TGAGGGACTC CTGTCTGCCA 5651 AGGGCCGTAT TGAGTACTTT CCACAACCTA ATTTAATCCA CACTATACTG 5701 TGAGATTAAA AACATTCATT AAAATGTTGC AAAGGTTCTA TAAAGCTGAG 5751 AGACAAATAT ATTCTATAAC TCAGCAATCC CACTTCTAGA TGACTGAGTG 5801 TCCCCACCCA CCAAAAAACT ATGCAAGAAT GTTCAAAGCA GCTTTATTTA 5851 CAAAAGCCAA AAATTGGAAA TAGCCCGATT GTCCAACAAT AGAATGAGTT 5901 ATTAAACTGT GGTATGTTTA TACATTAGAA TACCCAATGA GGAGAATTAA 5951 CAAGCTACAA CTATACCTAC TCACACAGAT GAATCTCATA AAAATAATGT 6001 TACATAAGAG AAACTCAATG CAAAAGATAT GTTCTGTATG TTTTCATCCA 6051 TATAAAGTTC AAAACCAGGT AAAAATAAAG TTAGAAATTT GGATGGAAAT 6101 TACTCTTAGC TGGGGGTGGG CGAGTTAGTG CCTGGGAGAA GACAAGAAGG 6151 GGCTTCTGGG GTCTTGGTAA TGTTCTGTTC CTCGTGTGGG GTTGTGCAGT 6201 TATGATCTGT GCACTGTTCT GTATACACAT TA TGCTTCAA AATAACTTCA 6251 CATAAAGAAC ATCTTATACC CAGTTAATAG ATAGAAGAGG AATAAGTAAT 6301 AGGTCAAGAC CACGCAGCTG GTAAGTGGGG GGGCCTGGGA TCAAATAGCT 6351 ACCTGCCTAA TCCTGCCCTC TTGAGCCCTG AATGAGTCTG CCTTCCAGGG 6401 CTCAAGGTGC TCAACAAAAC AACAGGCCTG CTATTTTCCT GGCATCTGTG 6451 CCCTGTTTGG CTAGCTAGGA GCACACATAC ATAGAAATTA AATGAAACAG 6501 ACCTTCAGCA AGGGGACAGA GGACAGAATT AACCTTGCCC AGACACTGGA 6551 AACCCATGTA TGAACACTCA CATGTTTGGG AAGGGGGAAG GGCACATGTA 6601 AATGAGGACT CTTCCTCATT CTATGGGGCA CTCTGGCCCT GCCCCTCTCA 6651 GCTACTCATC CATCCAACAC ACCTTTCTAA GTACCTCTCT CTGCCTACAC 6701 TCTGAAGGGG TTCAGGAGTA ACTAACACAG CATCCCTTCC CTCAAATGAC 6751 TGACAATCCC TTTGTCCTGC TTTGTTTTTC TTTCCAGTCA GTACTGGGAA 6801 AGTGGGGAAG GACAGTCATG GAGAAACTAC ATAAGGAAGC ACCTTGCCCT 6851 TCTGCCTCTT GAGAATGTTG ATGAGTATCA AATCTTTCAA ACTTTGGAGG 6901 TTTGAGTAGG GGTGAGACTC AGTAATGTCC CTTCCAATGA CATGAACTTG 6951 CTCACTCATC CCTGGGGGCC AAATTGAACA ATCAAAGGCA GGCATAATCC 7001 AGCTATGAAT TCTAGGATCG ATCCAGACAT GATAAGATAC ATTGATGAGT 7051 TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA 7101 ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA 7151 AGTTAACAAC AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG 7201 TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTATGGCT 7251 GATTATGATC TCTAGTCAAG GCACTATACA TCAAATATTC CTTATTAACC 7301 CCTTTACAAA TTAAAAAGCT AAAGGTACAC AATTTTTGAG CATAGTTATT 7351 AATAGCAGAC ACTCTATGCC TGTGTGGAGT AAGAAAAAAC AGTATGTTAT 7401 GATTATAACT GTTATGCCTA CTTATAAAGG TTACAGAATA TTTTTCCATA 7451 ATTTTCTTGT ATAGCAGTGC AGCTTTTTCC TTTGTGGTGT AAATAGCAAA 7501 GCAAGCAAGA GTTCTATTAC TAAACACAGC ATGACTCAAA AAACTTAGCA 7551 ATTCTGAAGG AAAGTCCTTG GGGTCTTCTA CCTTTCTCTT CTTTTTTGGA 7601 GGAGTAGAAT GTTGAGAGTC AGCAGTAGCC TCATCATCAC TAGATGGCAT 7651 TTCTTCTGAG CAAAACAGGT TTTCCTCATT AAAGGCATTC CACCACTGCT 7701 CCCATTCATC AGTTCCATAG GTTGGAATCT AAAATACACA AACAATTAGA 7751 ATCAGTAGTT TAACACATTA TACACTTAAA AATTTTATAT TTACCTTAGA 7801 GCTTTAAATC TCTGTAGGTA GTTTGTCCAA TTATGTCACA CCACAGAAGT 7851 AAGGTTCCTT CACAAAGATC CGGGACCAAA GCGGCCATCG TGCCTCCCCA 7901 CTCCTGCAGT TCGGGGGCAT GGATGCGCGG ATAGCCGCTG CTGGTTTCCT 7951 GGATGCCGAC GGATTTGCAC TGCCGGTAGA ACTCCGCGAG GTCGTCCAGC 8001 CTCAGGCAGC AGCTGAACCA ACTCGCGAGG GGATCGAGCC CGGGGTGGGC 8051 GAAGAACTCC AGCATGAGAT CCCCGCGCTG GAGGATCATC CAGCCGGCGT 8101 CCCGGAAAAC GATTCCGAAG CCCAACCTTT AC TAGAAGGC GGCGGTGGAA 8151 TCGAAATCTC GTGATGGCAG GTTGGGCGTC GCTTGGTCGG TCATTTCGAA 8201 CCCCAGAGTC CCGCTCAGAA GAACTCGTCA AGAAGGCGAT AGAAGGCGAT 8251 GCGCTGCGAA TCGGGAGCGG CGATACCGTA AAGCACGAGG AAGCGGTCAG 8301 CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC CAACGCTATG 8351 TCCTGATAGC GGTCCGCCAC ACCCAGCCGG CCACAGTCGA TGAATCCAGA 8401 AAAGCGGCCA TTTTCCACCA TGATATTCGG CAAGCAGGCA TCGCCATGGG 8451 TCACGACGAG ATCCTCGCCG TCGGGCATGC GCGCCTTGAG CCTGGCGAAC 8501 AGTTCGGCTG GCGCGAGCCC CTGATGCTCT TCGTCCAGAT CATCCTGATC 8551 GACAAGACCG GCTTCCATCC GAGTACGTGC TCGCTCGATG CGATGTTTCG 8601 CTTGGTGGTC GAATGGGCAG GTAGCCGGAT CAAGCGTATG CAGCCGCCGC 8651 ATTGCATCAG CCATGATGGA TACTTTCTCG GCAGGAGCAA GGTGAGATGA 8701 CAGGAGATCC TGCCCCGGCA CTTCGCCCAA TAGCAGCCAG TCCCTTCCCG 8751 CTTCAGTGAC AACGTCGAGC ACAGCTGCGC AAGGAACGCC CGTCGTGGCC 8801 AGCCACGATA GCCGCGCTGC CTCGTCCTGC AGTTCATTCA GGGCACCGGA 8851 CAGGTCGGTC TTGACAAAAA GAACCGGGCG CCCCTGCGCT GACAGCCGGA 8901 ACACGGCGGC ATCAGAGCAG CCGATTGTCT GTTGTGCCCA GTCATAGCCG 8951 AATAGCCTCT CCAC CCAAGC GGCCGGAGAA CCTGCGTGCA ATCCATCTTG 9001 TTCAATCATG CGAAACGATC CTCATCCTGT CTCTTGATCA GATCTTGATC 9051 CCCTGCGCCA TCAGATCCTT GGCGGCAAGA AAGCCATCCA GTTTACTTTG 9101 CAGGGCTTCC CAACCTTACC AGAGGGCGCC CCAGCTGGCA ATTCCGGTTC 9151 GCTTGCTGTC CATAAAACCG CCCAGTCTAG CTATCGCCAT GTAAGCCCAC 9201 TGCAAGCTAC CTGCTTTCTC TTTGCGCTTG CGTTTTTCCCT TGTCCAGATA 9251 GCCCAGTAGC TGACATTCAT CCGGGGTCAG CACCGTTTCT GCGGACTGGC 9301 TTTCTACGTG TTCCGCTTCC TTTAGCAGCC CTTGCGCCCT GAGTGCTTGC 9351 GGCAGCGTGA AG SEQ ID NO: 31: Part of the humanized heavy chain amino acid sequence designated VHE-N73D EVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TADKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS SEQ ID NO: 32: Part of the humanized heavy chain amino acid sequence designated VHQ-N73D QVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TADKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS SEQ ID NO: 33: Nucleotide sequence encoding amino acid sequence SEQ ID NO: 8 GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA CTCTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA CCAGGTCAGGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC GGCCAGGGGACCAAGCTTGAAATCAAA SEQ ID NO: 34: Nucleotide sequence encoding the amino acid sequence SEQ ID NO: 31 GAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAGACAAATCCATCAGCACA GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC CTC A SEQ ID NO: 35 Nucleotide sequence encoding the amino acid sequence SEQ ID NO: 32 CAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAGACAAATCCATCAGCACA GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA SEQ ID No 36 Nucleotide sequence of expression vector LCVLISp20 1 CTAGAGTCCT AGAGAGGTCT GGTGGAGCCT GCAAAAGTCC AGCTTTCAAA 51 GGAACACAGA AGTATGTGTA TGGAATATTA GAAGATGTTG CTTTTACTCT 101 TAAGTTGGTT CCTAGGAAAA ATAGTTAAAT ACTGTGACTT TAAAATGTGA 151 GAGGGTTTTC AAGTACTCAT TTTTTTAAAT GTCCAAAATT TTTGTCAATC 201 AATTTGAGGT CTTGTTTGTG TAGAACTGAC ATTACTTAAA GTTTAACCGA 251 GGAATGGGAG TGAGGCTCTC TCATACCCTA TTCAGAACTG ACTTTTAACA 301 ATAATAAATT AAGTTTAAAA TATTTTTAAA TGAATTGAGC AATGTTGAGT 351 TGGAGTCAAG ATGGCCGATC AGAACCAGAA CACCTGCAGC AGCTGGCAGG 401 AAGCAGGTCA TGTGGCAAGG CTATTTGGGG AAGGGAAAAT AAAACCACTA 451 GGTAAACTTG TAGCTGTGGT TTGAAGAAGT GGTTTTGAAA CACTCTGTCC 501 AGCCCCACCA AACCGAAAGT CCAGGCTGAG CAAAACACCA CCTGGGTAAT 551 TTGCATTTCT AAAATAAGTT GAGGATTCAG CCGAAACTGG AGAGGTCCTC 601 TTTTAACTTA TTGAGTTCAA CCTTTTAATT TTAGCTTGAG TAGTTCTAGT 651 TTCCCCAAAC TTAAGTTTAT CGACTTCTAA AATGTATTTA GAACTCATTT 701 TCAAAATTAG GTTATGTAAG AAATTGAAGG ACTTTAGTGT CTTTAATTTC 751 TAATATATTT AGAAAACTTC TTAAAATTAC TCTATTATTC TTCCCTCTGA 801 TTATTGGTCT CCATTCAATT CTTTTCCAAT ACCCGAAGCA TTTACAGTGA 851 CTTTGTTCAT GATCTTTTTT AGTTGTTTGT TTTGCCTTAC TATTAAGACT 901 TTGACATTCT GGTCAAAACG GCTTCACAAA TCTTTTTTCAA GACCACTTTC 951 TGAGTATTCA TTTTAGGAGA AATACTTTTT TTTTAAATGA ATGCAATTAT 1001 CTAGGACCTG CAGGCATGCT GTTTTCTGTC TGTCCCTAAC ATGCCCTGTG 1051 ATTATCCGCA AACAACACAC CCAAGGGCAG AACTTTGTTA CTTAAACACC 1101 ATCCTGTTTG CTTCTTTCCT CAGGAACTGT GGCTGCACCA TCTGTCTTCA 1151 TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG 1201 TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT 1251 GGATAACGCC CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGG 1301 ACAGCAAGGA CAGCACCTAC AGCCTCAGCA GCACCCTGAC GCTGAGCAAA 1351 GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA CCCATCAGGG 1401 CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAGAGGG 1451 AGAAGTGCCC CCACCTGCTC CTCAGTTCCA GCCTGACCCC CTCCCATCCT 1501 TTGGCCTCTG ACCCTTTTTC CACAGGGGAC CTACCCCTAT TGCGGTCCTC 1551 CAGCTCATCT TTCACCTCAC CCCCCTCCTC CTCCTTGGCT TTAATTATGC 1601 TAATGTTGGA GGAGAATGAA TAAATAAAGT GAATCTTTGC ACCTGTGGTT 1651 TCTCTCTTTC CTCATTTAAT AATTATTATC TGTTGTTTTA CCAACTACTC 1701 AATTTCTCTT ATAAGGGACT AAATATGTAG TCATCCTAAG GCGGGATATC 1751 GAGATCTGAA GCTGATCCAG ACATGATAAG ATACATTGAT GAGTTTGGAC 1801 AAACCACAAC TAGAATGCAG TGAAAAAAAT GCTTTATTTG TGAAATTTGT 1851 GATGCTATTG CTTTATTTGT AACCATTATA AGCTGCAATA AACAAGTTAA 1901 CAACAACAAT TGCATTCATT TTATGTTTCA GGTTCAGGGG GAGGTGTGGG 1951 AGGTTTTTTA AAGCAAGTAA AACCTCTACA AATGTGGTAT GGCTGATTAT 2001 GATCTCTAGT CAAGGCACTA TACATCAAAT ATTCCTTATT AACCCCTTTA 2051 CAAATTAAAA AGCTAAAGGT ACACAATTTT TGAGCATAGT TATTAATAGC 2101 AGACACTCTA TGCCTGTGTG GAGTAAGAAA AAACAGTATG TTATGATTAT 2151 AACTGTTATG CCTACTTATA AAGGTTACAG AATATTTTTC CATAATTTTC 2201 TTGTATAGCA GTGCAGCTTT TTCCTTTGTG GT GTAAATAG CAAAGCAAGC 2251 AAGAGTTCTA TTACTAAACA CAGCATGACT CAAAAAACTT AGCAATTCTG 2301 AAGGAAAGTC CTTGGGGTCT TCTACCTTTC TCTTCTTTTT TGGAGGAGTA 2351 GAATGTTGAG AGTCAGCAGT AGCCTCATCA TCACTAGATG GCATTTCTTC 2401 TGAGCAAAAC AGGTTTTCCT CATTAAAGGC ATTCCACCAC TGCTCCCATT 2451 CATCAGTTCC ATAGGTTGGA ATCTAAAATA CACAAACAAT TAGAATCAGT 2501 AGTTTAACAC ATTATACACT TAAAAATTTT ATATTTACCT TAGAGCTTTA 2551 AATCTCTGTA GGTAGTTTGT CCAATTATGT CACACCACAG AAGTAAGGTT 2601 CCTTCACAAA GATCCGGACC AAAGCGGCCA TCGTGCCTCC CCACTCCTGC 2651 AGTTCGGGGG CATGGATGCG CGGATAGCCG CTGCTGGTTT CCTGGATGCC 2701 GACGGATTTG CACTGCCGGT AGAACTCCGC GAGGTCGTCC AGCCTCAGGC 2751 AGCAGCTGAA CCAACTCGCG AGGGGATCGA GCATCCCCCA TGGTCTTATA 2801 AAAATGCATA GCTTTAGGAG GGGAGCAGAG AACTTGAAAG CATCTTCCTG 2851 TTAGTCTTTC TTCTCGTAGA CTTCAAACTT ATACTTGATG CCTTTTTCCT 2901 CCTGGACCTC AGAGAGGACG CCTGGGTATT CTGGGAGAAG TTTATATTTC 2951 CCCAAATCAA TTTCTGGGAA AAACGTGTCA CTTTCAAATT CCTGCATGAT 3001 CCTTGTCACA AAGAGTCTGA GGTGGCCTGG TTGATTCATG GCTTCCTGGT 3051 AAACAGAACT GCCTCCGACT ATCCAAACCA TGTCTACTTT ACTTGCCAAT 3101 TCCGGTTGTT CAATAAGTCT TAAGGCATCA TCCAAACTTT TGGCAAGAAA 3151 ATGAGCTCCT CGTGGTGGTT CTTTGAGTTC TCTACTGAGA ACTATATTAA 3201 TTCTGTCCTT TAAAGGTCGA TTCTTCTCAG GAATGGAGAA CCAGGTTTTC 3251 CTACCCATAA TCACCAGATT CTGTTTACCT TCCACTGAAG AGGTTGTGGT 3301 CATTCTTTGG AAGTACTTGA ACTCGTTCCT GAGCGGAGGC CAGGGTCGGTTCTT GCCAATCCCC ATATTTTGGG ACACGGCGAC GATGCAGTTC 3401 AATGGTCGAA CCATGATGGC AGCGGGGATA AAATCCTACC AGCCTTCACG 3451 CTAGGATTGC CGTCAAGTTT GGGGGTACCG AGCTCGAATT AGCTTTTTGC 3501 AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC ACTACTTCTG GAATAGCTCA 3551 GAGGGCCCCAG GCGGCCTCGG CCTCTGCATA AATAAAAAAA ATTAGTCAGC 3601 CATGGGGCGG AGAATGGGCG GAACTGGGCG GAGTTAGGGG CGGGATGGGC 3651 GGAGTTAGGG GCGGGACTAT GGTTGCTGAC TAATTGAGAT GCATGCTTTG 3701 CATACTTCTG CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CTGGTTGCTG 3751 ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 3801 GACTTTCCAC ACCCTAACTG ACACACATTC CACAGCTGCC TCGCGCGTTT 3851 CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 3901 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG 3951 TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG_4001_CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT 4051 ACTGAGAGTG CACCATATGC GGCCGCATAT GCGGTGTGAA ATACCGCACA 4101 GATGCGTAAG GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC 4151 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA 4201 CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 4251 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC 4301 GCGTTGCTGG CGTTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 4351 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 4401 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 4451 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC 4501 GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC 4551 GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC 4601 GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 4651 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 4701 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 4751 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG 4801 AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG 4851 GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT 4901 CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 4951 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 5001 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 5051 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC 5101 TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG 5151 TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT 5201 GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT 5251 AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 5301 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 5351 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTG CAGGCATCGT 5401 GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 5451 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC 5501 TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC 5551 ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 5601 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 5651 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA CACGGGATAA 5701 TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT 5751 CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG 5801 ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 5851 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 5901 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 5951 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT 6001 TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC 6051 GAAAAGTGCC ACCTGACGTC TAAGAAACCA TTATTATCAT GACATTAACC 6101 TATAAAAATA GGCGTATCAC GAGGCCCTTT CGTCTTCAAG AATTCAGCTG 6151 CTCGAGGAAG AGCTCAAACC CATGCTACTC TCTGGCTTGA TGGAAGCAAC 6201 GCTTTCATAG CTGAGCTGTC ATAAATAATA AA GAGATTTT TTTATTAATA 6251 TTGAAAAGAT GGGTTATTTA TGTAAGACTC TGTCTTCATT TTAAAAACCA 6301 CACCTTCCAG TAGTATTCTG TTACTGTTCT GGCAATCACT GTGATCAAGA 6351 AGCTACACGG TGAGTTGTGC TTCTCAGTCC TAAGGGATAC ATCTACAAGA 6401 GGCTCCCATA CTCGAAGCTC AGGAAACATT GTAGAAAAGG AGGCAAAAGA 6451 CTGACAGAGC CAGAGGACCA AGAAATTTGT TGTGAGGTTG TGTCTCCTAC 6501 TAACAATATA AGCAATATCT ATAAATTGTT GATATCATGG CTACTAAAAT 6551 GTGAGTTGAA CGAGGAGGAC ACAAATGAAC ATGACAATCA GAATGAGGCC 6601 TCTCACCTGC AAAAAACACT ATAGAGAAGC AGATAAAGCT GTCAGCAGAAGCACC TCCTTATAGA AGAAGCCTAC CAGGTTTGAT ATATCAGCCT 6701 TGAAAACCTA CATAGTATTT ACATTATATC GAGTCTATGA GACATATTTA 6751 GTAATGCATA TGTATGTGTG TGTGTGCATG TATGTGTGTA AATACATATG 6801 TTCATAGAAA AATGTGTAAA AAGAGATCAT GAATTTAAGA GAGAACTGGG 6851 ACAATTTTTT TCAGGGAGTT GTAATCAGGA AAGTTAAGGG AAAA? TGTTG 6901 TAATTAAAAT TCAGGCTCAG AAACAAACAA AGGAAAAGAA AAAAAAACAA 6951 CAACAACAAC AAAAAAACAA AACAAAGGAG AAGCTGTATG GCCACAATAG_7001_CATCTACAGC TAACTGTGAA AGGATAATGG AACAAGTTAT GTACTGCCTA 7051 GAGCAGTATG ATGCCTAAAT CATCTCGACA TGGAGGAAAA TAGAACAAAG 7101 ACACTCTACA TAGACTATGA TAGAAATCAA AATAAGGTGT AAGACATAGA 7151 ACATTAGTTT TGTTTGTTGT TCAAAGAGAC TCACATTCCC ACAAAAAAAT 7201 CTGTGGGATT TTACAGGTCT GCAATAAGCT GCTGACCTGA TGATTTCTGC 7251 AGCTGTGCCT ACCCTTTGCT GATTTGCATG TACCCAAAGC ATAGCTTACT 7301 GACATGAGGA TTTCTTCATA GTCAGGTCAC ACCCTTTGCT GGAGTCAGAA 7351 TCACACTGAT CACACACAGT CATGAGTGTG CTCACTCAGG TCCTGGCGTT 7401 GCTGCTGCTG TGGCTTACAG GTAATGAAGA CAGCACTAGA ATTTTATTGA 7451 GCTTCCTGTA CACTGTGCTG CTTGTCTCTG TGAAAATTCT CTTGTGAATT 7501 AATCATGTGG GGATCTGTTT TCAATTTTTC AATTGTAGGT ACGCGTTGTG 7551 ACATTCTGCT GACCCAGTCT CCAGCCACCC TGTCTCTGAG TCCAGGAGAA 7601 AGAGCCACTC TCTCCTGCAG GGCCAGTCAG AACATTGGCA CAAGCATACA 7651 GTGGTATCAA CAAAAACCAG GTCAGGCTCC AAGGCTTCTC ATAAGGTCTT 7701 CTTCTGAGTC TATCTCTGGG ATCCCTTCCA GGTTTAGTGG CAGTGGATCA 7751 GGGACAGATT TTACTCTTAC CATCAGCAGT CTGGAGCCTG AAGATTTTGC 7801 AGTGTATTAC TGTCAACAAA GTAATACCTG GCCATTCACG TTCGGCCAGG 7851 GGACCAAGCT TGAAATCAAA CGTAAGTAGA ATCCAAAGTC TCTTTCTTCC 7901 GTTGTCTATG TCTGTGGCTT CTATGTCTAA AAATGATGTA TAAAATCTTA 7951 CTCTGAAACC AGATTCTGGC ACTCTCCAAG GCAAAGATAC AGAGTAACTC 8001 CGTAAGCAAA GCTGGGAATA GGCTAGACAT GTTCTCTGGA GAATGAATGC 8051 CAGTGTAATA ATTAACACAA GTGATAGTTT CAGAAATGCT CTAGTT SEQ ID NO: 37 Nucleotide sequence of expression vector HCVHEN73DSp20 1 ctagagaggt ctggtggagc ctgcaaaagt ccagctttca aaggaacaca gaagtatgtg 61 tatggaatat tagaagatgt tgcttttact cttaagttgg ttcctaggaa aaatagtaa 121 atactgtgac tttaaaatgt gagagggttt tcaagtactc atttttttaa atgtccaaaa 181 tttttgtcaa tcaatttgag gtcttgttg tgtagaactg acattactta aagtttaacc 241 gaggaatggg agtgaggctc tctcataccc tattcagaac tgacttttaa caataataaa 301 ttaagtttaa aatattttta aatgaattga gcaatgttga gttggagtca agatggccga 361 tcagaaccag aacacctgca gcagctggca ggaagcaggt catgtggcaa ggctatttgg 421 ggaagggaaa ataaaaccac taggtaaact tgtagctgtg gtttgaagaa gtggttttga 481 aacactctgt ccagccccac caaaccgaaa gtccaggctg agcaaaacac cacctggga 541 atttgcattt caataataag ttgaggattc agccgaaact ggagaggtcc tcttttaact 601 tattgagttc aaccttttaa ttttagcttg agtagttcta gtttccccaa acttaagttt 661 atcgacttct aaaatgtatt taagctttct ggggcaggcc aggcctgacc ttggctttgg 721 ggcagggagg gggctaaggt gaggcaggtg gcgccagcca ggggcacagc caatgcccat 781 gagcccagac actggacgct gaacctcgcg gacagttaag aacccagggg cctctgcgcc 841 ctgggcccag ctctgtccca caccgcggtc acatggcacc acctctcttg cagcctccac 901 caagggccca tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc 961 ggccctgggc tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc 1021 aggcgccctg accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta 1081 ctccctcagc agcgtggtga ccgtgccctc cagcagcttg ggcacccaga cctacatctg 1141 cacaagccca caacgtgaat gcaacaccaa ggggcacagg agagttggtg agaggccagc 1201 acagggaggg agggtgtctg ctggaagcca ggctcagcgc tcctgcctgg acgcatcccg 1261 gctatgcagt cccagtccag ggcagcaagg caggccccgt ctgcctcttc acccggaggc 1321 ctctgtcccc cccactcatg ctcagggaga gggtcttctg gctttttccc caggctctgg 1381 gcaggcacag gctaggtgcc cctaacccag gccctgcaca caagggccca ggccctgggc 1441 tcagacctgc caagagccat atccgggagg accctgcccc tgacctaagc ccaccccaaa 1501 ggccaaactc tccactccct cagcacgtac accttctctc ctcccagatt 'ccagtaactc 1561 ccaatcttct ctctgcagag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc 1621 caggtaagcc agcccaggcc tcgccctcca gctcaaggcg ggacaggtgc cctagagtag 1681 cctgcatcca gggacaggcc ccagccgggt gctgacacgt ccacctccat ctcttcctca 1741 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 1801 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 1861 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1921 agcagtacaa ccgcgggagg cgtgtggtca cagcacgtac gcgtcctcac cgtcctgcac 1981caggactggc tgaatggcaa ggagtacaag tgcaaggtct cccatcgaga aaaccatctc ccacgaagac 2041 cccccatccc caaagccaaa 2101 ggtgggaccc gtggggtgcg agggccacat ggacaggtgc cggctcggcc caccctctgc cctgagagtg accgctgtac caacctctgt 2161 cccagcatgg cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat 2221 gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc 2281 cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct 2341 ggactccgac ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca 2401 gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca 2461 gaagagcctc tccctgtccc cgggtaaatg agtgcgacgg ccggcaagcc cccgctcccc 2521 gggctctcgc ggtcgcacga ggatgcttgg cacgtacccc gtctacatac ttcccaggca 2581 aaataaagca cccagcatgg cccaccactg ccctgggccc ctgcgagact gtgatggttc 2641 tttccacggg tcaggccgag tctgaggcct gagtggcatg agggaggcag agcgggtccc 2701 cactggccca actgtcccca ggctgtgcag gtgtgcctgg gccgcctagg gtggggctca 2761 gccctcggca gccaggggct gggtggggga tttccacggg tggccctccc tccagcagca 2821 gctgccctgg gctgggccac gaagagcctt agggccacat ggggacagac acacagcccc 2881 tgcctctgta ggagactgtc ctgtcctgtg agcgccctgt cctccgaccc cgatgcccac 2941 tcgggggcat gcctagtcca tgcgcgtagg gacaggccct ccctcaccca tctaccccca 3001 cggcactaac ccctgggccc cctgcccagc ctcgcacccg catggggaca caaccgactc 3061 cgggtaaatg cactctcggg ccctgtggag ggactggtgc agatgcccac acacacactc 3121 agcccaggcc cgttcaacaa accccgcact gaggttggtc gagcgggagt gcggccagag 3181 cctgcctcgg ccgtcaggga ggactcccgg gctcactcga aggaggtgcc accatt TCAG 3241 ctttggtagc ttttcttctt cttttaaatt ttctaaagct cattaattgt ctttgatgtt 3301 tgacaataaa tcttttgtga atatcctttt taagtcttgt acttcgtgat gggagccgcc 3361 ttcctgtgtc cacgcgcctc ctgcccccgg tgggaagcac ggtcaggagg aggctggtcc 3421 agctgcacct cgggggctcc ctgcactcgc cacgcgcctc ctgcagccac acgcattgcc 3481 cgagcgaccc tccctggccc ctgtcactac atggacccct ggggcttctc ctcttttcta 3541 catggatgca gtttctcctc ctgctgggca cggtgctgcc tgccctggtc actctgcggg 3601 tccagggaaa ggacagggcc gctgggtcga ggctgggagc tggctcaggc ttcccaggca 3661 gagccacagg gagggccttc cagaaccaac catggtccga agcgagaggt gggtgtcaga 3721 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 3781 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta ttttaaagca ttataagctg 3841 gttaacaaca caataaacaa acaattgcat tcattttatg tttcaggttc agggggaggt 3901 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatct 3961 ctagtcaagg cactatacat caaatattcc ttattaaccc ctttacaaat taaaaagcta 4021 aaggtacaca atttttgagc atagcagaca atagttatta ctctatgcct gtgtggagta 4 081 agaaaaaaca gtatgttatg attataactg ttatgcctac ttataaaggt tacagaatat 4141 ttttttggaa ttttccataa tagcagtgca gctttttcct ttgtggtgta aatagcaaag 4201 ttctattact caagcaagag tgactcaaaa aaacacagca aacttagcaa ttctgaagga 4261 aagtccttgg ggtcttctac ctttctcttc ttttttggag gagtagaatg ttgagagtca 4321 gcagtagcct catcatcact agatggcatt tcttctgagc aaaacaggtt ttcctcatta 4381 aaggcattcc accactgctc ccattcatca gttccatagg ttggaatcta aaatacacaa 4441 acaattagaa tcagtagttt aacacattat acacttaaaa attttatatt taccttagag 4501 ctttaaatct ctgtaggtag tttgtccaat tatgtcacac cacagaagta aggttccttc 4561 acaaagatcc ggaccaaagc ggccatcgtg cetccccact cctgcagttc gggggcatgg 4621 atgcgcggat agccgctgct ggtttcctgg atgccgacgg attataactg ccggtagaac 4681 tccgcgaggt cgtccagcct caggcagcag ctgaaccaac tcgcgagggg atcgagcccg 4741 gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc 4801 ttccgaagcc cggaaaacga caacctttca tagaaggcgg cggtggaatc gaaatctcgt 4861 gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga 4921 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 4981 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 5041 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 5101 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 5161 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 5221 gtccagatca gatgctcttc tcctgatcga ttccatccga caagaccggc gtacgtgctc 5281 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agcgtatgca agccggatca 5341 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 5401 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 5461 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 5521 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 5581 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 5641 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 5701 caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 5761 agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 5821 agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagct 5881 to tcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg ttttcccttg 5941 tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt 6001 tctacgtgtt ccgcttcctt tagcagccct tgcgccctga gtgcttgcgg cagcgtgaag 6061 ctttttgcaa aagcctaggc ctccaaaaaa tacttctgga gcctcctcac atagctcaga 6121 ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga 6181 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt 6241 ttgagatgca tgctgactaa tgctttgcat acttctgcct gctggggagc ctggggactt 6301 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga 6361 gcctggggac tttccacacc ctaactgaca cacattccac agctgcctcg cgcgtttcgg 6421 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 6481 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 6541 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg 6601 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc 6661 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc 6721 tcggtcg ttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 6781 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 6841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 6901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 6961 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 7021 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 7081 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 7141 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 7201 gacttatcgc cactggcagc agccactggt aaagggaata gcagagcgag gtatgtaggc 7261 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 7321 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 7381 ggagaaaaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 7441 gatctcaaga agaaaaaaag agatcctttg atcttttcta cggggtctga cgctcagtgg 7501 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 7561 atccttttaa at taaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 7621 tctgacagtt accaatgctt aatgattgag gcacctatct cagcgatctg tctatttcgt 7681 tcatccatag ttgcctgact ccccgtcgtg tagataacta gggcttacca cgatacggga 7741 gtgctgcaat tctggcccca gataccgcga gacccacgct caccggctcc agatttatca 7801 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 7861 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 7921 ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg 7981 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 8041 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 8101 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 8161 tgcttttctg tgactggtga gtactcaacc aagtcattct ggaaatgttg tatgcggcga 8221 ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta 8281 tcattggaaa aaagtgctca acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 8341 gttcgatgta ttgagatcca acccactcgt gcacccaact gatcttcagc atcttttact 8401 ttcaccagcg tttctgggtg agaaaaaaaa atgccgcaaa gtaaggagaa aaagggaata 8461 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 8521 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 8581 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 8641 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc 8701 'gagctcggta cccatcagcc aaaaagcatg cctgccacac aacatcaatt tctggaaaac 8761 attatttcta gctacactta ttttttgttt gtagaacagc atcacctata gccaaaaaga 8821 agaaaaaaaa gtggcatcta ggagaaaaaa atcacaaaga aatgattgag aggcataata 8881 aaaattatca aaaaattatg agttttacga ttttattttt ttccaagttg aaatcatagg 8941 cacagtgaca gtggctttaa aggaatgtgc atgctgccat tatggtgctc tgcctaaaat 9001 ggttggagcc ttgtcatgct acagagaaac tgtcatacag cagggggtgc caaatttcca 9061 tattttttta tatcattgag CAGG tgcaca gaagaccaga aagcactttc tatcaggctg 9121 gccttcctct tcctttccag tatgaagcaa aaactgccaa tgaaactagc aattgttaaa 9181 ttcctttttc aaacagtatt tgtgctatca gaacatagtg cattcaaaag tctagcctga 9241 gagaacaacc cagttttatt cattcctcct actacctctc tcattcccac tgtttgtgtt 9301 ctccctccca ttttaattgt ctatctagtc caaactaagc gtccacatta acacgatcca 9361 aacaacatgt ttttatttta agtcaaatac aagacacctt taatatcagc ccttgttcat 9421 aatcgtgctt ctagtgactt aatgtacatg tcacactgta ctgttgggtt ttgtgtctca 9481 tcatgaacaa tgttgtgaag gtattaagtg gagagtaagc agaattagat tcctctaatg 9541 atgcacaccc acactaagag cagaaataat attaaaaata gaaaaaaaag ttttaattga 9601 gatttcaaat acccaggtat gagctgcagt ttcttcaagt taaagcatcg aggttgtcag 9661 ttacactatt acaggaaaca tatgcagagt ttttatttta gtatattagt tttcacatat 9721 ggggaattgc tattaaacta tatttttttt tcaaatgctt accattgtaa atgagtttgt 9781 gactttgtgt aggtgagtgc acatgactct ggatgcctaa gaggactgaa gaagttggag 9841 ttataggtag ttttatttta cttgactgtt cagtgctaaa aatacaactg aggtccttta 9901 aactgctgtt catgaacttc ttaattgata tatctcatga gatctctaaa ctatttttatacgt ttcaccattt tcactgtaac gatttttatg ttttatatta atgtaactat 10021 atgacacttc ccaaaatccc catattcaca attgaactgt ttcaaagttt taccttgact 10081 tatgggaaat gaaaacccac attttataat tttaaaatga aatgtttatt ttttattttt 10141 caaggaaaga gcaaatttca ttagtcactg gtgtgtgaga gcagaggagc ataagagttc 10201 tccattatga aggaatagaa ttctggaggc aaggaagaac tgatgccaag gtttcagtat 10261 tccactggaa aagagcagta aggataaagt cactacatct gagcacagag caggacatct 10321 acataatgag tggtcactaa tgggccactg ttacactgtt atatgtataa ggctcaagaa 10381 tgagcactga ggctgtaagg tgtatgggtg aggacatcag gatgtaaacc cagctcaggt 10441 gaggacagca agaggactca cagtcagcat gaactaataa acatcagata agataaggca 10501 caagctcagc tatatagggt aagggatctt tgtaaatctg attgtgcatc cagtctagtt 10561 caatgtgact taggaagccc agtcatatgc aaatctagag aagactttag agtagaaatc 10621 tgaggctcac ctcacatacc agcaagcgag tgaccagtta gtattaagtc accacttctt 10681 agacatcatg gcttgggtgt ggaccttgcc attcctgatg gcagctgccc aaagtaagac 10741 agagttccaa attaaaaata agcagttcca ggggaattga tgaatactca ccttcctgtg 10801 ttcttttcac aggtgtccag gcagaggtgc agctggtgga gtcaggagcc gaagtgaaaa 10861 agcctggggc tgttgtgaag gtgtcctgca aggcctctgg atacacattc actaattata 10921 ttatccactg ggtgaagcag gagcctggtc agggccttga atggattgga tat tttaatc 10981 cttacaatca tggtactaag tacaatgaga agttcaaagg cagggccaca ctaactgcag 11041 acaaatccat cagcacagcc tacatggagc tcagcagcct gcgctctgag gacactgcgg 11101 tctactactg tgcaagatca cctggtttga ggaccctatg cacctggggc caagggacca 11161 cggtcaccgt ctcctcaggt aagaatggcc actctagggc ctttgttttc tgctgctgcc 11221 tgtgggattt catgagcatt gcaaagttgt cctcgggaca tgttccgagg ggacctgggc 11281 ggactggcca ggaggggacg ggcactgggg tgccttgagg atctgggagc ctctgtggat 11341 tttccgatgc ctttggaaaa tgggactgag gttgggtgcg tctgagacag taactcagcc 11401 tgggggcttg gtgaagatcg ccgcacagca gcgagtccgt gaaatatctt atttagactt 11461 gtgaggtgcg ctgtgtgtca atttacatct taaatccttt attggctgga aagagaattg 11521 ttggagtggg tgaatccagc caggagggac gcggggggat cea SECIDNO: 38 S ecu nce of nuc leo t of the expression vector HCVHQN73DSp20 1 CTAGAGAGGT CTGGTGGAGC CTGCAAAAGT CCAGCITTCA AAGGAACACA GAAGTATGTG 61 TAICGAATAT TAGAAGATGT TGCITTTACR CTTAAGTTGG TTCCTAGGAA AAATAGTIAA 121 ATACTGTGAC TITAAAATGT GAGM3GGITT TCAAGTACTC ATITTTTTAA AIGTCCAAAA 181 TG? TTC? A TC? AT? GAG GTCGGGTGIG TGTAGAACGG ACATIACGGA A? GT? A? CC 241 Gagga? TGGG AGTGAGGCTC TCGCATACCC TATGCAG ? AC TGACG? TA? CA? TAATAA? 3rd? TGA? GT? A? AATAT? TTA AAIG ?? TIG? GCA? TGTGG? GTGGG? GTCA AGATGGCCGA 361 TC? GAACC? G AACACCIGCA GCAGCTGGCA GGAAGCAGGT CATGIGGCAA GGCTA? TTGG 421 GGA? GGGA? A ATA ACCAC TAGGTAAACG TGTAGCTGTG GT? GAAGAA GTGGT? GA 481 AACACICTGT CCAGCCCCAC CAAACCGA? A GTCCAGGCTG AGCAAAACAC CACCTGGGTA 541 ATTGGCATTT CTAAAATAAG TGG? GG? TGC AGCCG? AACT GGAGAGGTCC TCITTTAACG 601 TATGGAGTGC AACCT? TAA TGGTAGCGTG AGTAGTTCGA GTTTCCCCAA ACGGAAGTT 661 AT03ACTTCT AAAA1GTA? T TAAGCTTTCT GGGGCAGGCC AGGCCTGACC TIGGCITTGG 721 GGCAGGGAGG GGGCTA? GGT GAGGCAGGTG GCGCCAGCCA GGTGCAC? CC CAATGCCCAT 781 GAGCCCAGAC CTGGACGCT GAACCTCGCG GACAGTTAAG AACCCAGGGG CCTCTGCGCC 841 CTGGGCCCAG CTCIGTCCCA C? CCGCGGTC ACATGGCACC ACCTCGC? G CAGCCTCCAC 901 CA? GGGCCCA TCGGTCGTCC CCCTGGCACC CGCCTCC? AG AGCACCGCTG GGGGCACAGC 961 GGCCCTGGGC TGCCTGGTCA AGGACGACGG CCCCGAACCG GTGACGGTGT CGTGGAACGC 1021 AGGGGCCCIG ACCAGOGGCG 1GCACACCTT CCCGGC? GTC CTACAGTCCT CAGGAC1CTA 1081 CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC CAGCAGCTTG GGCACCCAGA CCTACATCTG 1141 CAACGI? A? T CACA? GCCCA GC? ACACCA? GGIGGACAAG GAGTIGGTG AGAGGCCAGC 1201 ACAGGGAGGG AGGGTGTCIG CIGGAA000A GGCTCAGGGC TCCIGCCIGG ACGCATCCCG 1261 GCTA1GCAGT CCCAGTCCAG GGCAGCAAGG CñGGCCCCGT CIGCC1CITC ACCCGGAGGC 1321 CTCTGCCCGC CCCACTCATG CTCAGGGAGA GGGTCTTCTG GCT1TTTCCC CAGGCTCTGG 1381 GCAGGCACAG GCTAGGTGCC CCTAACCCAG GCCCTGCACA CAAAGGGGCA GGTGCTGGGC 1441 TC? G? CCGGC CAAGAGCCAT ATCCGGGAGG CCCTGCCCC TGACCGAAGC CCCCCCAAA 1501 GGCCAAACTC TCCACTCCCT CAGCTCGGAC ACCTTCTCTC CTCCCAGATT CCAGTAACTC 1561 CCAATCnCT CTCTGCAGAG CCCAAATCTT GIGACAAAAC TCACACATGC CCACCGTGCC 1621 CAGGTAAGCC AGCCCAGGCC TCGCCCTCCA GCTCAAGGCG GGACAGGTGC CCTAGAGTAG_1681_ CCTGCATCCA GGGACAGGCC CCAGCCGGGT GCTGACACGT CCACCTCCAT CTCTTCCTCA 1741 GCACCTGAAC TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC 1801 CTCATGATCT CCCGGACCCC TGAGGTCAC? TGCGTGGTGG 1GGACGTGAG CCACGAAGAC 1861 CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG 1921 CCGCGGGAGG AGCAGTACAA CAGCACGTAC OGTGTGGTCA GOGTCCTCAC OGTCCTGCAC 1981 CAGGACIGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC 2041 CCCATOGAGA AAACCATCTC CAAAGCCAAA GGTGGGACCC G GGGGIGCG AGGGCCACAT 2101 GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACCGCTGTAC CAACCTCTGT 2161 CCCTACAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT 2221 GACCAAGAAC CAGGTCAGCC GACCTGCCT GGTCAAAGGC TTCTATCCCA GGGACAT03C 2281 CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACIAC AAGACCACGC CTCCCGTGCT 2341 GGACTCCGAC GGCTCCTTCT TCCTCTATAG CAAGCTCACC GTGGACAAGA GCAGGIGGCA 2401 GCAGGGGAAC GTCITCrCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA 2461 GAAGAGCCTC TCCCTGTCCC CGGGTAAATG AGTGCGAOGG CCGGCAAGCC CCCGCTCCCC 2521 GGGCTCTCGC GGTCGCACGA GGATGCTTGG CACGTACCCC GTCTACATAC TTCCCAGGCA 2581 CCCAGCATGG AAATAAAGCA CCCACCACIG CCCTGGGCCC CTGCGAGACT GIGATGGTTC 2641 TGTCCACGGG TCAGGCCGAG TCTGAGGCCT GAGTGGCATG GGGAGGCAG GCGGGTCCC 2701 ACTGTCCCCA CACIGGCCCA GGCTGTGCAG GTGTGCCTGG GCCGCCTAGG GIGGGGCTCA 2761 GCCAGGGGCT GCCCTCGGCA GGGTGGGGGA TTTGCCAGCG TGGCCCTCCC TCCAGCAGCA 2821 GCTGCCCTGG GCTGGGCCAC GAGAAGCCCT AGGAGCCCCT GGGGACAGAC ACACAGCCCC 2881 TGCCTCTGTA GGAGACTGTC CTGTCCTGTG AGCGCCCTGT CCTCCGACCC OGATGCCCAC 2941 TCGGGGGCAT GCCTAGTCCA TGCGCGTAGG GACAGGCCCT CCCTCACCCA TCTACCCCCA 3001 CGGCACTAAC CCCTGGCAGC CCIGCCCAGC CTCGCACCCG CAIGGGGACA CAACCGACTC 3061 CGGGGACATG CACTCTCGGG CCCTGTGGAG GGACTGGTGC AGATGCCCAC ACACACACTC 3121 AGCCCAGACC OGTTCAACAA ACCCCGCACT GAGGTTGGTC GAGCGGGAGT GCGGCCAGAG 3181 CCTGCCTCGG CCGTCAGGGA GGACTCCCGG GCTCACTCGA AGGAGGIGCC ACCATTTCAG 3241 CTTTGGTAGC TTTTCTTCTT CTTTTAAATT TTCTAAAGCT CATTAATTGT CTTTGATGTT 3301 TCTTTTGTGA TGACAATAAA ATATCCTTTT TAAGTCTTGT ACTTOGIGAT GGGAGCCGCC 3361 TGCCGGTGTC CACGCGCCTC CTGCCCCCGG TGGGA? GCAC GGTCAGGAGG GGCTGGTCC 3421 AGCTGCACCT QGGGGGCTCC CTGCACTCGC CCCCCGCCTC CIGCAGCCAC ACGCATTGCC 3481 GGAGGGACCC TCCCTGGCCC CTGTCACTAC ATGGACCCCT GGGGCTTCTC CTCTTTTCTA 3541 CATGGATGCA GTTTCTCCTC CTGCTGGGCA CGGIGCTGCC TGCCCTGGTC ACTCTGCGGG 3601 GGACAGGGCC TCCAGGGAAA GCTGGGTOGA GGCIGGGAGC TGGCTCAGGC TGGCCAGGCA 3661 GAGCCACAGG GAGGGCCTTC CAGAACCAAC CATGGTCCGA AGCGAGAGGT GGGTGTCAGA 3721 TCCAGACATG ATAAGATACA TTGA1GAGTT TGGACAAACC ACAACIAGAA TGCAGIGAAA 3781 AAAATGC? T ATTTGTGAAA TTTGTGATGC TATTGCTTTA TITGTAACCA TTATAAGCIG 3841 CA? TA? ACA? GTTAACAACA ACAATTGCAT TC? T? TATG TTTCAGGTTC AGGGGGAGGT 3901 GTGGGAGGTG TTTTAA? GCA AGTAAAACCT CTACAAAGGT GGTATGGCGG ATIATGATCT 3961 CTAGTCA? GG CACTATACAT CAAATATTCC TTATTAACCC CITTACAAAT TAAAAAGCTA 4021 AAGGTACACA ATTTTTGAGC ATAGTTATTA ATAGCAGACA CTCTATGCCT GTGTGGAGTA 4081 AGAAAAAACA GTATGTTATG ATTATAACTG TTATGCCTAC TTATAAAGGT TACAGAATAT 4141 TTTTCCATAA TITGCGTGTA TAGCAGTGCA GCTTTTTCCT TIGTGGTGTA A? TAGCAA? G 4201 CAAGCAAGAG TTCTATTACT AAACACAGCA TGACTCAAAA AACTTAGCAA TTCTGAAGGA 4261 AAGTCCTTGG GGTCTTCTAC CTTTCTCTTC TITTTTGGAG GAGTAGAATG TTGAGAGTCA 4321 GCAGTAGCCT CATCATCACT AGATGGCATT TCTTCTGAGC AAAACAGGTT TTCCTCATTA 4381 AAGGCATTCC ACCACIGCTC CCATTCATCA GTTCCATAGG TTGGAATCTA AAATACACAA 4441 ACAATTAGAA TCAGTAGTTT AACACATAT ACACTTAAAA ATTTTATATT TACCTTAGAG 4501 CTTTAAATCT CTGTAGGTAG TTTGTCCAAT TATGTCACAC CACAGAAGTA AGGTTCCTTC 4561 ACAAAGATCC GGACCAAAGC GGCCATCGT3 CCTCCCCACT CCIGCAG TC GGGGGCATGG 4621 ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG ATGCCGACGG ATTTGCACTG CCGGTAGAAC 4681 TCCGCGAGGT CGTCCAGCCT CAGGCAGCAG CTGAACCAAC TCGCGAGGGG ATCGAGCCCG 4741 GGGTGGGCGA AGAACTCCAG CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC 4801 CGGAAAACGA TTCCGAAGCC CAACCTTTCA TAGAAGGCGG CGGTGGAATC GAAATCTCGT 4861 GA TGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTQ3AACC CCAGAGTCCC GCTCAGAAGA 4921 ACTOGTCAAG AAGGCGATAG AAGGCGATGC GCIGCGAATC GGGAGCGGCG ATACCGTAAA 4981 GCACGAGGAA GCGGTCAGCC CATTCGCCGC CAAGCTCTTC AGCAATATCA CGGGTAGCCA 5041 ACGCTATGTC CTGATAGCGG TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA 5101 AGCGGCCATT TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC ACGACGAGAT 5161 CCTCGCCGTC GGGCAIGCGC GCCTTGAGCC TGGCGAACAG TTCGGCTGGC GG3AGCCCCT 5221 GATGCTCTTC GTCCAGATCA TCCTGATQ3A CAAGACCGGC TTCCATCCGA GTACGTGCTC 5281 GCTGGATGCG ATGTTTOGCT TGGTGGTCGA ATGGGCAGGT AGCCGGATCA AGCGTATGCA 5341 GCCGCOGCAT TGCATCAGCC ATGATGGATA CITTCTCGGC AGGAGCAAGG TGAGAIGACA 5401 GGAGATCCTG CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT TCAGTGACAA 5461 aGTOGAGCAC AGCIGCGCAA GGAACGCCCG TCGTGGCCAG CCACGATAGC CGCGCIGCCT 5521 CGTCCTGCAG TTCATTCAGG GCACCGGACA GGTCGGTCIT GACAAAAAGA ACCGGGCGCC 5581 CCTGCGCTGA CAGCCGGAAC ACGGCGGCAT CAGAGCAGCC GATTGTCTGT TGIGCCCAGT 5641 CATAGCCGAA TAGCCTCTCC ACCCAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT 5701 CAATCATG CG AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC CTGCGCCATC 5761 AGATCCITGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA GGGCTTCCCA ACCTTACCAG 5821 AGGGOGCCCC AGCIGGCAAT TCCGG TCGC TTGCTGTCCA TAAAACCGCC CAGTCTAGCT 5881 ATOGCCATGT AAGCCCACTG CAAGCTACCT GCTTTCTCTT TGCGCTTGCG TTTTCCCTTG 5941 TCCAGATAGC CCAGTAGCTG ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT 6001 TCTACGTGTT CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG CAGOGIGAAG 6061 CTTGGTGCAA AAGCCTAGGC CTCCAAA ?? A GCCTCCTCAC TACTTCGGGA ATAGCTCAGA 6121 GGCCGAGGGG GCCTCGGCCT CIGCATAAAT AAAAAAAA.TT AGTCAGCCAT GGGGCGGAGA 6181 ATGGGOGGAA CTGGGCGGAG TTAGGGGCGG GATGGGCGGA GTTAGGGGGG GGACTATGGT 6241 TGCTGACTAA TTGAGATGCA TGCITTGCAT ACITCTGCCT GCIGGGGAGC CTGGGGACTT 6301 TCCACACCTG GTTGCTGACT AATIGAGATG CATGCTTTGC ATACTTCTGC CIGCTGGGGA 6361 GCCTGGGGAC TTTCCACACC CTAACTGACA CACATTCCAC AGCTGCCTCG CGCGTTTCGG 6421 TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA 6481 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGIGTTG GCGGGTGTOG 6541 GGGCGCAGCC ATGACCCAGT CAOGTAGCGA TAGCGGAGTG TATACTGGCT TAACIATGOG 6601 GCATCAGAGC AGATTGTACT GAGAGTGCAC CATAIGCGGT GTGAAATACC GCACAGATGC 6661 GTA? GGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT CGCGCACIGA CTCGCGGCGC 6721 TCGGTCGTTC GGCIGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC 6781 ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGGTAAA? AGGCCGCGTT GCIGGCGTTT TTCCATAGGC TCCGCCCCCC IGACGAGCAT 6901 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG 6961 GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCIGCC GCTTACCGGA 7021 TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG 7081 TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT 7141 CAGCCCGACC GCIGCGCCTT ATCCGGTAAC TATOGTCTTG AGTCCAACCC GGTAAGACAC 7201 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC 7261 GGGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT 7321 GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTOSGAAAAA GAGTTGGTAG CTCTTGATCC 7381 GGCAAACAAA CCACCGCTGG TAGCGGTGGT '1'1'1'1'i'lGTTT GCAAGCAGCA GATTAG_3_CGC 7441 AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCITTTCTA CGGGGTCTGA CGCTCAGTGG 7501 A? sG? AAACT CACGTTAAGG GATTTTGGTC AIGAGATTAT CAAAAAGGAT CTTCACCTAG_7561_ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG 7621 TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTOGT 7681 TCATCCATAG T GGCCTGACT CCCCGTCGTG TAGATAACTA CGATAC3GGA GGGCTTACCA 7741 TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA 7801 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC 7861 TCCATCCAGT CTATTA? TTG TIGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT 7921 TTGCGCAACG TTGTTGCCAT TGCTGCAGGC ATOGIGGTGT CAGGCTCGTC GTTTGGTATG 7981 GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC 8041 AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG 8101 TTATCACTCA TGGTTATGGC AGCACTGCAT A? TTCTCTTA CTGTCATGCC ATCCGTAAGA 8161 TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA 8221 CCGAGTTGCT CTTGCCCGGC GTCAACAOGG GATAATACCG CGCCACATAG CAGAACTTTA 8281 AAAGTGCTCA TCATTGGAAA ACGTTCTTOG GGGCGAAAAC TCTCAAGGAT CTTACCGCIG 8341 TTGAGATCCA GTT03ATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT 8401 TTCACCAGCG TTTCIGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA 8461 AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT 8521 TATCAGGGTT ATTGTCT CAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA 8581 ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT 8641 ATCATGACAT TAACCTATAA AAATAGGCGT ATCAOGAGGC CCTTTCGTCT TCAAGAATTC GAGCTOGGTA 8701 CCCATCAGCC AAAAAGCATG CCTGCCACAC AACATCAATT TCTGGAAAAC 8761 GCTACACTTA ATTATTTCTA GTAGAACAGC TCTTTGGTTT GCCAAAAAGA ATCACCIATA 8821 GTGGCATCIA AGCACAAAAA GGAGAAAAAA ATCACAAAGA AATGATIGAG AGGCATAATA 8881 AAAATTATCA AAAAATTATG AGTTTTACGA TITCATCTTT TTCCAAGTTG AAATCATAGG 8941 GTGGCTTTAA CACAG1GACA AGGAATGTGC ATGCTGCCAT TATGGTGCTC TGCCTAAAA.T 9001 GGTTGGAGCC TTGTCA1GCT ACAGAGAAAC TGTCATACAG CAGGGGGTGC CAA? TTTCCA 9061 TATITTTTTA TATCATTGAG CAGGTGCACA GAAGACCAGA AAGCACTTTC TATCAGGCTG 9121 GCCTTCCTCT TCCITTCCAG TATGAAGCAA AAACIGCCAA TGAAACTAGC AATTGTTAAA 9181 TGCCTTTTTC AAACAGTATG TGTGCTATCA GAACATAGTG CATTCAAAAG TCGAGCCTGA 9241 GAGAACAACC CAGTTTTATT CATTCCTCCT ACTACCTCTC TCATTCCCAC TGTTTGTGTT 9301 CTCCCTCCCA TTTTAATTGT CTATCTAGTC CAAACTAAGC ACACGATCCA GTCCACATIA 9361 AACAACATGT TTTCACITTA AGTCAAATAC AAGACACCTT TAATATCAGC CCITGTTCAT 9421 AATCGTGCTT CTAGTGACTT AAIGTACATG TCACACTGTA CTGTTGGGTT TTGTGTCTCA 9481 TCATGAACAA TGTTGIGAAG GTATTAAGTG GAGAGTAAGC AGAATTAGAT TCCTCTAAIG 9541 ATGCACACCC ACACTAAGAG CAGAAATAAT ATTAAAAATA GAAAAAAAAG TTTTACATGA 9601 GATTTCAAAT ACCCAGGTAT GAGCIGCAGT TTCITCAAGT TAAAGCATCG AGGTTGTCAG 9661 TTACACTATT ACAGGAAACA TATGCAGAGT TITTATTTTA GTATATTAGT TTTCACATAT 9721 GTGGAATTAC TATTAAACTA TTCTTTCTTT TCAAATGCTT ACCATTGTAA AIGAGTTTGT 9781 GACTTTGTGT AGGTGAGTGC ACATGACTCT GGATGCCTAA GAGGACTGAA GAAGTTGGAG 9841 TTATAGGTAG TTTTATTCTA CTTGACTGTT CAGTGCTAAA AATACAACTG AGGTCCTTTA 9901 AACTGCTGTT CAIGAACTTC TTAATTGATA TATCTCATGA GATCTCTAAA CTAL'ITITAT 9961 TATGACACGT TTCACCATIT TCACTGTAAC GATITTTATG TTITATATTA ATGTAACTAT 10021 ATGACACTTC CCAAAATCCC CATATTCACA ATTGAACTGT TTCAAAGTTT TACCTTGACT 10081 TAIGGGAAAT GAAAACCCAC ATTTTATAAT TTTAAAATGA AATGTTTATT TTATATTTCT 10141 GCAAATTTCA CAAGGAA? GA TTAGTCACIG GTGTGTGAGA GCAGAGGAGC ATAAGAGTTC 10201 AGGAATAG? A TCCATTATGA TTCTGGAGGC AAGGAAGAAC TGATGCCAAG GTTTCAGTAT 10261 AAGAGCAGTA TCCACTGGAA AGGATAAAGT CACTACATCT GAGCACAGAG CAGGACATCT 10321 ACATAATGAG TGGTCACTAA TGGGCCACIG TTACACTGTT ATATGTATAA GGCTCAAGAA 10381 IGAGCACTGA GGCTGTAAGG TGTATGGGTG AGGACATCAG GATGTAAACC CAGCTCAGGT 10441 AGAGGACTCA GAGGACAGCA CAGTCAGCAT GAACTAATAA ACATCAGATA AGATAAGGCA 10501 CAAGCTCAGC TATATAGGGT AAGGGATCTT TGTAAATCTG ATTGGCATC CAGTCTAGTT 10561 C ?? TGTGACT TAGGAAGCCC AGTCATATGC AAATCTAGAG AAGAC? TAG AGTAGAATC 10621 TGAGGCTCAC CTCACATACC AGCAAGCGAG TGACCAGTTA GTCTTAAGGC ACCAC1TCTT 10681 AGACATCATG GCTTGGGTGT GGACCTTGCC ATGCCTGATG GCAGCTGCCC AAGTAAGAC 10741 ATCAGA ?? A? AGAGTTCCAA GGGGA? TTGA AGCAGTTCCA TGAATACTCA CCTTCCTGTG 10801 TCG? TCAC AGGTGTCCAG GCACAGGTGC AGCTGGTGGA GTCAGGAGCC GAAGTGA? AA 10861 AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AGGCCTCTGG ATACACATTC ACTAATTATA 10921 TTATCCACTG GGTGAAGCAG GAGCCTGGTC AGGGCCTTGA ATGGATTGGA TATTTTAATC 10981 CTTACAATCA TGGTACTAAG TACAATGAGA AGTTCAAAGG CAG000CACA CTAACTGCAG 11041 ACAAATCCAT CAGCACAGCC TACATGGAGC TCAGCAGCCT GCGCTCTGAG GACACTGCGG 11101 TCTACTACTG GCAAGATCA GGACCCTATG CCTGGTTTGA CACCTGGGGC CAAGGGACCA 11161 CGGTCACCGT CTCCTCAGGT AAGAATGGCC ACTCTAGGGC (CT 11221 TGTTTTC TGCTGCTGCC TGTGGGATTT CAGAGCATT GCAAAGTTGT CCTCGGGACA TGTTCCGAGG GGACCTGGGC 11281 GGACTGGCCA GGAGGGGACG GGCACTGGGG TGCCTTGAGG ATCTGGGAGC CTCTGTGGAT 11341 TTTCCGATGC CTTTGGAAAA IGGGACIGAG GTTGGGTGCG TCIGAGACAG TAACTCAGCC 11401 TGGGGGCTTG GTGAAGATCG CCGCACAGCA GCGAGTCCGT GAAATATCTT ATTTAGACIT 11461 GTGAGGTGCG CTGTGTGTCA ATTTACATCT TAAATCCTTT ATTGGCTGGA AAGAGAATTG 11521 TGGGAGIGGG TGAATCCAGC CAGGAGGGAC GCGGGGGGAT CCA SEQ ID NO: 39 Nucleotide sequence of expression vector LCVL2Sp20 of 1 CTAGAGTCCT AGAGAGGTCT GGTGGAGCCT GCAAAAGTCC AGCTTTCAAA 51 GGAACACAGA AGTATGTGTA TGGAATATTA GAAGATGTTG CTTTTACTCT 101 TAAGTTGGTT CCTAGGAAAA ATAGTTAAAT ACTGTGACTT TAAAATGTGA 151 GAGGGTTTTC AAGTACTCAT TTTTTTAAAT GTCCAAAATT TTTGTCAATC 201 AATTTGAGGT CTTGTTTGTG TAGAACTGAC ATTACTTAAA GTTTAACCGA 251 GGAATGGGAG TGAGGCTCTC TCATACCCTA TTCAGAACTG ACTTTTAACA 301 ATAATAAATT AAGTTTAAAA TATTTTTAAA TGAATTGAGC AATGTTGAGT 351 TGGAGTCAAG ATGGCCGATC AGAACCAGAA CACCTGCAGC AGCTGGCAGG 401 AAGCAGGTCA TGTGGCAAGG CTATTTGGGG AAGGGAAAAT AAAACCACTA 451 GGTAAACTTG TAGCTGTGGT TTGAAGAAGT GGTTTTGAAA CACTCTGTCC 501 AGCCCGACCA AACCGAAAGT CCAGGCTGAG CAAAACACCA CCTGGGTAAT 551 TTGCATTTCT AAAATAAGTT GAGGATTCAG CCGAAACTGG AGAGGTCCTC 601 TTTTAACTTA TTGAGTTCAA CCTTTTAATT TTAGCTTGAG TAGTTCTAGT 651 TTCCCCAAAC TTAAGTTTAT CGACTTCTAA AATGTATTTA GAACTCATTT 701 TCAAAATTAG GTTATGTAAG AAATTGAAGG ACTTTAGTGT CTTTAATTTC 751 TAATATATTT AGAAAACTTC TTAAAATTAC TCTATTATTC TTCCCTCTGA 801 TTATTGGTCT CCATTCAATT CTTTTCCAAT ACCCGAAGCA TTTACAGTGA 851 CTTTGTTCAT GATCTTTTTT AGTTGTTTGT TTTGCCTTAC TATTAAGACT 901 TTGACATTCT GGTCAAAACG GCTTCACAAA TCTTTTTTCAA GACCACTTTC 951 TGAGTATTCA TTTTAGGAGA AATACTTTTT TTTTAAATGA ATGCAATTAT 1001 CTAGGACCTG CAGGCATGCT GTTTTCTGTC TGTCCCTAAC ATGCCCTGTG 1051 ATTATCCGCA AACAACACAC CCAAGGGCAG AACTTTGTTA CTTAAACACC 1101 ATCCTGTTTG CTTCTTTCCT CAGGAACTGT GGCTGCACCA TCTGTCTTCA 1151 TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG 1201 TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT 1251 GGATAACGCC CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGG 1301 ACAGCAAGGA CAGCACCTAC AGCCTCAGCA GCACCCTGAC GCTGAGCAAA 1351 GCAGACTACG AGAAACACAA AGTCTACGCC TGCGAAGTCA CCCATCAGGG 1401 CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG TGTTAGAGGG 1451 AGAAGTGCCC CCACCTGCTC CTCAGTTCCA GCCTGACCCC CTCCCATCCT 1501 TTGGCCTCTG ACCCTTTTTC CACAGGGGAC CTACCCCTAT TGCGGTCCTC 1551 CAGCTCATCT TTCACCTCAC CCCCCTCCTC CTCCTTGGCT TTAATTATGC 1601 TAATGTTGGA GGAGAATGAA TAAATAAAGT GAATCTTTGC ACCTGTGGTT 1651 TCTCTCTTTC CTCATTTAAT AATTATTATC TGTTGTTTTA CCAACTACTC 1701 AATTTCTCTT ATAAGGGACT AAATATGTAG TCATCCTAAG GCGGGATATC 1751 GAGATCTGAA GCTGATCCAG ACATGATAAG ATACATTGAT GAGTTTGGAC 1801 AAACCACAAC TAGAATGCAG TGAAAAAAAT GCTTTATTTG TGAAATTTGT 1851 GATGCTATTG CTTTATTTGT AACCATTATA AGCTGCAATA AACAAGTTAA 1901 CAACAACAAT TGCATTCATT TTATGTTTCA GGTTCAGGGG GAGGTGTGGG 1951 AGGTTTTTTA AAGCAAGTAA AACCTCTACA AATGTGGTAT GGCTGATTAT 2001 GATCTCTAGT CAAGGCACTA TACATCAAAT ATTCCTTATT AACCCCTTTA 2051 CAAATTAAAA AGCTAAAGGT ACACAATTTT TGAGCATAGT TATTAATAGC 2101 AGACACTCTA TGCCTGTGTG GAGTAAGAAA AAACAGTATG TTATGATTAT 2151 AACTGTTATG CCTACTTATA AAGGTTACAG AATATTTTTC CATAATTTTC 2201 TTGTATAGCA GTGCAGCTTT TTCCTTTGTG GTGTAAATAG CAAAGCAAGC 2251 AAGAGTTCTA TTACTAAACA CAGCATGACT CAAAAAACTT AGCAATTCTG 2301 AAGGAAAGTC CTTGGGGTCT TCTACCTTTC TCTTCTTTTT TGGAGGAGTATGAG AGTCAGCAGT AGCCTCATCA TCACTAGATG GCATTTCTTC 2401 TGAGCAAAAC AGGTTTTCCT CATTAAAGGC ATTCCACCAC TGCTCCCATT 2451 CATCAGTTCC ATAGGTTGGA ATCTAAAATA CACAAACAAT TAGAATCAGT 2501 AGTTTAACAC ATTATACACT TAAAAATTTT ATATTTACCT TAGAGCTTTA 2551 AATCTCTGTA GGTAGTTTGT CCAATTATGT CACACCACAG AAGTAAGGTT 2601 CCTTCACAAA GATCCGGACC AAAGCGGCCA TCGTGCCTCC CCACTCCTGC 2651 AGTTCGGGGG CATGGATGCG CGGATAGCCG CTGCTGGTTT CCTGGATGCC 2701 GACGGATTTG CACTGCCGGT AGAACTCCGC GAGGTCGTCC AGCCTCAGGC 2751 AGCAGCTGAA CCAACTCGCG AGGGGATCGA GCATCCCCCA TGGTCTTATA 2801 AAAATGCATA GCTTTAGGAG GGGAGCAGAG AACTTGAAAG CATCTTCCTG 2851 TTAGTCTTTC TTCTCGTAGA CTTCAAACTT ATACTTGATG CCTTTTTCCT 2901 CCTGGACCTC AGAGAGGACG CCTGGGTATT CTGGGAGAAG TTTATATTTC 2951 CCCAAATCAA TTTCTGGGAA AAACGTGTCA CTTTCAAATT CCTGCATGAT 3001 CCTTGTCACA AAGAGTCTGA GGTGGCCTGG TTGATTCATG GCTTCCTGGT 3051 AAACAGAACT GCCTCCGACT ATCCAAACCA TGTCTACTTT ACTTGCCAAT 3101 TCCGGTTGTT CAATAAGTCT TAAGGCATCA TCCAAACTTT TGGCAAGAAA 3151 ATGAGCTCCT CGTGGTGGTT CTTTGAGTTC TCTACTGAGA ACTATATTAA 3201 TTCTGTCCTT TAAAGGTCGA TTCTTCTCAG GAATGGAGAA CCAGGTTTTC 3251 CTACCCATAA TCACCAGATT CTGTTTACCT TCCACTGAAG AGGTTGTGGT 3301 CATTCTTTGG AAGTACTTGA ACTCGTTCCT GAGCGGAGGC CAGGGTCGGT 3351 CTCCGTTCTT GCCAATCCCC ATATTTTGGG ACACGGCGAC GATGCAGTTC 3401 AATGGTCGAA CCATGATGGC AGCGGGGATA AAATCCTACC AGCCTTCACG 3451 CTAGGATTGC CGTCAAGTTT GGGGGTACCG AGCTCGAATT AGCTTTTTGC 3501 AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC ACTACTTCTG GAATAGCTCA 3551 GAGGGCCGAG GCGGCCTCGG CCTCTGCATA AATAAAAAAA ATTAGTCAGC 3601 CATGGGGCGG AGAATGGGCG GAACTGGGCG GAGTTAGGGG CGGGATGGGC 3651 GGAGTTAGGG GCGGGACTAT GGTTGCTGAC TAATTGAGAT GCATGCTTTG 3701 CATACTTCTG CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CTGGTTGCTG 3751 ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 3801 GACTTTCCAC ACCCTAACTG ACACACATTC CACAGCTGCC TCGCGCGTTT 3851 CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA CAGCTTGTCT 3901 GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG 3951 TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG_4001_CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT 4051 ACTGAGAGTG CACCATATGC GGCCGCATAT GCGGTGTGAA ATACCGCACA 4101 GATGCGTAAG GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC 4151 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA 4201 CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 4251 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC 4301 GCGTTGCTGG CGTTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 4351 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 4401 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 4451 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC 4501 GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC 4551 GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC 4601 GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 4651 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 4701 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 4751 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG 4801 AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG 4851 GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT 4901 CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 4951 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 5001 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 5051 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC 5101 TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG 5151 TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT 5201 GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT 5251 AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 5301 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 5351 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTG CAGGCATCGT 5401 GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 5451 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC 5501 TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC 5551 ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 5601 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 5651 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA CACGGGATAA 5701 TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT 5751 CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG 5801 ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 5851 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 5901 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 5951 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT 6001 TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC 6051 GAAAAGTGCC ACCTGACGTC TAAGAAACCA TTATTATCAT GACATTAACC 6101 TATAAAAATA GGCGTATCAC GAGGCCCTTT CGTCTTCAAG AATTCAGCTG 6151 CTCGAGGAAG AGCTCAAACC CATGCTACTC TCTGGCTTGA TGGAAGCAACATAG CTGAGCTGTC ATAAATAATA AAGAGATTTT TTTATTAATA 6251 TTGAAAAGAT GGGTTATTTA TGTAAGACTC TGTCTTCATT TTAAAAACCA 6301 CACCTTCCAG TAGTATTCTG TTACTGTTCT GGCAATCACT GTGATCAAGA 6351 AGCTACACGG TGAGTTGTGC TTCTCAGTCC TAAGGGATAC ATCTACAAGA 6401 GGCTCCCATA CTCGAAGCTC AGGAAACATT GTAGAAAAGG AGGCAAAAGA 6451 CTGACAGAGC CAGAGGACCA AGAAATTTGT TGTGAGGTTG TGTCTCCTAC 6501 TAACAATATA AGCAATATCT ATAAATTGTT GATATCATGG CTACTAAAAT 6551 GTGAGTTGAA CGAGGAGGAC ACAAATGAAC ATGACAATCA GAATGAGGCC 6601 TCTCACCTGC AAAAAACACT ATAGAGAAGC AGATAAAGCT GTCAGCAGAA 6651 GAGGCGCACC TCCTTATAGA AGAAGCCTAC CAGGTTTGAT ATATCAGCCT 6701 TGAAAACCTA CATAGTATTT ACATTATATC GAGTCTATGA GACATATTTA 6751 GTAATGCATA TGTATGTGTG TGTGTGCATG TATGTGTGTA AATACATATG 6801 TTCATAGAAA AATGTGTAAA AAGAGATCAT GAATTTAAGA GAGAACTGGG 6851 ACAATTTTTT TCAGGGAGTT GTAATCAGGA AAGTTAAGGG AAAAATGTTG 6901 TAATTAAAAT TCAGGCTCAG AAACAAACAA AGGAAAAGAA 'AAAAAAACAA 6951 CAACAACAAC AAAAAAACAA AACAAAGGAG AAGCTGTATG GCCACAATAG_7001_CATCTACAGC TAACTGTGAA AGGATAATGG AACAAGTTAT GTACTGCCTA 7051 GAGCAGTATG ATGCCTAAAT CATCTCGACA TGGAGGAAAA TAGAACAAAG 7101 ACACTCTACA TAGACTATGA TAGAAATCAA AATAAGGTGT AAGACATAGA 7151 ACATTAGTTT TGTTTGTTGT TCAAAGAGAC TCACATTCCC ACAAAAAAAT 7201 CTGTGGGATT TTACAGGTCT GCAATAAGCT GCTGACCTGA TGATTTCTGC 7251 AGCTGTGCCT ACCCTTTGCT GATTTGCATG TACCCAAAGC ATAGCTTACT 7301 GACATGAGGA TTTCTTCATA GTCAGGTCAC ACCCTTTGCT GGAGTCAGAA 7351 TCACACTGAT CACACACAGT CATGAGTGTG CTCACTCAGG TCCTGGCGTT 7401 GCTGCTGCTG TGGCTTACAG GTAATGAAGA CAGCACTAGA ATTTTATTGA 7451 GCTTCCTGTA CACTGTGCTG CTTGTCTCTG TGAAAATTCT CTTGTGAATT 7501 AATCATGTGG GGATCTGTTT TCAATTTTTC AATTGTAGGT ACGCGTTGTG 7551 ACATTCTGCT GACCCAGTCT CCAGCCACCC TGTCTCTGAG TCCAGGAGAA 7601 AGAGCCACTT TCTCCTGCAG GGCCAGTCAG AACATTGGCA CAAGCATACA 7651 GTGGTATCAA CAAAAAACAA ATGGTGCTCC AAGGCTTCTC ATAAGGTCTT 7701 CTTCTGAGTC TATCTCTGGG ATCCCTTCCA GGTTTAGTGG CAGTGGATCA 7751 GGGACAGATT TTACTCTTAC CATCAGCAGT CTGGAGCCTG AAGATTTTGC 7801 AGTGTATTAC TGTCAACAAA GTAATACCTG GCCATTCACG TTCGGCCAGG 7851 GGACCAAGCT TGAAATCAAA CGTAAGTAGA ATCCAAAGTC TCTTTCTTCC 7901 GTTGTCTATG TCTGTGGCTT CTATGTCTAA AAATGATGTA TAAAATCTTA 7951 CTCTGAAACC AGATTCTGGC ACTCTCCAAG GCAAAGATAC AGAGTAACTC 8001 CGTAAGCAAA GCTGGGAATA GGCTAGACAT GTTCTCTGGA GAATGAATGC 8051 CAGTGTAATA ATTAACACAA GTGATAGTTT CAGAAATGCT CTAGTT SEQ ID NO: 40 Nucleotide sequence of the expression vector HCVHESp20 1 CTAGAGAGGT CTGGTGGAGC CTGCAAAAGT C AG YYTCA AAGGAACACA 51 GAAGTATGTG TATGGAATAT TAGAAGATGT TGCTTTTACT CTTAAGTTGG 101 TTCCTAGGAA AAATAGTTAA ATACTGTGAC TTTAAAATGT GAGAGGGTGT 151 TCAAGTACTC ATTTTTTTAA ATGTCCAAAA TTTTTGTCAA TCAATTTGAG 201 GTCTTGTTTG TGTAGAACTG ACATTACTTA AAGTTTAACC GAGGAATGGG 251 AGTGAGGCTC TCTCATACCC TATTCAGAAC TGACTTTTAA CAATAATAAA 301 TTAAGTTTAA AATATTTTTA AATGAATTGA GCAATGTTGA GTTGGAGTCA 351 AGATGGCCGA TCAGAACCAG AACACCTGCA GCAGCTGGCA GGAAGCAGGT 401 CATGTGGCAA GGCTATTTGG GGAAGGGAA? ATAAAACCAC TAGGTAAACT 451 TGTAGCTGTG GTTTGAAGAA GTGGTTTTGA AACACTCTGT CCAGCCCCAC 501 CAAACCGA ?? GTCCAGGCIG AGCAAAACAC CACCTGGGTA ATGTGCATTT 551 CTAAAATAAG TTGAGGATTC AGCCGAAACT GGAGAGGTCC TCTTTTAACT 601 TATTGAGTTC AACCTTTTAA TTTTAGCTTG AGTAGTTCTA GTTTCCCCAA 651 ACTTA? GTGT ATCGACTTCT AAAATGTATT TA? GC? TC GGGGCAGGCC 701 AGGCCTGACC TTGGCTITGG GGCAGGGAGG GGGCTAAGGT GAGGCAGGTG 751 GCGCCAGCCA GG1GCACACC CAATGCCCAT GAGCCCAGAC ACTGGACGCT 801 GAACCTCGCG GACAGTTAAG AACCCAGGGG CCTCTGCGCC CTGGGCCCAG 851 CTCTGTCCCA CACCGCGGTC ACATGGCACC ACCTCTCTTG CAGCCTCCAC 901 CAAGGGCCCA TCGGTCTTCC CCCTGGCACC CTCCTCCAAG AGCACCTCTG 951 GGGGCACAGC GGCCCIGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG 1001 GTGACGGTGT CGGGAACTC AGGCGCCCTG ACCAGCGGCG T3CACACCTT 1051 CCCGGCTGTC CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA 1101 CCGTGCCCTC CAGCAGCTTG GGCACCCAGA CCTACATCTG CAACGTGAAT 1151 CACAAGCCCA GCAACACCAA GGTGGACAAG AGAGTTGGTG AGAGGCCAGC 1201 ACAGGGAGGG AGGGTGTCTG CTGGAAGCCA GGCTCAGCGC TCCTGCCTGG 1251 ACGCATCCCG GCTATGCAGT CCCAGTCCAG GGCAGCAAGG CAGGCCCCGT 1301 CTGCCTCTTC ACCCGGAGGC CTCTGCCCGC CCCACTCATG CTCAGGGAGA 1351 GGGTCTTCTG GCTTTTTCCC CAGGCTCTGG GCAGGCACAG GCTAGGTGCC 1401 CCTAACCCAG GCCCTGCACA CAAAGGGGCA GGTGCTGGGC TCAGACCTGC 1451 CAAGAGCCAT ATCCGGGAGG ACCCTGCCCC TGACCTAAGC CCACCCCAAA 1501 GGCCAAACTC TCCACTCCCT CAGCTCGGAC ACCTTCTCTC CTCCCAGATT 1551 CCAGTAACTC CCAATCTTCT CTCTGCAGAG CCCAAATCTT GTGACAAAAC 1601 TCACACATGC CCACCGTGCC CAGGTAAGCC AGCCCAGGCC TCGCCCTCCA 1651 GCTCAAGGCG GGACAGGTGC CCTAGAGTAG CCTGCATCCA GGGACAGGCC 1701 CCAGCCGGGT GCTGACACGT CCACCTCCAT CTCTTCCTCA GCACCTGAAC 1751 TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC 1801 CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG T3GACGTGAG 1851 CCACGAAGAC CCIGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC 1901- 1951 CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 2001 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 2051 AAACCATCTC CAAAGCCAAA GGTGGGACCC GTGGGGTGCG AGGGCCACAT 2101 GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACCGCTGTAC 2151 CAACCTCTGT CCCTACAGGG CAGCCCCGAG A ACCACAGGT GTACACCCTG 2201 CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT 2251 GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG 2301 GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGCT GGACTCCGAC 2351 GGCTCCTTCT TCCTCTATAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA 2401 GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC 2451 ACTACACGCA GAAGAGCCTC TCCCTGTCCC CGGGTAAATG AGTGCGACGG 2501 CCGGCAAGCC CCCGCTCCCC GGGCTCTCGC GGTCGCACGA GGATGCTTGG 2551 CACGTACCCC GTCTACATAC TTCCCAGGCA CCCAGCATGG AAATAAAGCA 2601 CCCACCACTG CCCTGGGCCC CTGCGAGACT GTGATGGTTC TTTCCACGGG 2651 TCAGGCCGAG TCTGAGGCCT GAGTGGCATG AGGGAGGCAG AGCGGGTCCC 2701 ACTGTCCCCA CACIGGCCCA GGCTGTGCAG GTGTGCCTGG GCCGCCTAGG 2751 GTGGGGCTCA GCCAGGGGCT GCCCTCGGCA GGGTGGGGGA TTTGCCAGCG 2801 TGGCCCTCCC TCCAGCAGCA GCTGCCCTGG GCTGGGCCAC GAGAAGCCCT 2851 AGGAGCCCCT GGGGACAGAC ACACAGCCCC TGCCTCTGTA GGAGACTGTC 2901 CTGTCCTGTG AGCGCCCTGT CCTCCGACCC CGATGCCCAC TCGGGGGCAT 2951 GCCTAGTCCA TGCGCGTAGG GACAGGCCCT CCCTCACCCA TCTACCCCCA 3001 CGGCACTAAC CCC TGGCAGC CCTGCCCAGC CTCGCACCCG CATGGGGACA 3051 CAACCGACTC CGGGGACATG CACTCTCGGG CCCTGTGGAG GGACTGGTGC 3101 AGATGCCCAC ACACACACTC AGCCCAGACC CGTTCAACAA ACCCCGCACT 3151 GAGGTTGGTC GAGCGGGAGT GCGGCCAGAG CCTGCCTCGG CCGTCAGGGA 3201 GGACTCCCGG GCTCACTCGA AGGAGGTGCC ACCATTTCAG CTTTGGTAGC 3251 TTTTCTTCTT CT? TAAATG TTCTAAAGCT CATTA? TTGT CIGTGATGTG 3301 TCTTTTTGTGA TGACAATAAA ATATCCT? T TAAGTCTTGT ACTTCGTGAT 3351 GGGAGCCGCC TTCCTGTGTC CACGCGCCTC CTGCCCCCGG T3GGAAGCAC 3401 GGTCAGGAGG AGGCTGGTCC AGCTGCACCT CGGGGGCTCC CTGCACTCGC 3451 CCCCCGCCTC CTGCAGCCAC ACGCATTGCC CGAGCGACCC TCCCTGGCCC 3501 CTGTCACTAC ATGGACCCCT GGGGCTTCTC CTCTTTTCTA CATGGATGCA 3551 GTTTCTCCTC CIGCTGGGCA CGGTGCIGCC TGCCCTGGTC ACTCTGCGGG 3601 GGACAGGGCC TCCAGGGAAA GCTGGGTCGA GGCTGGGAGC TGGCTCAGGC 3651 TGGCCAGGCA GAGCCACAGG GAGGGCCITC CAGAACCAAC CATGGTCCGA 3701 AGCGAGAGGT GGGTGTCAGA TCCAGACATG ATAAGATACA TTGATGAGTT 3751 TGGACAAACC ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA 3801 TTTGTGATGC TATTGCITTA TTTGTAACCA TTATAAGCTG CAATA? ACAA 3851 GTTAACAACA ACAATTGCAT TCATTITATG TTCAGGTTC AGGGGGAGGT 3901 GTGGGAGGTT TTTTAAAGCA AGTAAAACCT CTACAAATGT GGTATGGCTG 3951 ATTATGATCT CTAGTCAAGG CACTATACAT CAAATATTCC TTATTAACCC 4001 CTTTACAAAT TA? AAAGCTA AAGGTACACA ATTTT GAGC ATAGTTATTA 4051 ATAGCAGACA CTCTATGCCT GTGTGGAGTA AGAAAAAACA GTATGTTATG 4101 ATTATAACTG TTATGCCTAC TTATAAAGGT TACAGAATAT TTTTCCATAA 4151 TTTTCTTGTA TAGCAGTGCA GCTTTTTCCT TTG TGGTGTA AATAGCAAAG 4201 CAAGCAAGAG TTCTATTACT AAACACAGCA TGACTCAAAA AACTTAGCAA 4251 TTCTGAAGGA AAGTCCTTGG GGTCTTCTAC CITTCTCTTC TlTiTTGGAG 4301 GAGTAGAATG TTGAGAGTCA GCAGTAGCCT CATCATCACT AGATGGCATT 4351 TCTTCTGAGC AAAACAGGTT TTCCTCATTA AAGGCATTCC ACCACTGCTC 4401 CCATTCATCA GTTCCATAGG TTGGAATCTA AAATACACAA ACAATTAGAA 4451 TCAGTAGTTT AACACATTAT ACACTTAAAA ATTTTATATT TACCTTAGAG 4501 CTTTAAATCT CTGTAGGTAG TTTGTCCAAT TATGTCACAC CACAGAAGTA 4551 AGGTTCCTTC ACAAAGATCC GGACCAA? GC GGCCATCGTG CCTCCCCACT 4601 CCTGCAGTTC GGGGGCATGG ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG 4651 ATGCCGACGG ATTTGCACTG CCGGTAGAAC TCCGCGAGGT CGTCCAGCCT 4701 CAGGCAGCAG CTGAACCAAC TCGCGAGGGG ATCGAGCCCG GGGTGGGCGA 4751 AGAACTCCAG CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC 4801 CGGAAAACGA TTCCGAAGCC CAACCOTCA TAGAAGGCGG CGGTGGAATC 4851 GAAATCTCGT GATGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTCGAACC 4901 CCAGAGTCCC GCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC 4951 GCTGCGAATC GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC 5001 CATTCGCCGC CAAGCTCTTC AGCAATATCA CGGGTAGCCA ACGCTATGTC 5051 CTGATAGCGG TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA 5101 AGCGGCCATT TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC 5151 ACGACGAGAT CCTCGCCGTC GGGCATGCGC GCCTTGAGCC TGGCGAACAG 5201 TTCGGCTGGC GCGAGCCCCT GATGCTCTTC GTCCAGATCA TCCTGATCGA 5251 CAAGACCGGC TTCCATCCGA GTACGTGCTC GCTCGATGCG ATGTTTCGCT 5301 TGGTGGTCGA ATGGGCAGGT AGCCGGATCA AGCGTATGCA GCCGCCGCAT 5351 TGCATCAGCC ATGATGGATA CTTTCTCGGC AGGAGCAAGG TGAGATGACA 5401 GGAGATCCTG CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT 5451 TCAGTGACAA CGTCGAGCAC AGCTGCGCAA GGAACGCCCG TCGTSGCCAG 5501 CCACGATAGC CGC GCTGCCT CGTCCTGCAG TTCATTCAGG GCACCGGACA 5551 GGTGGGTCTT GACAAAAAGA ACCGGGCGCC CCTGCGCTGA CAGCCGGAAC 5601 ACGGCGGCAT CAGAGCAGCC GATTGTCTGT TGTGCCCAGT CATAGCCGAA 5651 TAGCCTCTCC ACCCAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT 5701 CAATCATGCG AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC 5751 CTGCGCCATC AGATCCTTGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA 5801 GGGCTTCCCA ACCTTACCAG AGGGCGCCCC AGCTGGCAAT TCCGGTTCGC 5851 TTGCTGTCCA TAAAACCGCC CAGTCTAGCT ATCGCCATGT AAGCCCACTG 5901 CAAGCTACCT GCTGTCTCTT TGCGCTTGCG TTTTCCCTTG TCCAGATAGC 5951 CCAGTAGCTG ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT 6001 TCTACGTGTT CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG 6051 CAGCGTGAAG CTUT'iTGCAA AAGCCTAGGC CTCCAAAAAA GCCTCCTCAC 6101 TACTTCTGGA ATAGCTCAGA GGCCGAGGCG GCCTCGGCCT CTGCATAAAT 6151 AAAA? AAATT AGTCAGCCAT GGGGCGGAGA ATGGGCGGAA CTGGGCGGAG 6201 TTAGGGGCGG GATGGGCGGA GTTAGGGGCG GGACTATGGT TGCTGACTAA 6251 TTGAGATGC? TGCTTGC? T ACTTCTGCCT GCTGGGGAGC CTGGGGACIT 6301 TCCACACCTG GTTGCT3ACT AATTGAGATG CATGCTTTGC ATACTTCTGC 6351 CTGCTGGGGA GCCTGGGGAC TTTCCACACC CTAACIGACA CACATTCCAC 6401 AGCTGCCTCG CGGGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA 6451 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC 6501 AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC 6551 ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT TAACTATGCG 6601 GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC 6651 GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT 6701 CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCIGCGGCG AGCGGTATCA 6751 GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 6801 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 6851 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT 6901 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA 6951 AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC 7001 CGACCCTGCC GCTTACCGGA TACCTGTCCG CC TTTCTCCC TTCGGGAAGC 7051 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 7101 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA A CCCCCCGTT CAGCCCGACC 7151 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC 7201 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG 7251 GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT A? CTACGGCT 7301 ACACTAGA? G GACAGTATTT GGTATCTGCG CTCGGCTGA? GCCAGTTACC 7351 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG 7401 TAGCGGTGGT T1TTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG 7451 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 7501 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT 7551 CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA 7601 GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG 7651 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT 7701 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 7751 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA 7801 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCIGCAAC 7851 TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA 7901 GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGC 7951 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC 8001 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 8051 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG 8101 TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC 8151 ATCCGTAAGA TGCTTTTCIG TG ACTGGTGA GTACTCAACC AAGTCATTCT 8201 GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAACACGG 8251 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA 8301 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 8351 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT 8401 TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA 8451 AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT 8501 TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC 8551 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT 8601 TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT 8651 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAAGAATTC 8701 GAGCTCGGTA CCCATCAGCC AAAA? GC? TG CCTGCCACAC AACATCAATT 8751 TCTGGAAAAC GCTACACTTA ATTATTTCTA GTAGAACAGC TCTTTGGTTT 8801 GCCAAAAAGA ATCACCTATA GTGGCATCTA AGCACAAAAA GGAGAAAAAA 8851 ATCACAAAGA AATGATTGAG AGGCATAATA AAAATTATCA AAAAATTATG 8901 AGTTTTACGA TTTCATCTTT TTCCAAGTTG AAATCATAGG GTGGCTTTAA 8951 CACAGTGACA AGGAATGGC ATGCTGCCAT TATGGTGCTC TGCCTAAAAT 9001 GGTTG GAGCC TTGTCATGCT ACAGAGAAAC TGTCATACAG CAGGGGGTGC 9051 CAAATTTCCA TAT1TTTTTA TATCATTGAG CAGGTGCACA GAAGACCAGA 9101 AAGCACTTTC TATCAGGCTG GCCTTCCTCT TCCTTTCCAG TATGAAGCAA 9151 AAACTGCCA? TGAAACTAGC AATTGTTA? TGCCTGTTTC AAACAGTATT 9201 TGTGCTATC? GAACATAGTG CATTCAAA? G TCTAGCCTGA GAGA? CAACC 9251 CAGTTTTATT CATTCCTCCT ACTACCTCTC TCATTCCCAC TGTTTGTGTT 9301 CTCCCTCCCA TTTTAATTGT CTATCTAGTC CAAACTA? GC ACACGATCCA 9351 GTCCACATTA AACAACATGT TTTC? CTTTA AGTCAAATAC AAGACACCTT 9401 TAATATCAGC CCTTGTTCAT AATCGTGCTT CTAGTGACTT AATGTACATG 9451 TCACACTGTA CTGTTGGGTT TTGTGTCTCA TCATGAACAA TGTTGTGAAG 9501 GTATTAAGTG GAGAGTAAGC AGAATTAGAT TCCTCTAATG ATGCACACCC 9551 ACACTAAGAG CAGAAATAAT ATTAAAAATA GAAAAAAAAG TTTTACATGA 9601 GATTTCAAAT ACCCAGGTAT GAGCTGCAGT TTCTTCAAGT TAAAGCATCG 9651 AGGTTGTCAG TTACACTATT ACAGGAAACA TATGCAGAGT TTTTATTTTA 9701 GTATATTAGT TTTCACATAT GTGGAATTAC TATTAAACTA TTCTTTCTTT 9751 TCAAATGCTT ACCATTGTAA ATGAGTTTGT GACTTTGTGT AGGTGAGTGC 9801 ACATGACGCT GGATGCCTA? GAGGACGGA? GAAGTTGGAG TATAGGTAG 9851 TTTTATTCTA CTTGACTGTT CAGTGCTAAA AATACAACTG AGGTCCTTTA 9901 A? CTGCTGTT CATGAACTTC TTAATTGATA TATCTCATGA GATCGCTAAA 9951 CTATTTTTAT TATGACACGT TTCACCATTT TCACTGTAAC GATT TTTATG 10001 TTTTATATTA ATGTAACTAT ATGACACTTC CCAAAATCCC CATATTCACA 10051 ATTGAACTGT TTCAAAGTTT TACC TGACT TATGGGAAAT GAAAACCCAC 10101 ATTTTATAAT TTTAAAATGA AATGTTTATT TTATATTTCT GCAAATTTCA 10151 CAAGGAAAGA TTAGTCACTG GTGTGTGAGA GCAGAGGAGC ATAAGAGTTC 10201 AGGAATAG ?? TCCATTATGA TTCTGGAGGC A? GGA? SAGs TGATGCCAAG 10251 GITTCAGTAT AAGAGCAGTA TCCACTGGAA AGGATAAAGT CACTACATCT 10301 GAGCACAGAG CAGGACATCT ACATAATGAG TGGTCACTAA TGGGCCACTG 10351 TTACACTGTT ATATGTATAA GGCTCAAGAA TGAGCACTGA GGCTGTAAGG 10401 TGTATGGGTG AGGACATCAG GATGTAAACC CAGCTCAGGT AGAGGACTCA 10451 GAGGACAGCA CAGTCAGCAT GAACTAATAA ACATCAGATA AGATAAGGCA 10501 CAAGCTCAGC TATATAGGGT AAGGGATCTT TGTAAATCTG ATTGTGCATC 10551 CAGTCTAGTT CAATGTGACT TAGGAAGCCC AGTCATATGC AAATCTAGAG 10601 AAGACTTTAG AGTAGAAATC TGAGGCTCAC CTCACATACC AGCAAGCGAG 10651 TGACCAGTTA GTCTTAAGGC ACCACTTCTT AGACATCATG GCTTGGGTGT 10701 GGACCTTGCC ATTCCTGATG GCAGCTGCCC AAAGTAAGAC ATCAGAAAAA 10751 AGAGTTCCAA GGGGAATTGA AGCAGTTCCA TGAATACTCA CCTTCCTGTG 10801 TTCTTTTCAC AGGTGTCCAG GCAGAGGTGC AGCTGGTGGA GTCAGGAGCC 10851 GAAGTGAAAA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AGGCCTCTGG 10901 ATACACATTC ACTAATTATA TTATCCACTG GGGAAGCAG GAGCCTGGTC 10951 AGGGCCTTGA ATGGATTGGA TATTTTAATC CTTACAATCA TGGTACTAAG 11001 TACAATGAGA AGTTCAAAGG CAGGGCCACA CTA ACTGCAA ACAAATCCAT 11051 CAGCACAGCC TACATGGAGC TCAGCAGCCT GCGCTCTGAG GACACTGCGG 11101 TCTACTACTG TGCAAGATCA GGACCCTATG CCTGGTTTGA CACCTGGGGC 11151 CAAGGGACCA CGGTCACCGT CTCCTCAGGT AAGAATGGCC ACTCTAGGGC 11201 CTTTGTTTTC TGCTGCTGCC TGTGGGATTT CATGAGCATT GCAAAGTTGT 11251 CCTCGGGACA TGTTCCGAGG GGACCTGGGC GGACTGGCCA GGAGGGGACG 11301 GGCACTGGGG TGCCTTGAGG ATCTGGGAGC CTCTGTGGAT TTTCCGATGC 11351 CTTTGGAAAA TGGGACTG? G GTTGGGTGCG TCTGAGACAG TAACTCAGCC 11401 TGGGGGCTTG GTGAAGATCG CCGCACAGCA GCGAGTCCGT GAAATATCTT 21451 ATTTAGACTT GTGAGGTGCG CTGTGTGTCA ATTTACATCT TAAATCCTTT 11501 ATTGGCTGGA AAGAGAATTG TTGGAGTGGG TGAATCCAGC CAGGAGGGAC 11551 GCGGGGGGGAT CCA SEQ ID NO: 41 Nucleotide sequence of the expression vector HCVHQSp20 1 CTAGAGAGGT CTGGTGGAGC CTGCAAAAGT CCAGCTTTCA AAGGAACACA 51 GAAGTATGTG TATGGAATAT TAGAAGATGT TGCTTTTACT CTTAAGTTGG 101 TTCCTAGGAA AAATAGTTAA ATACTGTGAC TTTAAAATGT GAGAGGGTTT 151 TCAAGTACTC ATTTTTTTTAA ATGTCCAAAA TTTTTGTCAA TCAATTTGAG 201 GTCTTGTTTG TGTAGAACTG ACATTACTTA AAGTTTAACC GAGGAATGGG 251 AGTGAGGCTC TCTCATACCC TATTCAGAAC TGACTTTTAA CAATAATAAA 301 TTAAGTTTAA AATATTTTTA AATGAATTGA GCAATGTTGA GTTGGAGTCA 351 AGATGGCCGA TCAGAACCAG AACACCTGCA GCAGCTGGCA GGAAGCAGGT 401 CATGTGGCAA GGCTATTTGG GGAAGGGAAA ATAAAACCAC TAGGTAAACT 451 TGTAGCTGTG GTTTGAAGAA GTGGTTTTGA AACACTCTGT CCAGCCCCAC 501 CAAACCGAAA GTCCAGGCTG AGCAAAACAC CACCTGGGTA ATTTGCATTT 551 CTAAAATAAG TTGAGGATTC AGCCGAAACT GGAGAGGTCC TCTTTTAACT 601 TATTGAGTTC AACCTTTTAA TTTTAGCTTG AGTAGTTCTA GTTTCCCCAA 651 CTTAAGTTT ATCGACTTCT AAAATGTATT TAAGCTTTCT GGGGCAGGCC 701 AGGCCTGACC TTGGCTTTGG 000AGGGAGG GGGCTAAGGT GAGGCAGGTG 751GCGCCAGCCA GGTGCACACC CAATGCCCAT GAGCCCAGAC ACTGGACGCT 801 GAACCTCGCG GACAGTTAAG AACCCAGGGG CCTCTGCGCC CTGGGCCCAG 851 CTCTGTCCCA CACCGCGGTC ACATGGCACC ACCTCTCTTG CAGCCTCCAC 901 CAAGGGCCCA TCGGTCTTCC CCCTGGCACC CTCCTCCAAG AGCACCTCTG 951 GGGGCACAGC GGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG 1001 GTGACGGTGT CGTGGAACTC AGGCGCCCTG ACCAGCGGCG TGCACACCTT 1051 CCCGGCTGTC CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA 1101 CCGTGCCCTC CAGCAGCTTG GGCACCCAGA CCTACATCTG CAACGTGAAT 1151 CACAAGCCCA GCAACACCAA GGTGGACAAG AGAGTTGGTG AGAGGCCAGC 1201 ACAGGGAGGG AGGGTGTCTG CTGGAAGCCA GGCTCAGCGC TCCTGCCTGG 1251 ACGCATCCCG GCTATGCAGT CCCAGTCCAG GGCAGCAAGG CAGGCCCCGT 1301 CTGCCTCTTC ACCCGGAGGC CTCTGCCCGC CCCACTCATG CTCAGGGAGA 1351 GGGTCTTCTG GCTTTTTCCC CAGGCTCTGG GCAGGCACAG GCTAGGTGCC 1401 CCTAACCCAG GCCCTGCACA CAAAGGGGCA GGTGCTGGGC TCAGACCTGC 1451 CAAGAGCCAT ATCCGGGAGG ACCCTGCCCC TGACCTAAGC CCACCCCAAA 1501 GGCCAAACTC TCCACTCCCT CAGCTCGGAC ACCTTCTCTC CTCCCAGATT 1551 CCAGTAACTC CCAATCTTCT CTCTGCAGAG CCCAAATCTT GTGACAAAAC 1601 TCACACATGC CCACCGTGCC CAGGTAAGCC AGCCCAGGCC TCGCCCTCCA 1651 GCTCAAGGCG GGACAGGTGC CCTAGAGTAG CCTGCATCCA GGGACAGGCC 1701 CCAGCCGGGT GCTGACACGT CCACCTCCAT CTCTTCCTCA GCACCTGAAC 1751 TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC 1801 CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG 1851 CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG 1901 TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC 1951 CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 2001 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 2051 AAACCATCTC CAAAGCCAAA GGTGGGACCC GTGGGGTGCG AGGGCCACAT 2101 GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACCGCTGTAC 2151 CAACCTCTGT CCCTACAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG 2201 CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT 2251 GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG 2301 GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGCT GGACTCCGAC 2351 GGCTCCTTCT TCCTCTATAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA 2401 GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC 2451 ACTACACGCA GAAGAGCCTC TCCCTGTCCC CGGGTAAATG AGTGCGACGG 2501 CCGGCAAGCC CCCGCTCCCC GGGCTCTCGC GGTCGCACGA GGATGCTTGG 2551 CACGTACCCC GTCTACATAC TTCCCAGGCA CCCAGCATGG AAATAAAGCA 2601 CCCACCACTG CCCTGGGCCC CTGCGAGACT GTGATGGTTC TTTCCACGGG 2651TCAGGCCGAG TCTGAGGCCT GAGTGGCATG AGGGAGGCAG AGCGGGTCCC 2701 ACTGTCCCCA CACTGGCCCA GGCTGTGCAG GTGTGCCTGG GCCGCCTAGG 2751 GTGGGGCTCA GCCAGGGGCT GCCCTCGGCA GGGTGGGGGA TTTGCCAGCG 2801 TGGCCCTCCC TCCAGCAGCA GCTGCCCTGG GCTGGGCCAC GAGAAGCCCT 2851 AGGAGCCCCT GGGGACAGAC ACACAGCCCC TGCCTCTGTA GGAGACTGTC 2901 CTGTCCTGTG AGCGCCCTGT CCTCCGACCC CGATGCCCAC TCGGGGGCAT 2951 GCCTAGTCCA TGCGCGTAGG GACAGGCCCT CCCTCACCCA TCTACCCCCA 3001 CGGCACTAAC CCCTGGCAGC CCTGCCCAGC CTCGCACCCG CATGGGGACA 3051 CAACCGACTC CGGGGACATG CACTCTCGGG CCCTGTGGAG GGACTGGTGC 3101 AGATGCCCAC ACACACACTC AGCCCAGACC CGTTCAACAA ACCCCGCACT 3151 GAGGTTGGTC GAGCGGGAGT GCGGCCAGAG CCTGCCTCGG CCGTCAGGGA 3201 GGACTCCCGG GCTCACTCGA AGGAGGTGCC ACCATTTCAG CTTTGGTAGC 3251 TTTTCTTCTT CTTTTAAATT TTCTAAAGCT CATTAATTGT CTTTGATGTT 3301 TCTTTTGTGA TGACAATAAA ATATCCTTTT TAA GTCTTGT ACTTCGTGAT 3351 GGGAGCCGCC TTCCTGTGTC CACGCGCCTC CTGCCCCCGG TGGGAAGCAC 3401 GGTCAGGAGG AGGCTGGTCC AGCTGCACCT CGGGGGCTCC CTGCACTCGC 3451 CCCCCGCCTC CTGCAGCCAC ACGCATTGCC CGAGCGACCC TCCCTGGCCC 3501 CTGTCACTAC ATGGACCCCT GGGGCTTCTC CTCTTTTCTA CATGGATGCA 3551 GTTTCTCCTC CTGCTGGGCA CGGTGCTGCC TGCCCTGGTC ACTCTGCGGG 3601 GGACAGGGCC TCCAGGGAAA GCTGGGTCGA GGCTGGGAGC TGGCTCAGGC 3651 TGGCCAGGCA GAGCCACAGG GAGGGCCTTC CAGAACCAAC CATGGTCCGA 3701 GCGAGAGGT GGGTGTCAGA TCCAGACATG ATAAGATACA TTGATGAGTT 3751 TGGACAAACC ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA 3801 TTTGTGATGC TATTGCTTTA TTTGTAACCA TTATAAGCTG CAATAAACAA 3851 GTTAACAACA ACAATTGCAT TCATTTTATG TTTCAGGTTC AGGGGGAGGT 3901 GTGGGAGGTT TTTTAAAGCA AGTAAAACCT CTACAAATGT GGTATGGCTG 3951 TTATGATCT CTAGTCAAGG CACTATACAT CAAATATTCC TTATTAACCC 4001 CTTTACAAAT TAAAAAGCTA AAGGTACACA ATTTTTGAGC ATAGTTATTA 4051ATAGCAGACA CTCTATGCCT GTGTGGAGTA AGAAAAAACA GTATGTTATG 4101 ATTATAACTG TTATGCCTAC TTATAAAGGT TACAGAATAT TTTTCCATAA 4151 TTTTCTTGTA TAGCAGTG CA GCTTTTTCCT TTGTGGTGTA AATAGCAAAG 4201 CAAGCAAGAG TTCTATTACT AAACACAGCA TGACTCAAAA AACTTAGCAA 4251 TTCTGAAGGA AAGTCCTTGG GGTCTTCTAC CTTTCTCTTC TTTTTTGGAG 4301 GAGTAGAATG TTGAGAGTCA GCAGTAGCCT CATCATCACT AGATGGCATT 4351 TCTTCTGAGC AAAACAGGTT TTCCTCATTA AAGGCATTCC ACCACTGCTC 4401 CCATTCATCA GTTCCATAGG TTGGAATCTA AAATACACAA ACAATTAGAA 4451 TCAGTAGTTT AACACATTAT ACACZTAAAA ATTTTATATT TACCTTAGAG 4501 CTTTAAATCT CTGTAGGTAG TTTGTCCAAT TATGTCACAC CACAGAAGTA 4551AGGTTCCTTC ACAAAGATCC GGACCAAAGC GGCCATCGTG CCTCCCCACT 4601 CCTGCAGTTC GGGGGCATGG ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG 4651 TGCCGACGG ATTTGCACTG CCGGTAGAAC TCCGCGAGGT CGTCCAGCCT 4701 CAGGCAGCAG CTGAACCAAC TCGCGAGGGG ATCGAGCCCG GGGTGGGCGA 4751 AGAACTCCAG CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC 4801 CGGAAAACGA TTCCGAAGCC CAACCTTTCA TAGAAGGCGG CGGTGGAATC 4851 GAAATCTCGT GATGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTCGAACC 4901 CCAGAGTCCC GCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC 4951 GCTGCGAATC GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC 5001 CATTCGCCGC CAAGCTCTTC AGCAATATCA CGGGTAGCCA ACGCTATGTC 5051 CTGATAGCGG TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA 5101 GCGGCCATT TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC 5151 ACGACGAGAT CCTCGCCGTC GGGCATGCGC GCCTTGAGCC TGGCGAACAG 5201 TTCGGCTGGC GCGAGCCCCT GATGCTCTTC GTCCAGATCA TCCTGATCGA 5251 CAAGACCGGC TTCCATCCGA GTACGTGCTC GCTCGATGCG ATGTTTCGCT 5301 TGGTGGTCGA ATGGGCAGGT AGCCGGATCA AGCGTATGCA GCCGCCGCAT 5351 TGCATCAGCC ATGATGGATA CTTTCTCGGC AGGAGCAAGG TGAGATGACA 5401 GGAGATCCTG CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT 5451 TCAGTGACAA CGTCGAGCAC AGCTGCGCAA GGAACGCCCG TCGTGGCCAG 5501 CCACGATAGC CGCGCTGCCT CGTCCTGCAG TTCATTCAGG GCACCGGACA 5551 GGTCGGTCTT GACAAAAAGA ACCGGGCGCC CCTGCGCTGA CAGCCGGAAC 5601 ACGGCGGCAT CAGAGCAGCC GATTGTCTGT TGTGCCCAGT CATAGCCGAA 5651 TAGCCTCTCC ACC CAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT 5701 CAATCATGCG AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC 5751 CTGCGCCATC AGATCCTTGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA 5801 GGGCTTCCCA ACCTTACCAG AGGGCGCCCC AGCTGGCAAT TCCGGTTCGC 5851 TTGCTGTCCA TAAAACCGCC CAGTCTAGCT ATCGCCATGT AAGCCCACTG 5901 CAAGCTACCT GCTTTCTCTT TGCGCTTGCG TTTTCCCTTG TCCAGATAGC 5951 CCAGTAGCTG ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT 6001 TCTACGTGTT CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG 6051 CAGCGTGAAG CTTTTTGCAA AAGCCTAGGC CTCCAAAAAA GCCTCCTCAC 6101 TACTTCTGGA ATAGCTCAGA GGCCGAGGCG GCCTCGGCCT CTGCATAAAT 6151 AAAAAAATT AGTCAGCCAT GGGGCGGAGA ATGGGCGGAA CTGGGCGGAG 6201 TTAGGGGCGG GATGGGCGGA GTTAGGGGCG GGACTATGGT TGCTGACTAA 6251 TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC CTGGGGACTT 6301 TCCACACCTG GTTGCTGACT AATTGAGATG CATGCTTTGC ATACTTCTGC 6351 CTGCTGGGGA GCCTGGGGAC TTTCCACACC CTAACTGACA CACATTCCAC 6401 AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA 6451 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC6501 AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC 6551 ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT TAACTATGCG 6601GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC 6651GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT 6701 CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA 6751 GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 6801 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 6851 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT 6901 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA 6951 AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC 7001 CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 7051 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 7101 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC 7151 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC 7201GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG 7251 GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT 7301 ACACTAGAAG GACAGTATTT GGTATCTGCG CTCT GCTGAA GCCAGTTACC 7351 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG 7401 AGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG 7451 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 7501 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT 7551 CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA 7601 GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG 7651GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT 7701 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 7751 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA 7801 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC 7851 TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA 7901 GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGC, 7951 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC 8001 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 8051 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG 8101 TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC 8151 ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT 8201 GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAACACGG 8251 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA 8301 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 8351 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT 8401 TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA 8451 AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT 8501 TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC 8551 ATATTTGAAT GTA7TTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT 8601 TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT 8651 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAAGAATTC 8701GAGCTCGGTA CCCATCAGCC AAAAAGCATG CCTGCCACAC AACATCAATT 8751 TCTGGAAAAC GCTACACTTA ATTATTTCTA GT AGAACAGC TCTTTGGTTT 8801 GCCAAAAAGA ATCACCTATA GTGGCATCTA AGCACAAAAA GGAGAAAAAA 8851 ATCACAAAGA AATGATTGAG AGGCATAATA AAAATTATCA AAAAATTATG 8901 AGTTTTACGA TTTCATCTTT TTCCAAGTTG AAATCATAGG GTGGCTTTAA 8951 CACAGTGACA AGGAATGTGC ATGCTGCCAT TATGGTGCTC TGCCTAAAAT 9001 GGTTGGAGCC TTGTCATGCT ACAGAGAAAC TGTCATACAG CAGGGGGTGC 9051 CAAATTTCCA TATTTTTTTA TATCATTGAG CAGGTGCACA GAAGACCAGA 9101 AAGCACTTTC TATCAGGCTG GCCTTCCTCT TCCTTTCCAG TATGAAGCAA 9151 AAACTGCCAA TGAAACTAGC AATTGTTAAA TTCCTTTTTC AAACAGTATT 9201 TGTGCTATCA GAACATAGTG CATTCAAAAG TCTAGCCTGA GAGAACAACC 9251 CAGTTTTATT CATTCCTCCT ACTACCTCTC TCATTCCCAC TGTTTGTGTTCCA TTTTAATTGT CTATCTAGTC CAAACTAAGC ACACGATCCA 9351 GTCCACATTA AACAACATGT TTTCACTTTA AGTCAAATAC AAGACACCTT 9401 TAATATCAGC CCTTGTTCAT AATCGTGCTT CTAGTGACTT AATGTACATG 9451 TCACACTGTA CTGTTGGGTT TTGTGTCTCA TCATGAACAA TGTTGTGAAG 9501 GTATTAAGTG GAGAGTAAGC AGAATTAGAT TCCTCTAATG ATGCACACCC 9551 ACACTAAGAG CAGAAATAAT ATTAAAAATA GAA? AAAAAG TTTTACATGA 9601 GATTTCAAAT ACCCAGGTAT GAGCTGCAGT TTCTTCAAGT TAAAGCATCG 9651 AGGTTGTCAG TTACACTATT ACAGGAAACA TATGCAGAGT TTTTATTTTA 9701 GTATATTAGT TTTCACATAT GTGGAATTAC TATTAAACTA TTCTTTCTTT 751 TCAAATGCTT ACCATTGTAA ATGAGTTTGT GACTTTGTGT AGGTGAGTGC 9801 ACATGACTCT GGATGCCTAA GAGGACTGAA GAAGTTGGAG TTATAGGTAG_851_TTTTATTCTA CTTGACTGTT CAGTGCTAAA AATACAACTG AGGTCCTTTA 9901 AACTGCTGTT CATGAACTTC TTAATTGATA TATCTCATGA GATCTCTAAA 9951 CTATTTTTAT TATGACACGT TTCACCATTT TCACTGTAAC GATTTTTATG 10001 TTTTATATTA ATGTAACTAT ATGACACTTC CCAAAATCCC CATATTCACA 10051 ATTGAACTGT TTCAAAGTTT TACCTTGACT TATGGGAAAT GAAAACCCAC 10101 ATTTTATAAT TTTAAAATGA AATGTTTATT TTATATTTCT GCAAATTTCA 10151 CAAGGAAAGA TTAGTCACTG GTGTGTGAGA GCAGAGGAGC ATAAGAGTTC 10201 AGGAATAGAA TCCATTATGA TTCTGGAGGC AAGGAAGAAC TGATGCCAAG 10251 GTTTCAGTAT AAGAGCAGTA TCCACTGGAA AGGATAAAGT CACTACATCT 10301 GAGCACAGAG CAGGACATCT ACATAATGAG TGGTCACTAA TGGGCCACTG 10351 TTACACTGTT ATATGTATAA GGCTCAAGAA TGAGCACTGA GGCTGTAAGG 10401 TGTATGGGTG AGGACATCAG GATGTAAACC CAGCTCAGGT AGAGGACTCA 10451 GAGGACAGCA CAGTCAGCAT GAACTAATAA ACATCAGATA AGATAAGGCA 10501 CAAGCTCAGC TATATAGGGT AAGGGATCTT TGTAAATCTG ATTGTGCATC 10551 CAGTCTAGTT CAATGTGACT TAGGAAGCCC AGTCATATGC AAATCTAGAG 10601 AGACTTTAG AGTAGAAATC TGAGGCTCAC CTCACATACC AGCAAGCGAG 10651 TGACCAGTTA GTCTTAAGGC ACCACTTCTT AGACATCATG GCTTGGGTGT 10701 GGACCTTGCC ATTCCTGATG GCAGCTGCCC AAAGTAAGAC ATCAGAAAAA 10751 AGAGTTCCAA GGGGAATTGA AGCAGTTCCA TGAATACTCA CCTTCCTGTG 10B01 TTCTTTTCAC AGGTGTCCAG GCACAGGTGC AGCTGGTGGA GTCAGGAGCC GAAGTGAAAA 10851 10901 AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AGGCCTCTGG ATACACATTC ACTAATTATA TTATCCACTG GGTGAAGCAG GAGCCTGGTC AGGGCCTTGA ATGGATTGGA 10951 11001 TATTTTAATC CTTACAATCA TGGTACTAAG TACAATGAGA AGTTCAAAGG CAGGGCCACA CTAACTGCAA ACAAATCCAT CAGCACAGCC TACATGGAGC 11051 11101 TCAGCAGCCT GCGCTCTGAG GACACTGCGG TCTACTACTG TGCAAGATCA GGACCCTATG CCTGGTTTGA CACCTGGGGC CAAGGGACCA CGGTCACCGT 11151 11201 CTCCTCAGGT AAGAATGGCC ACTCTAGGGC CTTTGTTTTC TGCTGCTG CC TGTGGGATTT CATGAGCATT GCAAAGTTGT 11251 CCTCGGGACA TGTTCCGAGG GGACCTGGGC GGACTGGCCA GGAGGGGACG 11301 GGCACTGGGG TGCCTTGAGG ATCTGGGAGC CTCTGTGGAT TTTCCGATGC 11351 CTTTGGAAAA TGGGACTGAG GTTGGGTGCG TCTGAGACAG TAACTCAGCC 11401 TGGGGGCTTG GTGAAGATCG CCGCACAGCA GCGAGTCCGT GAAATATCTT 11451 ATTTAGACTT GTGAGGTGCG CTGTGTGTCA ATTTACATCT TAAATCCTTT 11501 ATTGGCTGGA AAGAGAATTG TTGGAGTGGG TGAATCCAGC CAGGAGGGAC 11551 GCGGGGGGAT CCA EXAMPLE 9 Efficacy In vitro humanized DC45RO / RB binding antibodies VHE / humV1 v VHQ / humV1 In order to determine the effectiveness of the humanized CD45R0 / RB humanized antibodies, VHE / humV1 and VHQ / humV1 compared to the chimeric antibody, the ability of the antibody to induce apoptosis in human T cells was analyzed. the ability to inhibit the proliferation of human T cells.

Cells and reagents Peripheral blood mononuclear cells (PBMC) were isolated from leukapheresis samples from healthy human donors with known blood type, but unknown HLA type through centrifugation in Ficoll-Hypaque (Pharmacia LKB). The PBMC is used as the stimulators that were first consumed for T and NK cells using ferromagnetic beads coated with CD3 (Miltenyi). The pearls and pollution cells were removed through a magnetic field. The PBMC consumed from the T cells was used as cells that respond after irradiation (50 Gy). CD4 + T cells were used as responding cells in MLR and were isolated from PBMC with a negative CD4 T cell selection team (Miltenyi). The cells obtained were analyzed by FACScan or FACSCalibur (Becton Dickinson &Co., CA) and the purity of the obtained cells is > 75% Cells were suspended in RPM11640 medium supplemented with 10% FCS heat inactivated, penicillin, streptomycin and L-glutamine.

Apoptosis assays Human PBMC was cultured from three healthy volunteer donors in growth medium (RPM11640 + 10% FCS) overnight (< 16 hours) in the presence of chiral linker mAb CD45R0 / RB, humanized antibodies (VHE / humV1 and VHQ / humV1) or anti-LPS control mAb. If indicated, an interlacing reagent, a fragment of goat anti-human IgG F (ab ') 2 (Cat. No. 109-006-098, JacksonLab) was included at a concentration μg / ml being twice as high than the concentration of anti-CD45 antibodies in the sample. The concentration of PBS in all the cavities introduced through the reagents of the antibody was kept constant between all the samples, mainly 20% of the samples (v / v) for samples without an interlacer or 40% (v / v) for samples with interleaver. Previous experiments show that the amount of PBS does not affect the reading. After overnight culture in the presence of the antibodies, the samples were subjected to flow cytometric analysis and stained with the apoptosis marker AnnexinV-FtTC (Cat. No. 556419, BD / Pharmingen) and the cell marker T CD2-PE (Cat. No. 556609, BD / Pharmingen). The samples were run on a Becton Dickinson FACSCalibur instrument and the data was analyzed using the CelIQuest Pro software. For the collected data, the curves were adjusted using the Origin v7.0300 software. The equation used for the adjustment is Y = + A2 ("Sigmoid Logistics") 1 + (x / x0) p Ax final value (to adjust the sessions established for "shared" and "floated") A2: initial value (to adjust the sessions established for "shared" and "floated") p: energy X0: ED50; IC50 (see below). In the absence of the interleaver, VHE / humV1 is more effective, with an ED50 value of 148 + 71 nM, followed by VHQ / humV1 with 377 ± 219 nM. The CD45RB / RO binding chimeric antibody is less effective with an ED50 value of 2440 + 1205 nM. In the presence of an interlayer antiserum, the ED50 values are exchanged dramatically towards a higher efficiency through at least two orders of magnitude. In addition, the presence of the interlayer allowed higher levels of apoptosis at very high antibody concentrations, now reaching up to 80%, while the absence of the interlayer only allowed up to 50% of apoptosis. In the presence of the interleaver, the curves (antibody concentration /% apoptosis) are bi-modal with two levels: the first level was reached at low antibody concentrations (-5nM), where the level of apoptosis corresponds to the maximum level obtained in the absence of the interleaver. The second level was reached at very high antibody concentrations (approximately 500 nM) and apoptosis was observed within 70-80% of the T cell population. Both humanized CD45R0 / RB binding mAbs are equally effective and better or equal compared to the CD45R0 / RB chimeric binding mAb with respect to its ability to induce apoptosis in primary human T cells.

Mixed Lymphocyte Reaction Assays 1 × 10 5 PBMC or 5 × 10 4 CD4 + cells were mixed with PBMC consumed by irradiation 1 × 10 5 or 5 × 10 4 T cells (50 Gy) in each well of 96-well culture plates in the presence or absence of different concentrations of mAb. The mixed cells were cultured for 5 days and proliferation was determined by pulsing the cells with 3H-thymidine during the last 16-20 hours of the culture. The MLR inhibition at each antibody concentration was expressed as percent inhibition as described in Example 2. The effect of increasing concentrations of HEV / humV1 and VHQ / humV1 on MLR was evaluated in three responders: stimulator combinations. All antibodies inhibited the MLR in a dose-dependent manner. IC 0 values (see below) are similar for VHE / humV1 (7 ± 7 nM) and.VHQ / humV1 (39 ± 54 nM) of humanized Ab. Both humanized antibodies are more potent in the inhibition of MLR than the parent chimeric antibody (IC50 of 347 + 434 nM). As is usually seen with MLR experiments, the variability of the donor is high in these experiments.

VHE-N73D / humV1 To direct the biological effect of VHE-N73D / humV1 that induces apoptosis in peripheral blood mononuclear cells (PBMC), the target PBMC was incubated overnight in the presence of various concentrations of HEV-N73D / humV1 and was analyzed substantially for attachment of the AnnexinV apoptosis marker by flow cytometry analysis: the human PBMC was incubated overnight in 1 ml of tissue culture medium containing various concentrations of either VHE- N73D / humV1, or another variant of the humanized linker molecule CD45RO / RB VHE / humV1, or chimeric anti-CD45RO / RB mAb, or an isotope IgG 1 control mAb. In addition, goat anti-human Ig F interlaced F (ab ') 2 fragments were added at 2x mass concentration of each mAb, mimicking the interlacing of CD45RO / RB binding molecules through Fe receptors. , what can happen in vivo. The next day, the cells were washed through centrifugation for 10 minutes at 400 times the standard gravity force (g) and the medium was removed. Cells were resuspended in pH regulator FACS (PBS containing 1% v / v of FBS, 0. 1% w / v EDTA and 0.1% w / v sodium azide) and seeded in bottom microtitre plates V of 96 cavities at a cell density of 1 x 105 cells per well. Each cell sample was incubated for 30 minutes at 4 ° C in 50 μl of pH regulator FACS containing 100 μg / ml of normal mouse serum to block non-specific binding sites in cells and with CD2 conjugated with phycoerythrin (PE) ) to identify human cells. After washing twice via centrifugation, the cells were resuspended in 100 μl of calcium competent AnnexinV staining pH buffer (Vendor BD / Pharmingen kit 556419) containing 1: 100 v / v of AnnexinV labeled with FITC. After incubation for 15 minutes at room temperature in the dark, 7-amino actinomycin D (7-AAD) was added to a final concentration of 1 μg / ml and analyzed using a FACSCalibur flow cytometer (Becton Dickinson). The ED50 values for the effectiveness of the antibody to induce apoptosis can be calculated from an analysis of the amount of apoptosis (= FITC fluorescence intensity of AnnexinV) induced as a function of antibody concentration using the Origin 7.5 software with the adjustment equation of Sigmoidal curve / Logistics. After these analyzes, a two-phase effect of all the CD45RO / RB binding molecules tested was observed: at very low concentrations of the antibody, a low level of less than 30% of T-cell apoptosis was found. The ED50 value for reaching this level of apoptosis was calculated as 0.31 + 0.13 nM for VHE-N73D / humV1. At higher antibody concentrations, apoptosis can be induced in up to 70% of T cells. The ED50 value to reach this highest level of apoptosis was calculated as 352 + 83 nM for HEV-N73D / humV1. In summary, it was found that VHE-N73D / humV1 also induces apoptosis in human T cells, which can be improved through entanglement.

EXAMPLE 10 Specificity of the CD45RB / RO binding molecule Chimeric CD45RB / RO binding molecule The CD45 molecule was expressed in all leukocytes. However, different CD45 isoforms were expressed through the various subgroups of leukocytes. In order to determine the reactivity of the leukocyte subgroup of the CD45RB / RO antibody chimeric antibody immunofluorescently labeled molecule of human leukocytes with subgroup-specific markers and the subsequent immunofluorescent labeling was carried out with a chimeric antibody conjugate of CD45RB binding / RO with dye, followed by flow cytometry analysis. Briefly, specific subgroups of a newly isolated preparation of human peripheral blood mononuclear cells (PBMC), human platelets, human peripheral blood neutrophils or haematopoietic stem cells derived from spinal cord were identified through incubation with antibodies coupled to phycoerythrin against CD2 (T lymphocytes), CD14 (monocytes), CD19 (B lymphocytes), CD34 (stem cells), CD42a (platelets), CD56 (natural killer cells) or CD66b (granuloclints). Simultaneous binding of a FITC-labeled chimeric CD45RB / RO binding molecule was detected in T lymphocytes, monocytes, stem cells, natural killer cells and granulocytes, but not in platelets or B lymphocytes.VHE-N7'3D / humV1 In order to determine the reactivation of the subgroup of leukocytes VHE-N73D / humV1 immunofluorescently labeled human leukocytes with specific markers of the subgroup and the simultaneous immunofluorescent labeling with a VHE-N73D / humV1 conjugated with dye was carried out, followed by flow cytometric analysis. Briefly, specific subgroups of a newly isolated preparation of human peripheral blood mononuclear cells (PBMC) or human peripheral blood neutrophils were identified depending on incubation with phycoerythrin-coupled antibodies against CD3, CD4, CD8 (T lymphocytes), CD14 ( monocytes), CD16 (natural killer cells and monocytes), CD19 (B lymphocytes), or CD66b (granulocytes). The simultaneous binding of VHE-N73D / humV1 labeled with FITC was detected in T lymphocytes, monocytes, natural killer cells and granulocytes, but not in B lymphocytes. In this way, the VHE-N73D / humV1 molecules do not react with human B lymphocytes.

EXAMPLE 11 In vitro induction of suppressor T cells (regulatory T cells) and functionally paralyzed T cells To demonstrate the ability of a CD45RO / RB chimeric binding antibody to induce suppressor T cells, the antibody is included at various concentrations during the generation of CD8 + T cell lines reactive with antigen (MP1) matrix protein 1 of hemophilic influenza. . These lines are generated through the repeated co-culture of human CD8 + lymphocytes with human CD14 + monocytes pulsed with the antigen. Then, in CD14 + monocytes, they can be replaced with a line of leukocyte antigen-2 positive cells such as the MP1 antigen presenting a cell (APC). If said MP1-specific CD8 + T cells from a culture including the CD45RO / RB chimeric binding antibody are mixed with newly isolated human CD8 + T cells and this cell mixture is stimulated with the MP1 antigen in APC, the addition of CD8 + T cells of the culture in the presence of the binding molecule CD45RO / RB is able to reduce the production of IFN-? in a dose-dependent manner of antibody. No CD45RO / RB binding chimeric antibody is present during this IFN-α assay culture, indicating that pretreatment with the CD45RO / RB mAb mAb has induced CD8 + T cells capable of suppressing the activation of newly isolated T cells. Due to this induction of suppressor T regulatory cells through the chimeric binding antibody CD45RO / RB, the antibody can be useful in diseases where it is believed that a population of unregulated and / or activated T cells contributes to the pathology. Examples of such diseases include autoimmune diseases, transplant rejection, psoriasis, dermatitis, inflammatory bowel disease and allergies. To demonstrate the ability of a CD45RO / RB chimeric binding molecule to convert hyporesponsive (anergic) T cells to additional stimulation, ie, in functionally paralyzed T cells, the antibody is included during generation of CD8 + T cell lines reactive with the protein 1 of the hemophilic influenza antigen (MP1) matrix as mentioned above. Paralysis is evaluated by activating the T cells (previously exposed to the chimeric binding antibody CD45RO / RB) with the MP1 antigen presented through APC. No CD45RO / RB binding molecule is present in this culture. CD8 + T cells not exposed to the CD45RO / RB chimeric binding antibody previously produce IFN-γ. depending on the mentioned stimulus. In contrast, CD8 + T cells pretreated with the CD45RO / RB chimeric binding antibody are markedly reduced in the non-existent production of this cytokine in response to antigen challenge, demonstrating the ability of the CD45RO / RB chimeric binding antibody to functionally paralyze cells Human T Because of this induction of functional T cell hyporesponsiveness through the CD45RO / RB binding molecule, the antibody can be used in diseases, such as autoimmune diseases, rejection to transplantation, psoriasis, dermatitis, inflammatory bowel disease or allergies, wherein a population of activated T cells is believed to contribute to the pathology.

EXAMPLE 12 In vivo skin studies of SCID-hu mice The therapeutic benefit of CD45RB / RO binding chimeric antibody in skin inflammation was tested using SCID mice. Human skin from normal individuals was transplanted into SCID mice (SCID-hu skin) and the inflammatory process was mimicked through the adoptive transfer of mononuclear leukocytes isolated from human individuals unrelated to the human skin donor.

Human adult skin transplantation in SCID mice (Skin of SCID-hu mice) Two small pieces of human adult skin (1 cm2) (obtained from the Regional Tissue Bank of Eastern Hungary; WHRTB, Gyor) consisting of complete epidermis, the papillary dermis and part of the reticular dermis, were transplanted on the upper right and left back sides of SCIDC CB 17 mice / GbmsTac-Pr mice with Lystba csc (Taconic, Germantown, NY ) in replacement of mouse skin. The quality of the grafts was monitored for 5-6 weeks after transplantation and the successfully transplanted mice (skin from SCID-hu mice, generally> 85%) were selected for in vivo testing of the CD45RB / RO binding chimeric antibody.

Grafting mononuclear cells in SCID mice Mononuclear leukocytes (Spl) were isolated from spleen biopsies of human adults (WHRTB, Gyor) after cell suspension (using a cell dissociation molecular sieve equipped with a 50 mesh size) ) and standard density gradient procedures. The aliquots of 5 x 108 SpI were resuspended in 1.5 ml of RPMI-10% FCS and injected peritoneally (i.p.), on day 0 of the experiment, into the skin of the SCID-hu mice. These SpI numbers have been found in previous experiments as being sufficient to induce a lethal xeno-GvHD in > 90% of mice within 4-6 weeks after cell transfer.

Skin antibody treatment of SCID-hu mice The skin of SCID-hu mice, reconstituted with human SpI, was treated with the chimeric binding antibody CD45RB / RO or control mAb anti-LPS on day 0, immediately after the injection of the mononuclear cell, on days 3 and 7 and weekly intervals thereafter. Antibodies were distributed subcutaneously (s.c.) in 100 μl of PBS to a final concentration of 1 mg / kg of body weight (p.c.).

Evaluation of the anti-CD45 treatment The efficacy of the CD45RB / RO binding chimeric antibody is evaluated through the survival of the transplanted mice and by monitoring the rejection of skin grafts. The importance of the results is evaluated through the statistical method of survival analysis using the Log-rank test (Mantel method) with the help of Systat v10 software. At the end of the experiment, biopsies of human skin and liver, lung, kidney and spleen grafts were obtained from mice sacrificed for histological purposes. All mice were weighed at the beginning (before the cell transfer) and throughout the experiment (every two days) as an indirect estimate of their health status. Linear regression lines were generated using the body weight against the values of the days after the PBMC transfer obtained from each mouse and subsequently, their inclinations (control against mice treated with anti-CD45) were compared using the Mann-Whitney test non-parametric Results Human skin grafts are very well tolerated by SCID mice. Initially, the grafts experienced a period of keratinocyte hyperproliferation resulting in the formation of hyperkeratotic scabs. About 5 weeks after the transplant, the scabs fell off the grafts and revealed a tissue containing all the characteristic structures observed in normal human skin. During this process, human skin grafts were fused with the adjacent skin of the mouse and generated a network of newly created human vessels that connect the grafts to the underlying mouse tissue. The human SpI in circulation within the skin of the mouse SCID-hu (in experimental day 0, approximately 6 weeks after the skin transplant) if it infiltrated the skin grafts and after recognition the alloantigen molecules expressed in the human skin mounted an inflammatory response, which has similarity with the inflammatory process of the skin that occurs in psoriatic skin , and this in some cases completely destroys the graft. The treatment of these mice with the CD45RB / RO binding chimeric antibody suppresses the inflammatory process and prevents rejection of human skin grafts. In constaste, the sample obtained from the mouse treated with control shows a massive infiltration with multiple signs of necrosis and a dramatic destruction of the epidermis. This process is easily monitored at a glance and documented by a simple photograph of the mice. The skin of six of the six SCID-hu mice transferred with allogeneic SpI and treated with control anti-LPS mAb shows a strong inflammatory response to clearly visible to the naked eye 23 days after the mononuclear cell transfer. All mice show considerable lesions, including erythema, and escalating and pronounced pustules. In contrast, the skin grafts of all mice treated with the CD45RB / RO binding chimeric antibody have a normal appearance. The dramatic differences between the two groups of mice are specifically due to the treatment of the antibody and that the human skin of all mice looks identical at the beginning of the experiment. This aspect does not change until the second week after the cell transfer, the moment in which the control group initiated the development of skin lesions. The experiment was terminated on day 34 after the mononuclear cell transfer. At this time, one of the control mice is already dead (day 30) and four others were sacrificed (days 27, 27, 27 and 30) due to a strong xeno-GvHD. The pathological reactions observed in the mice treated with the control of the antibody also correlate with a loss of body weight in these animals.

In contrast, the group treated with the CD45RB / RO binding chimeric antibody exhibits a healthy state during the entire experiment time.

EXAMPLE 13 In vivo activities of a CD45Rb / RO binding chimeric antibody in a model of human islet cell transplantation Mice Female NOD / SCID (Charles River Laboratories, Calco, Italy) free of conditions were kept under a specific pathogen. The blood glucose level of the venous tail was quantified using the Glucometer Elite system (Bayer, Germany). Diabetes was induced in NOD / SCID mice through intravenous injection of 180 mg / kg streptozotocin (Sigma, St. Louis, MO). A diagnosis of diabetes is made after two consecutive glucose measurements higher than 250 mg / dL.

Preparations and transplantation of Isleta Pancreata is obtained from cadaveric multi-organ donors defying the heat. The islands were isolated according to the method described in Bertuzzi et al., Diabetes, 1999, 48: 1971-8. The purified islets were cultured in sterile flasks containing 25 ml of M199 medium (Seromed Biochrom, Berlin, Germany) supplemented with 10% FCS, 1% L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin ( half full). The islands were incubated at 30 ° C in 5% C02 and 95% humidified air. Mice were anesthetized with an intraperitoneal injection of avertin and aliquots of 1500 EI of human eggs were transplanted under the kidney capsule of the NOD / SCID recipient diabetic mice as described in Davalli AM et al., Diabetes, 1996, 45: 1161 -7. After transplantation, 50 x 106 of newly isolated PBMC were intraperitoneally injected to the NOD / SCID mice.

Treatment of transplanted mice The transplanted Hu-PBL-NOD / SCID mice were treated a.c. on day 0, +3 and +5 with 1 mg / kg of a CD45RB / RO binding chimeric antibody. The control mice were treated either with saline or with purified mAb of IgG (Vinci, Biochem, Italy).

Histological Analysis The poles of the kidney containing human islet graft were frozen in Tek tissue (Miles Lab., IN) and stored at -70 °. Frozen 5 μm thick sections were stained with biotinylated mAb against human insulin or human CD3 followed by streptavidin peroxidase conjugate. Diaminonbenzidine (DAKO, Carpenteria, CA) was used as a chromogen and hematozilyn as anti-dye. The lymphocyte infiltration of the grafts was evaluated in the frozen sections stained with hematoxylin and eosin.

Results Normal NOD / SCID mice transplanted with human islets remained normoglycemic until 100 days after transplantation, while the mean rejection time of hu-PBL-NOD / SCID mice transplanted with human islets is 3513 days. The short treatment of hu-PBL-NOD / SCID mice transplanted with human islets with a mAb of the present invention significantly prolongs the survival of human islets with a survival rate of > 70% on day 60 and 50% on day 100 after transplant. Histological analysis of human islet grafts carried out on day 100 after transplantation in hu-PBL-NOD / SCID mice shows a massive infiltration of CD3 +, CD4 + and CD8 + positive T cells in the control recipient mouse. In contrast, in the recipient mice treated with a CD45RB / RO binding chimeric antibody, a lower infiltration of human cells was observed. Positive insulin staining demonstrates that graft function in the hu-PBL-NOD / SCID receptor mice treated with the chimeric CD45RB / RO binding antibody compared to the control recipient mice. In hu-PBL-NOD / SCID mice treated with a CD45RB / RO binding chimeric antibody and with islet transplantation, lower amounts of IFN-α were detected. human serum compared to the control recipient mice. These data indicate that short-term treatment with a CD45RB / RO binding chimeric antibody leads to prolonged human islet allograft survival through the prevention of graft infiltration and inhibiting the leukocyte-mediated graft rejection reaction in vivo. .

Claims

1. A linker molecule comprising at least one antigen binding site, comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-lle-lle-His (NYIIH), said CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT).

2. A linker molecule according to claim 1, comprising: a) a first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-lle-lle-His (NYIIH), said CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT); and b) a second domain comprising in sequence the hypervariable regions CDR1 ', CDR2"and CDR3', CDR1 'has the amino acid sequence Arg-Ala-Ser-GIn-Asn-lle-Gly-Thr-Ser-lle-Gin ( RASQNIGTSIQ), CDR2 'has the amino acid sequence Ser-Ser-Ser-Glu-Ser-lle-Ser (SSSESIS) and CDR3' has the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe- Thr (QQSNTWPFT).

3. A linker molecule according to any of claims 1 or 2, which is a chimeric or humanized linker molecule.

4. A linker molecule according to any of claims 1 to 3, wherein the chimeric or humanized linker molecule is a chimeric or humanized monoclonal antibody.

5. A linker molecule according to any of claims 1 to 4, which comprises no more than one glycosylation site.

6. A linker molecule according to claim 5, wherein the glycosylation site is an N-glycosylation site.

7. A linker molecule according to any of claims 1 to 6, wherein no glycosylation site is present in the variable region.

8. A linker molecule according to any of claims 1 to 7, comprising a polypeptide of SEQ ID NO: 1 and / or a polypeptide of SEQ ID NO: 2.

9. A linker molecule according to any of claims 1 to 7, comprising a polypeptide of SEQ ID NO: 3 and / or a polypeptide of SEQ ID NO: 4.

A binding molecule according to any of claims 8 or 9 which is a chimeric monoclonal antibody.

11. A binding molecule that is a humanized antibody comprising a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8.

12. A binding molecule which is a humanized antibody comprising a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8.

13. A molecule of linkage which is a humanized antibody comprising: a polypeptide of SEQ ID NO: 9 and a polypeptide of SEQ ID NO: 7, a polypeptide of SEQ ID NO: 9 and a polypeptide of SEQ ID NO: 8, a polypeptide of SEQ ID NO: 10 and a polypeptide of SEQ ID NO: 10 NO: 7, - a polypeptide of SEQ ID NO: 10 and a polypeptide of SEQ ID NO: 8, - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 7, - a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 8, - a polypeptide of SEQ ID NO: 32 and a polypeptide of SEQ ID NO: 7, or - a polypeptide of SEQ ID NO: 32 and a polypeptide of SEQ ID NO: 7 NO: 8.

14. The isolated polynucleotides comprising polynucleotides encoding a linker molecule according to any of claims 1 to 13.

15. The polynucleotides according to claim 14, which encodes the amino acid sequence of CDR1, CDR2. and CDR3 according to claim 2 and / or polynucleotides encoding the amino acid sequence of CDR1 ', CDR2' and CDR3 'according to claim 2.

16. Polynucleotides encoding the amino acid sequence of CDR1', CDR2 ' and CDR3 ', according to said CDR1' have the amino acid sequence Arg-Ala-Ser-GIn-Asn-lle-Gly-Thr-Ser-lle-Gln (RASQNIGTSIQ), CDR2 'has the amino acid sequence Ser-Ser -Ser-Glu-Ser-lle-Ser (SSSESIS) and CDR3 'has the amino acid sequence Gln-GIn-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT).

17. Polynucleotides comprising a polynucleotide of SEQ ID NO: 5 and / or a polynucleotide of SEQ ID NO: 6.

18. Polynucleotides comprising polynucleotides that encode a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8 and / or a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10.

19. Polynucleotides comprising a polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 12 and / or a polynucleotide of SEQ ID NO: 13 or SEQ ID NO: 13 NO: 14.

20. Polynucleotides comprising polynucleotides that encode a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8 and / or a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32.

21. Polynucleotides that comprise a polynucleotide of SEQ ID NO: 34 or SEQ ID NO: 35 and / or a polynucleotide of SEQ ID NO: 33; SEQ ID NO: 14 or SEQ ID NO: 13.

22. An expression vector comprising polynucleotides according to any of claims 14 to 21.

23. An expression system comprising a polynucleotide according to any of claims 14. to 21, wherein said expression system or part thereof is capable of producing a polypeptide of any of claims 1 to 13, when said expression system or part thereof is present in a compatible host cell.

24. An expression system comprising a polynucleotide according to any of claims 14 to 21, wherein said expression system or part thereof is capable of producing a binding molecule comprising at least one antigen binding site. , which comprises in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 has the amino acid sequence Asn-Tyr-IIe-ile-His (NYIIH), said CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr -Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp -Thr (SGPYAWFDT), when said expression system or part thereof is present in a compatible host cell.

25. An isolated host cell comprising an expression system according to claim 23 or 24.

26. The use of a binding molecule or a humanized antibody according to any of claims 1 to 13 as a pharmaceutical.

27. The use according to claim 26 in the treatment and / or prophylaxis of autoimmune diseases, rejection to transplantation, psoriasis, dermatitis, inflammatory bowel disease and / or allergies.

28. The use according to claim 27 in the treatment and / or prophylaxis of graft-versus-host disease (GVHD).

29. The use of a binding molecule or a humanized antibody according to any of claims 1 to 13 in the preparation of a medicament for the treatment of rejection of pancreatic islet cell transplantation.

30. The use according to any of claims 26 to 29, wherein the binding molecule or a humanized antibody comprises a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO. : 7 or of SEQ ID NO: 8.

31. A pharmaceutical composition comprising a molecule or a humanized antibody according to any of claims 1 to 13 in association with at least one pharmaceutically acceptable carrier or diluent.

32. A method for the treatment and / or prophylaxis of diseases associated with autoimmune diseases, rejection to transplantation, psoriasis, dermatitis, inflammatory bowel disease and / or allergies, which comprises administering to a subject in need of such treatment and / or prophylaxis an effective amount of a humanized molecule or antibody according to any one of claims 1 to 13.

33. A method for the treatment and / or prophylaxis of psoriasis comprising administering to a subject in need of said treatment and / or prophylaxis an effective amount of a binding molecule or a humanized antibody according to any one of claims 1 to 13.

34. A method for the treatment and / or prophylaxis of graft-versus-host disease (GVHD) comprising administering to a subject in need of such treatment and / or prophylaxis an effective amount of a humanized molecule or antibody in accordance with any of claims 1 to 13.

35. A method for the treatment and / or prophylaxis of a disease associated with rejection of pancreatic islet cell transplantation comprising administering to a subject in need of such treatment and / or prophylaxis a The effective amount of a binding molecule or a humanized antibody according to any one of claims 1 to 13.

36. The method according to any of claims 32 to 35., wherein the binding molecule or the humanized antibody comprises a polypeptide of SEQ ID NO: 31 or SEQ ID NO: 32 and / or a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8.

37. The use of a binding molecule having a binding specificity for both CD45RO and CD45RB in medicine.

38. The use according to claim 37, wherein the binding molecule is a chimeric, humanized or fully human monoclonal antibody.

39. The use according to claim 37 or 38, wherein the binding molecule binds to a CD45RO isoform with a dissociation constant (Kd) of < 15nM.

40. The use according to any of claims 37 to 38, wherein the binding molecule is linked to an isoform CD45RB with a dissociation constant (Kd) < 15nM.

41. The use according to any of claims 37 to 40, wherein the binding molecule binds to the CD45 isoforms which - include the A and B epitopes, but not the C epitope of the CD45 molecule; and / or - includes the B epitope, but not the A and not the C epitope of the CD45 molecule; and / or - does not include any of the A, B or C epitopes of the CD45 molecule.

42. The use according to any of claims 37 to 41, wherein the binding molecule does not bind to the CD45 isoforms which - include all epitopes A, B, and C of the CD45 molecule; and / or - includes both B and C epitopes, but not the epitope, A of the CD45 molecule.

43. The use according to any of claims 37 to 42, wherein the binding molecule binds to its target epitope in PEER cells, and wherein said linkage is with a Kd < 15nM.