HK1208168B - Monoclonal antibodies for use in diagnosis and therapy of cancers and autoimmune disease - Google Patents

Description

Monoclonal antibodies for the diagnosis and treatment of cancer and autoimmune diseases

This application claims priority to U.S. Provisional Application Serial No. 61/669,967, filed on July 10, 2012, and U.S. Provisional Application Serial No. 61/702,916, filed on September 19, 2012, both of which are incorporated herein by reference in their entireties.

Background of the Invention

This invention was made with government support under P50CA100632 awarded by the National Cancer Institute/National Institutes of Health. The government has certain rights in this invention.

1. Technical Field

The present invention relates generally to the fields of cancer and immunotherapy. More particularly, it relates to immunodiagnostic and immunotherapeutic antibodies for the treatment and prevention of cancer and autoimmune diseases.

2. Background Technology

The immune system has long been implicated in cancer control; however, evidence for specific and effective immune responses in human cancers is lacking. In chronic myelogenous leukemia (CML), allogeneic bone marrow transplantation (BMT) or interferon-α2b (IFN-α2b) therapy have produced complete remissions, but the mechanisms of disease control are unknown and may involve immune anti-leukemic responses.

Based on evidence in the art, it is believed that lymphocytes play a role in mediating anti-leukemic effects. Studies have shown that allogeneic donor lymphocyte infusion (DLI) has been used to treat relapses of myeloid leukemia after allogeneic BMT (Giralt and Kolb, 1996; Kolb and Holler, 1997; Kolb et al., 1995; Kolb et al., 1996; Antin, 1993). In approximately 70% to 80% of patients with chronic myeloid leukemia (CML) in chronic phase (CP), lymphocyte infusion from the original bone marrow (BM) donor induces both hematological and cytogenetic responses (Kolb et al., 1996; Kolb and Holler, 1997). For AML, remission after DLI is generally not as durable as the remission obtained in chronic phase CML, which may reflect the rapid kinetics of tumor growth exceeding the kinetics of the immune response. In addition, most patients with myeloid forms of leukemia will die from the disease unless they can be treated with an allogeneic bone marrow transplant, in which the associated graft-versus-leukemia (GVL) effect cures the patient. However, graft-versus-host disease (GVHD) and transplant-related toxicities limit this treatment. It is believed that GVL can be separated from GHVD, and that targeting the immune response against leukemia-associated antigens will allow GVL to be transferred to patients without GVHD.

Therefore, if more antigens can be identified (i.e., leukemia antigens or antigens for other cancers) and if large numbers of the most potent antigen-specific cytotoxic T lymphocytes (CTLs) can be obtained, it will allow the development of leukemia-specific therapies, breast cancer-specific therapies, etc., using the antigens as targets for vaccines or for generating specific T cells for adoptive immunotherapy.

PR1 (a HLA-A2.1-restricted nonamer derived from proteinase 3 (P3) and elastase) was identified as a leukemia-associated antigen (Molldrem et al., 2000; Molldrem et al., 1996; Molldrem et al., 1997; Molldrem et al., 1999; Molldrem et al., 2003, each of which is incorporated herein by reference in its entirety). The discovery that PR1 is a leukemia-associated antigen has been independently confirmed by Burchert et al. (2002) and Scheibenbogen et al. (2002). CTLs specific for PR1 kill AML, CML, and MDS cells, but not normal bone marrow cells. In a recent phase I/II vaccine study, PR1 peptides were administered to AML, CML, and MDS patients, and PR1-specific CTL immunity was elicited in 47% of patients, while clinical responses were observed in 26% of patients. Therefore, this antigen provides an interesting platform for further studying anticancer immune responses and developing new therapeutic agents.

Summary of the Invention

Thus, according to the present invention, isolated and purified antibodies are provided that bind to VLQELNVTV (SEQ ID NO: 45) when bound by the HLA-A2 receptor, the antibodies having heavy chain CDRs comprising SEQ ID NOs: 3, 60, and 5 and light chain CDRs comprising SEQ ID NOs: 8, 9, and 10. The antibodies can be mouse antibodies, single-chain antibodies, bispecific antibodies, fused to non-antibody peptide or polypeptide segments, linked to diagnostic agents (e.g., fluorophores, chromophores, dyes, radioisotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents), linked to therapeutic agents (e.g., cytokines, chemotherapeutic agents, radiotherapeutic agents, hormones, antibody Fc fragments, TLR agonists, CpG-containing molecules, or immune co-stimulatory molecules), humanized antibodies, or combinations thereof. In addition to binding affinity for SEQ ID NO: 45, the bispecific antibody may also have binding affinity for B cells (CD19, CD20), NK cells, phagocytes (CD16), or monocytes (CD14). Particularly humanized antibodies may have light chain/heavy chain sequences of SEQ ID NOs: 40 and 38, or SEQ ID NOs: 42 and 38 and SEQ ID NOs: 42 and 44.

In another embodiment, a nucleic acid encoding the light chain CDRs encoded by SEQ ID NOs: 8, 9, and 10 is provided. The nucleic acid may encode SEQ ID NO: 7 or SEQ ID NO: 14, or may encode SEQ ID NO: 25 or SEQ ID NO: 27. The nucleic acid may further comprise a promoter sequence located 5' of the nucleic acid encoding the light chain CDRs, for example, a promoter sequence active in eukaryotic or prokaryotic cells. The nucleic acid may be located in a replicable vector (e.g., a non-viral or viral vector). The nucleic acid may further comprise a linker encoding segment, wherein the linker encoding segment is located between the CDR encoding segments, for example, a segment encoding a helix-turn-helix motif.

In another embodiment, an artificial antibody is provided, comprising a heavy chain encoding segment comprising CDRs comprising the sequences of SEQ ID NOs: 3, 60, and 5; and a light chain encoding segment comprising CDRs comprising the sequences of SEQ ID NOs: 8, 9, and 10. The CDRs can be linked by a synthetic linker. The heavy chain can be fused to a non-antibody peptide or polypeptide segment. The antibody can be linked to a diagnostic agent (e.g., a fluorophore, a chromophore, a dye, a radioisotope, a chemiluminescent molecule, a paramagnetic ion, or a spin trapping agent). The antibody can be linked to a therapeutic agent (e.g., a cytokine, a toxin, a chemotherapeutic agent, a radiotherapeutic agent, a hormone, an antibody Fc fragment, neutrophil elastase, proteinase 3, a TLR agonist, a CpG-containing molecule, or an immune co-stimulatory molecule). The light chain can comprise SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 25, or SEQ ID NO: 27, and/or the heavy chain comprises SEQ ID NO: 38 or SEQ ID NO: 44.

In yet another embodiment, a method for producing an antibody is provided, comprising: (a) introducing into a host cell (i) a nucleic acid sequence encoding a heavy chain comprising the CDRs set forth in SEQ ID NOs: 3, 60, and 5 and (ii) a nucleic acid sequence encoding a light chain comprising the CDRs set forth in SEQ ID NOs: 8, 9, and 10; and (b) culturing the host cell under conditions that support expression of the light and heavy chains. The method may further comprise isolating the antibody. The method may further comprise linking the antibody to a diagnostic or therapeutic agent.

In another embodiment, a method for detecting abnormal cells in a sample suspected of containing abnormal cells is provided, which includes contacting the sample with an antibody or artificial antibody as described above. The antibody or artificial antibody can be conjugated to a diagnostic agent (e.g., a fluorophore, a chromophore, a dye, a radioisotope, a chemiluminescent molecule, a paramagnetic ion, or a spin capture agent). The antibody or artificial antibody can be detected using a second binding agent (e.g., an anti-Fc receptor antibody). The sample can be (a) tumor tissue from the head and neck, brain, esophagus, breast, lung, liver, spleen, stomach, small intestine, large intestine, rectum, ovary, uterus, cervix, prostate, testicle, or skin tissue, or (b) fluid such as blood, lymph, urine, bone marrow aspirate, or nipple aspirate. The sample can come from a resected tumor bed. The method can also include making a treatment decision based on the presence, absence, or degree of detection, such as a decision to treat the subject with a peptide vaccine based on PR-1. The method can detect primary cancer cells, metastatic cancer cells, or myeloid dysplastic cells.

In another embodiment, a method for treating a subject suffering from cancer is provided, comprising administering an antibody or artificial antibody as described above to the subject. The antibody or artificial antibody can be conjugated with a therapeutic agent. The cancer can be a solid tumor, such as a head and neck tumor, a brain tumor, an esophageal tumor, a breast tumor, a lung tumor, a liver tumor, a spleen tumor, and a stomach tumor, a small intestine tumor, a colorectal tumor, a rectal tumor, an ovarian tumor, a uterine tumor, a cervical tumor, a prostate tumor, a testicular tumor, or a skin tumor. The cancer can be a blood cancer, such as a leukemia or a lymphoma. The therapeutic agent can be a cytokine, a toxin, a chemotherapeutic agent, a radiotherapeutic agent, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule, or an immune co-stimulatory molecule. The method can also include providing a second anticancer therapy to the subject, such as gene therapy, chemotherapy, radiotherapy, hormone therapy, toxin therapy, or surgery. The antibody or artificial antibody can be administered more than once to the subject. The cancer can be a recurrent cancer or a metastatic cancer. The antibody can be administered more than once to the subject.

In yet another embodiment, a method for treating a subject suffering from an autoimmune disease is provided, comprising administering to the subject an antibody or artificial antibody as described above. The autoimmune disease may be Wegener's granulomatosis, Churg-Strauss syndrome, or systemic small vessel vasculitis. The antibody or artificial antibody may be conjugated to a therapeutic agent (e.g., a toxin or an apoptosis-inducing agent). The method may further comprise providing the subject with a second anti-autoimmune therapy. The second anti-autoimmune therapy may be an anti-inflammatory agent. The antibody may be administered to the subject more than once.

Also provided is a method of inducing complement-mediated cytotoxicity in HLA-A2 cancer cells, comprising contacting the cancer cells with an antibody or artificial antibody as described above.

Another embodiment of the present invention provides a method for detecting abnormal cells in a sample suspected of containing abnormal cells, which includes contacting the sample with an antibody or artificial antibody as described above. The antibody or artificial antibody can be conjugated with a diagnostic agent (e.g., a fluorophore, a chromophore, a dye, a radioisotope, a chemiluminescent molecule, a paramagnetic ion, or a spin trapping agent). The antibody or artificial antibody can be detected using a second binding agent (e.g., an anti-Fc receptor antibody). The sample can be (a) tumor tissue from the head and neck, brain, esophagus, breast, lung, liver, spleen, stomach, small intestine, large intestine, rectum, ovary, uterus, cervix, prostate, testicle, or skin tissue, or (b) fluid such as blood, lymph, urine, bone marrow aspirate, or nipple aspirate. The sample can come from a resected tumor bed. The method can also include making a treatment decision based on the presence, absence, or degree of detection, such as a decision to treat the subject with a peptide vaccine based on PR-1. The method can detect primary cancer cells, metastatic cancer cells, or myeloid dysplasia cells.

In another embodiment, there is provided a method for treating an object suffering from cancer, which includes administering an antibody or artificial antibody as described above to the object. The antibody or artificial antibody can be conjugated with a therapeutic agent (e.g., a cytokine, a toxin, a chemotherapeutic agent, a radiotherapeutic agent, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule or an immune co-stimulatory molecule). The cancer can be a solid tumor, such as a head and neck tumor, a brain tumor, an esophageal tumor, a breast tumor, a lung tumor, a liver tumor, a spleen tumor and a stomach tumor, a small intestine tumor, a colorectal tumor, a rectal tumor, an ovarian tumor, a uterine tumor, a cervical tumor, a prostate tumor, a testicular tumor or a skin tumor. Alternatively, the cancer can be a blood cancer, such as a leukemia or a lymphoma. The cancer can be a recurrent cancer or a metastatic cancer. The method can also include providing a second anticancer treatment to the object, such as gene therapy, chemotherapy, radiotherapy, hormone therapy, toxin therapy or surgery. The antibody or artificial antibody can be administered more than once to the object.

In yet another embodiment, a method for treating a subject suffering from an autoimmune disease is provided, comprising administering to the subject an antibody or artificial antibody as described above. The autoimmune disease may be Wegener's granulomatosis, Churg-Strauss syndrome, or systemic vasculitis. The antibody or artificial antibody may be conjugated to a therapeutic agent (e.g., a toxin or an apoptosis-inducing agent). The method may further comprise providing the subject with a second anti-autoimmune therapy, such as an anti-inflammatory agent. The antibody may be administered to the subject more than once.

Additional methods include: (i) treating a subject having a myelodysplastic disorder, comprising administering to the patient an antibody or artificial antibody as described above; and (ii) inducing complement-mediated cytotoxicity in HLA-A2 cancer cells, comprising contacting the cancer cells with an antibody or artificial antibody as described above.

Hu1-8F4 and Hu2-8F4 are referred to as Hu8F4-1 and Hu8F4-2, respectively. In addition, in this document the term "Hu8F4" generally refers to the two humanized forms of 8F4 (Hu8F4-1 and Hu8F4-2).

As used in the specification herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the words "comprising/including", the words "a" or "an" may mean one or more than one. As used herein, "another" may mean at least another one or more.

Other objects and features of the present invention will become apparent from the following detailed description. However, it should be understood that although some preferred embodiments of the present invention are indicated, this description and specific examples are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art through this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 - Specificity of 8F4 for PR1/HLA-A2 . ELISA performed using recombinant peptide/HLA-A2 monomers loaded with PR1 or single-amino acid-modified PR1 analogs. To determine the amino acid positions within the PR1 sequence essential for optimal 8F4 binding, HLA-A2 monomers loaded with peptides containing alanine substitutions in PR1 (ALA1-ALA9) were coated onto microtiter wells at increasing concentrations. The wells were then incubated with a fixed concentration of 8F4 or the control antibody bb7.2 (a mouse IgG2a monoclonal antibody specific for the HLA-A*0201 allele). Binding was measured by ELISA using a peroxidase-conjugated goat anti-mouse antibody. 8F4 bound to HLA-A2 loaded with PR1 and to most PR1 analogs, but bound significantly less to an ALA1 analog (with a valine substitution at position 1 of the peptide) and did not bind to the control peptide pp65/HLA-A2. The control antibody bb7.2 bound equally well to HLA-A2 loaded with PR1 and pp65.

Figure 2 - Affinity of the 8F4 monoclonal antibody for PR1/HLA-A2 . Affinity measurements of peptide/HLA-A2 binding to 8F4 and bb7.2 antibodies were determined by surface plasmon resonance using a BIAcore 3000. Test antibodies were captured onto an anti-mouse antibody-coated surface. Analyte peptide/HLA-A2 was diluted to 100 nM and tested for binding to the antibody-coated surface in duplicate. The assay was performed at 25°C using PBS, 0.005% Tween-20, 0.1 mg/ml BSA, pH 7.4 as running buffer. To obtain binding affinities, the experimental data were fit to a first-order kinetic model (shown as the orange line in the figure), and the _KD for 8F4 and bb7.2 was subsequently determined.

Figure 3 - Specificity of 8F4 for HLA-A2+AML . Multiparameter flow cytometry of leukemia and normal PBMCs was performed using 8F4 and cell surface antibodies. PBMCs from AML patients and normal donors were gated on live cells using aqua, then stained with 8F4 (conjugated with ALEXA Fluor 647), bb7.2 (conjugated with FITC), and surface phenotypic antibodies for CD13 and CD33, and analyzed by flow cytometry. The following gating strategy was used: First, live cells in water were analyzed for CD13 and CD33 expression, and double-positive cells were analyzed for PR1/HLA-A2 (8F4) expression and total HLA-A2 expression (bb7.2). Negative quadrant gating was established using HLA-A2 negative AML control cells.

Figure 4A-B - 8F4 antibody induces complement-dependent cytotoxicity (CDC) in AML . Target cells were washed and resuspended in 10-well RPMI/HEPES at a concentration of 5 × ¹⁰ cells/ml. 20 microliters (μl) of antibody were mixed with 100 μl of cells in a 96-well plate and warmed to 37°C. Then, 20 μl of ice-cold standard rabbit complement (Cedarlane, Ontario, Canada) was added and incubated at 37°C for 90 minutes. Cytotoxicity was determined using the Cyto-Tox Glo cytotoxicity assay (Promega). Antibody-specific CDC (AB-CDC) was calculated as: AB-CDC = ((LC _+AB - _LC-AB ) / ( _Lmax - _Ls )) × 100%, where _LC+AB is target cell lysis in the presence of complement plus antibody; LT _+C is lysis in the presence of complement alone; _Lspont and _Lmax were measured before and after the addition of the cytotoxic agent digitonin to the cells according to the manufacturer's instructions. (Figure 4A) Incubation with 20 μg of 8F4 induced complement-dependent cytotoxicity in PBMCs and leukapheresis (LP) cells from patients with HLA-A2+ AML, but did not lyse control samples of PBMCs from HLA-A2-negative AML or PBMCs from HLA-A2+ normal donors. (Figure 4B) 8F4-mediated lysis of HLA-A2+ AML was antibody dose-dependent, whereas an isotype control antibody (IgG2a mouse anti-KLH) and human intravenous immunoglobulin (commercial IVIG) did not show AML lysis.

Figure 5 - 8F4 is specific for AML but not for normal PBMC . Surface staining of AML, PBMC, and T2 cells for PR1 and HLA-A2. Cells were stained with anti-PR1/HLA-A2 antibody (8F4)-alexa-647 (red) and FITC-conjugated anti-HLA-A2 (green), fixed with 1% paraformaldehyde, and then studied using confocal microscopy. T2 cells were pulsed with PR1 peptide (20 μg/ml) as a positive control and CMV peptide pp65 (20 μg/ml) as a negative control peptide. PR1/HLA-A2 expression was evident on the cell surface of AML and PR1-pulsed T2 cells, but not on HLA-A2+ PBMCs or on pp65-pulsed T2 cells. Dapi blue was used for nuclear staining.

Figure 6 - 8F4 antibody prevents engraftment of AML in an in vitro model . Primary HLA-A2+ leukemia cells ( ¹⁰⁶ ) were washed, resuspended in PBS (100 μl), mixed with 8F4 or isotype control antibody (20 μg) and injected intravenously into HLA-A2+ transgenic NOD/SCID mice irradiated with 200 cGy. Two weeks later, the mice were sacrificed, dissected, the tissues homogenized and analyzed by flow cytometry for the presence of leukemia cells using human and mouse cell surface markers. Flow cytometry results of cells isolated from mouse bone marrow (BM) are shown. Control (PBS-treated) and experimental animals receiving AML cells plus 8F4 (AML+8F4 antibody) showed no human leukemia cells in the BM. In contrast, animals receiving AML cells plus control antibody (AML+isotype control) showed human CD33+CD45+ cells in the bone marrow that had the same phenotype as the infused AML.

Figure 7 - Immunization strategy for obtaining anti-PR1/HLA-A2 antibodies . Schematic diagram of the MHC class I molecule. The MHC class is composed of a heavy chain and a β2 microglobulin chain. Peptide antigens bind to the groove of the MHC class I, flanked by the α1 and α2 helical domains of the chains.

Figure 8A-B - 8F4 antibody prevents engraftment of human AML in an HLA-A2Tg xenograft model . Primary HLA-A2+ AML cells ( ¹⁰⁶ ) were washed, resuspended in PBS (250 μl), mixed with 20 μg of 8F4 or isotype control antibody, and injected intravenously into sublethally irradiated (200 cGy) HLA-A2Tg NOD/SCID mice. The presence of leukemia in peripheral blood, bone marrow, and tissues was analyzed by histochemistry (Figure 8A) and flow cytometry (Figure 8B) at the indicated times. Irradiated mice without AML metastasis and pre-transfer AML cells served as negative and positive controls, respectively. (Figure 8A) AML infiltration in tissues of experimental mice injected with AML cells plus 8F4 (left panel), AML cells plus isotype control antibody (iso, center panel), and control mice without AML metastasis (right panel). (Fig. 8B) No AML cells were detected in the bone marrow (top two rows) and peripheral blood (bottom two rows) of non-transferred control mice and experimental mice treated with 8F4 (shown before transfer, left figure). Mice receiving AML cells mixed with isotype-matched control antibodies (iso) showed implantation of AML1 and AML5 two or four weeks after AML transfer. Flow cytometry analysis of AML implantation was performed using an extended panel (including mouse cell-specific markers (mCD45), 3-6 human markers (CD45, CD13, CD33, CD34, CD38, HLA-DR) and Live/DeadFixable Aqua (Invitrogen). All figures show viable mCD45- cells.

Figure 9A-C- Due to the expression of the conserved PR1 sequence on HLA-A2-expressing mouse hematopoietic cells, 8F4 induces transient (21 days) neutrophil reduction in HLA-A2 transgenic NOD/SCID. 200 μg (10 mg/kg) 8F4 or isotype control Ab were injected into HLA-A2Tg NOD/SCID mice. These animals showed that they presented endogenous PR1. After 9 days, bone marrow cells were harvested and stained with mAbs (B220-PE, Gr-1-PB, CD11b-APC, F4/80-PE-Cy7, CD3-FITC and LIVE/DEADFixable Aqua) against mouse antigens and examined by flow cytometry. (Figure 9A) The reduction of granulocytes is obvious in the scatter plot of bone marrow (left figure). There are Gr-1lo immature neutrophils, but there are many fewer Gr-1hi mature neutrophils in the bone marrow of 8F4-treated mice (middle figure). In addition, monocytes (SSCl0 CD11b+; lower right gate in the right panel) were reduced in 8F4-treated animals. (Figure 9B) Intravenous injection of 8F4 (5 mg/kg) induced a transient decrease in the absolute number of circulating mature granulocytes, macrophages, and monocytes in HLA-A2Tg NOD/SCID mice. Three weeks after treatment, all populations remained. The gates shown in Figure 9A were used to determine the frequency of each cell type as a percentage of viable cells. Error bars are standard deviations for n=2 animals per group. One representative experiment out of three is shown. (Figure 9C) Seven days after injection of 200 μg (10 mg/kg) of 8F4, no significant pathological changes were evident in the liver, lungs, spleen, kidneys, heart, or brain of HLA-A2Tg NOD/SCID mice. H&E sections of representative tissues from two mice are shown.

Figure 10A-B - 8F4 induces transient leukopenia in established human hematopoietic cells after transfer of human CD34+ cell-enriched umbilical cord blood into NOD/SCID mice . Fresh HLA-A2+ umbilical cord blood (CB) units (50-150 ml) were filtered using Histopaque 1077, washed with PBS, and then washed with CliniMACS buffer (0.5% HSA in PBS pH 7.2/1 mM EDTA, Miltenyi). ¹⁰⁸ cells were resuspended in 300 ml of MACS buffer, mixed with 100 ml of CD34 microbeads (Miltenyi), incubated at 4°C for 30 minutes, and washed. CD34 ⁺ cells labeled with magnetic beads were purified using 2LS columns (Miltenyi). CD34 ⁺ cells were eluted from the column, counted, and injected intravenously (iv) into irradiated (400 rad) NOD/SCID mice (1×10 ⁶ to 2.5×10 ⁶ cells/mouse). Control mice CB1-5 did not receive CB cells. (Figure 10A) Starting 4 weeks after transplantation, peripheral blood was collected from the mice weekly or every other week to monitor cord blood engraftment by FACS using mouse CD45, human CD45, and HLA markers. 9 to 12 weeks after transfer, mice were injected intravenously with 20 μg (1 mg/kg) 8F4 twice, with a 1-week interval between injections (dashed arrows). (Figure 10B) Four weeks after the second 8F4 injection, the mice were sacrificed. Human cell engraftment in blood, spleen, and bone marrow was analyzed as above.

Figure 11. Schematic structure of pCh8F4, pHu8F4-1, pHu8F4-2, and pHu8F4-2-AA (collectively referred to as expression vectors) . Continuing clockwise from the top SalI site, the plasmid contains the heavy chain transcription unit, which begins with the major immediate early promoter and enhancer (CMV promoter) of human cytomegalovirus (CMV) to initiate transcription of the antibody heavy chain gene. Following the CMV promoter are the VH exons (genomic sequences containing the human γ-1 heavy chain constant region), including the CH1, hinge, CH2, and CH3 exons and introns between them, and the CH3 exon is followed by a polyadenylation site. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter, followed by the VL exons and genomic sequences containing the human κ chain constant region exons (CL), preceded by a portion of introns, and followed by a polyadenylation site after the CL exons. Next, the light chain gene is followed by the SV40 early promoter (SV40 promoter), the E. coli xanthine guanine phosphoribosyltransferase gene (gpt), and a segment containing the SV40 polyadenylation site (SV40 poly (A) site). Finally, the plasmid contains a portion of the plasmid pUC19, which contains the bacterial-derived replication (pUC ori) and β-lactamase genes (β-lactamase).

Figure 12. Amino acid sequence alignment of 8F4 VH, humanized 8F4 (Hu8F4) VH, and human acceptor U96282 VH . Amino acid residues are shown in single-letter code. Numbers above the sequence indicate positions according to Kabat et al. (1991). CDR sequences defined by Kabat et al. (1991) are underlined. CDR residues in U96282 VH are omitted in this figure.

Figure 13. Amino acid sequence alignment of 8F4 VL, two forms of humanized 8F4 VL (Hu8F4 VL1 and VL2), and the human acceptor AY043146 VL. Amino acid residues are shown in single-letter code. The numbers above the sequence indicate the positions according to Kabat et al. (1991). The CDR sequences defined by Kabat et al. (1991) are underlined. The underlined residues in Hu8F4 VL1 are predicted to contact the CDRs and the corresponding mouse residues are retained at that position in Hu8F4 VL1. The CDR residues in AY043146 VL are omitted in this figure.

Figure 14. SDS PAGE analysis of purified 8F4 antibodies . Antibodies (5 μg each) were run on 4% to 20% SDS PAGE gels under reducing conditions. Invitrogen's SeeBlue Plus 2 prestained standard (Cat# LC5925) was used as a molecular weight standard (lanes 1, 7, and 8). Samples: 8F4.3.3 (lane 2), 8F4-4 (lane 3), Ch8F4 (lane 4), Hu8F4-1 (lane 5), Hu8F4-2 lot 9/9/10 (lane 6), Hu8F4-2 lot 1/23/11 (lane 9), and Hu8F4-2-AA (lane 10).

Figure 15. ELISA analysis of Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA binding to PR-1/HLA-A2 . Ch8F4, Hu8F-1, Hu8F4-2, and Hu8F-2-AA were tested for binding to PR-1/HLA-A2 at multiple concentrations, starting at 1 μg/ml and serially diluted 3-fold.

Figure 16A-C. Hu8F4 binding specificity and mechanism of action . (Figure 16A) Specificity assay. (Figure 16B) Affinity assay. (Figure 16C) CDC assay.

Figure 17. Treatment with Hu8F4 eliminates established AML . The assay measured the percentage of engraftment in AML transplants performed 3 weeks prior to antibody treatment.

Figure 18. 8F4 induces reversible pancytopenia: effects on normal hematopoietic progenitor cells in SCID mice.

Figure 19. 8F4 induces reversible pancytopenia: effects on normal blood cells in immunocompetent mice .

Figures 20A-C. Hu 8F4 delays breast cancer tumor growth and prolongs survival . (Figure 20A) Tumor-associated neutrophils in 231BrCA xenograft tumors. (Figure 20B) Primary tumor model. (Figure 20C) Metastatic tumor model.

Figures 21A-B. NE and P3 uptake by solid tumor cell lines . Cell lines representing solid tumors were incubated with (Figure 21A) NE (10 mg/ml) or (Figure 21B) P3 (10 mg/ml), then permeabilized and stained with anti-NE or anti-P3 Abs. Data are presented as the mean ± SEM fold increase in NE or P3 uptake relative to unpulsed cells in triplicate wells from two independent experiments. MDA-MB-231, breast cancer; MIAPaCa-2, pancreatic cancer; Mel 624 and Mel 526, melanoma; OVCAR3, ovarian adenocarcinoma; SW-620, colon adenocarcinoma.

Figure 22A-D. Breast cancer does not endogenously express P3 . mRNA was extracted from (Figure 22A) breast cancer cell lines and (Figure 22B) primary breast cancer tissue. PCR was performed using P3 primers, which showed a lack of P3 mRNA expression in breast cancer cell lines and primary breast cancer. Jurkat and HL-60 leukemia cell lines were used as negative and positive controls, respectively. Primary breast cancer cells from patient tissues were obtained by LCM of tumors obtained from patients during surgical resection (samples breast 1 to 3). Analysis of breast cancer cells was determined using mammaglobin-1 (MGB-1). β-actin and GAPDH were used as loading controls. (Figure 22C) Immunoblotting demonstrated the lack of P3 protein in WCLs from five different breast cancer cell lines. 20 mg of protein was loaded into the gel. Purified P3 (5 mg) was used as a positive control, and GAPDH was used as a loading control. (Figure 22D) Immunohistochemistry demonstrated the absence of P3 in patient breast cancer tissue (breast 3). The left image, H&E section (original magnification 3200), shows poor differentiation between carcinoma and mixed neutrophils. The right image, immunohistochemical staining for P3, shows positive staining (brown) for P3 in mixed neutrophils but not in breast cancer cells. The inset (original magnification 3400) shows rare tumor cells engulfing neutrophils. Both images were taken from the same patient and represent five tissues. Arrows indicate neutrophils.

Figure 23 A-C. P3 is taken in by breast cancer cell line and localized to lysosome compartment . (Figure 23 A) MDA-MB-231, MCF-7 and MCF-7-HER18 cell lines were incubated with soluble P3 (10 mg/ml) for 1, 4 and 24 hours, and then intracellular staining was performed with anti-P3Ab. The MFI of three replicate groups was measured and normalized to the MFI of unpulsed cells. MFI was plotted on the y-axis relative to the multiple increase of unpulsed cells. Data are mean values 6 SEM and represent two independent experiments. (Figure 23 B) MDA-MB-231 cells were incubated with soluble P3 or OVA (ova) that gradually increased dosage, and the intracellular uptake of P3 or OVA was analyzed by flow cytometry using anti-P3 or anti-OVAAb, respectively. Data are mean values 6 SEM from two replicates. (Figure 23C) MDA-MB-231 cells were cultured with soluble P3 (10 mg/ml) and then intracellularly stained for P3 (red) and LAMP-2 (green). Confocal microscopy images demonstrate the localization of P3 in lysosomal compartments 4 hours after uptake, as shown by the overlay image (yellow). Nuclei are shown in blue using DAPI. Scale bar, 5 mm.

Figure 24A-F. P3 uptake and cross-presentation of P3 and NE increase breast cancer susceptibility to killing by PR1-CTLs and anti-PR1/HLA-A2 . (Figure 24A) MDA-MB-231 breast cancer cells were incubated with soluble P3, irradiated PMNs, or PBMCs for 4 hours. The cells were permeabilized, stained with anti-P3 Ab, and analyzed by flow cytometry. For cell-associated uptake, light scatter observed on flow cytometry provided clear distinction between PBMCs, PMNs, and MDA-MB-231 cells. PBMCs and PMNs alone served as negative and positive controls, respectively. A Tukey test (*p<0.05) was performed following ANOVA using Prism 5.0 software. Data are mean ± SEM of two replicates. (Figure 24B) MDA-MB-231 breast cancer cells were incubated with soluble P3 or NE (10 mg/ml) at increasing time points and analyzed for PR1/HLA-A2 expression. The MFI of PR1/HLA-A2 was increased by the mean 6SEM of unpulsed cells as shown in two replicates. Tukey's test (*p=0.01, **p, 0.0001) was performed using Prism 5.0 software after ANOVA. (Figures 24C-D) MDA-MB-231 cells were cultured for 24 hours in a medium containing NE or P3 (10 mg/ml) and the Ag presentation inhibitors brefeldin A or lactacystin. The cells were then analyzed for PR1/HLA-A2 expression. The mean 6SEM of the MFI of PR1/HLA-A2 is shown in two replicates of a representative experiment. Tukey's test (*p, 0.01, **p, 0.0001) was performed using Prism 5.0 software after ANOVA. (Figure 24E) MDA-MB-231 cells were cultured overnight in a medium containing P3 or NE (10 mg/ml) loaded with calcein-AM and then co-cultured with PR1-CTL for 4 hours. Cytotoxicity was determined by measuring the released calcein-AM. Cells pulsed with NE or P3 showed higher killing than unpulsed MDA-MB-231 cells. Cells pulsed with PR1 and T2 cells without PR1 pulse were used as positive and negative controls, respectively. The data are the mean ± SEM of two replicate wells of a representative experiment. (Figure 24F) MDA-MB-231 cells were cultured with NE (10 mg/ml) or P3 (10 mg/ml) for 24 hours. The cells were then incubated with anti-PR1/HLA-A2 (8F4) Ab for 60 minutes before complement was added. Complement-dependent cytotoxicity was measured using calcein-AM release and demonstrated specific killing of MDA-MB-231 cells by 8F4Ab pulsed with NE or P3. Cytotoxicity data are the mean ± SEM of two replicate wells from a representative experiment. Unpaired t-tests (*p, 0.05) were performed using Prism 5.0 software.

Figure 25A-D. Detection of PR1/HLA-A2 and PR1-CTL in breast cancer and melanoma patients . (Figure 25A) Resected HLA-A2+ patient breast cancer tissue (breast 1 and 4) was stained with anti-PR1/HLA-A2 (8F4)-647 (red) and anti-CK7)-FITC (green) and then imaged using confocal laser microscopy. PR1/HLA-A2 appears to be expressed by breast cancer cells, as shown by co-staining with 8F4 and CK7. DAPI-blue is used to stain the nuclei. (Figure 25B) Serial sections of resected HLA-A2+ patient breast cancer tissue were stained with anti-CD45-647 (red) (left panel) or anti-CK7-FITC (green) and 8F4-647 (red) (right panel) and then imaged using confocal laser microscopy. PR1/HLA-A2 is expressed by breast cancer cells (8F4+/CK7+) in areas with minimal leukocytes (CD452), thereby determining the PR1/HLA-A2 expression of breast cancer cells. DAPI-blue is used to stain the nucleus. (Figure 25C) Box plots show PR1-CTL in peripheral blood of breast cancer (n=11) HLA-A2+ patients, melanoma (n=7) HLA-A2+ patients, and healthy (n=9) HLA-A2+ donors. Mann-Whitney U test (*p, 0.05) was performed using Prism 5.0 software. (Figure 25D) Excised HLA-A2+ (melanoma 1) and HLA-A22 (melanoma 2) patient tissues were stained with 8F4-647 (red) and anti-MITF-FITC (green) and then imaged using confocal laser microscopy. PR1/HLA-A2 appears to be expressed in an HLA-A2+ melanoma sample (Melanoma 1), as shown by co-staining with 8F4 and MITF. DAPI-blue was used to stain the nuclei. Scale bar, 20 mm.

Figure 26A-F. Cross-presentation of P3 and NE by melanoma cells increases susceptibility to PR1-CTLs . Double staining of primary melanoma patient samples for (Figure 26A) NE (brown) and MITF (pink) or (Figure 26B) P3 (brown) and MITF (pink) shows the absence of NE and P3 in melanoma. Images were acquired at an original magnification of 3100. Insets at an original magnification of 3400 show scattered NE- or P3-positive cells, which are most likely inflammatory cells. (Figure 26C) Western blots show the absence of NE and P3 in melanoma cell lines. The U-937 leukemia cell line was used as a positive control for NE and P3. Tubulin was used as a loading control. M = molecular weight (mw) marker. (Figures 26D-E) 526 HLA-A2+ melanoma cell lines were cultured with soluble NE (10 mg/ml) or P3 (10 mg/ml) at increasing time points and analyzed for NE and P3 uptake (Figure 26D) and cross-presentation (i.e., PR1/HLA-A2 expression) (Figure 26E). The fold increase in MFI relative to unpulsed cells for NE or P3 (Figure 26D) or PR1/HLA-A2 (Figure 26E) is shown on the y-axis. Tukey's test was performed following ANOVA using Prism 5.0 software (**p=0.0001, *p<0.05). Data represent the mean ± SEM of two replicates. (F) Calcein-AM cytotoxicity assay showing PR1-CTL killing of 526 HLA-A2+ melanoma cell lines pulsed with NE (10 mg/ml) and P3 (10 mg/ml) for 24 hours relative to unpulsed (Unp) Mel 526. Unpulsed (T2Unp) T2 cells and PR1-pulsed (T2PR1) T2 cells served as negative and positive controls, respectively. Data are mean ± SEM of duplicate wells from a representative experiment. Unpaired t-tests (*p, 0.05) were performed using Prism 5.0 software.

Description of Illustrative Embodiments

It has been shown that the PR-1 self-peptide (VLQELNVTV; SEQ ID NO: 45) is recognized by CD8+ cytotoxic T lymphocytes (CTLs) on HLA-A*0201 expressed on the leukemia cell membrane, and that PR1-specific CTLs specifically lyse myeloid leukemia cells without lysing normal bone marrow cells. Vaccination of HLA-A2+ patients with AML, CML, and MDS with the PR-1 peptide induced PR-1-CTL immunity in 58% of patients and a clinical response of interest in 13 of 66 patients (20%). Although these results are encouraging, high tumor burden remains an obstacle to successful vaccination.

Because the PR1 peptide is expressed only on the surface of myeloid leukemia cells and not in sufficient amounts on normal bone marrow cells, the present inventors sought to develop antibodies targeting PR1/HLA-A*0201 that could be used therapeutically to treat myeloid leukemia patients or to identify patients who may be susceptible to PR1-based immunotherapies (such as vaccines or adoptive T cell transfer). Because HLA-A2+ is the most commonly expressed HLA allele (40% of the general Caucasian population), antibody-based therapies targeting T cell epitopes are novel and have broad applicability. By immunizing immunocompetent BALB/c mice with recombinant PR1/HLA-A*0201 monomers, they generated an IgG2a-κ monoclonal antibody (8F4) specific for the combined PR1/HLA-A*0201 epitope. The 8F4 antibody was shown to have high affinity for PR1/HLA-A*0201 ( _KD = 9.9 nanomolar) and was shown to recognize only T2 target cells pulsed with PR-1 but not cells pulsed with a control peptide. 8F4 bound to HLA-A2+ AML, using both FACS and confocal microscopy to mark the cells, while not marking normal HLA-A2+ peripheral blood cells.

Furthermore, 8F4 induced dose-dependent complement-mediated cytotoxicity (CDC) in HLA-A2+ primary human leukemia cells but not in normal bone marrow cells. Remarkably, the 8F4 antibody specifically prevented human AML engraftment in the HLA-A2 transgenic NOD/SCID animal model, with only a single exposure to the antibody upon adoptive transfer into the animals. Furthermore, 8F4 delayed breast cancer tumor growth and prolonged survival, despite the fact that P3 and NE are not expressed in breast cancer cells. Taken together, these results demonstrate the generation of antibodies specific for the cell membrane-bound PR1/HLA-A*0201 epitope, an important T cell target antigen, that specifically target and eliminate human leukemias and solid tumors.

I. Definition

The phrase "isolated" or "biologically pure" refers to a material that is substantially or essentially free of components that normally accompany the material as it is found in its native state. Thus, an isolated peptide according to the present invention is preferably free of materials that normally associate with the peptide in its native environment.

The "major histocompatibility complex" or "MHC" is a group of genes that play a role in controlling the cellular interactions responsible for physiological immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes, see Paul (1993).

"Human leukocyte antigen" or "HLA" is a human class I or class II major histocompatibility complex (MHC) protein (see, eg, Stites, 1994).

As used herein, "HLA supertype or family" describes a collection of HLA molecules grouped based on shared peptide binding specificity. HLA class I molecules that share somewhat similar binding affinities for peptides with certain amino acid motifs are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA-xx-like supertype molecule (where xx represents a specific HLA type) are synonymous.

The term "motif" refers to a pattern of residues in a peptide of defined length (typically, a peptide of about 8 to about 13 amino acids for class I HLA motifs and about 6 to about 25 amino acids for class II HLA motifs) that is recognized by a specific HLA molecule. The peptide motif is typically different for each protein encoded by each human HLA allele and differs in the pattern of primary and secondary anchor residues.

An "abnormal cell" is any cell that is considered to have characteristics atypical for that cell type, including atypical growth, typical growth in an atypical location, or typical action on an atypical target. Such cells include cancer cells, benign proliferative or dysplastic cells, inflammatory cells, or autoimmune cells.

II. PR-1 and HLA Restriction

A.PR-1

The PR-1 self-peptide (VLQELNVTV; SEQ ID NO: 45) is derived from proteinase 3 (P3) and neutrophil elastase (NE), both of which are abnormally expressed in leukemias. It has been shown to be recognized by CD8+ cytotoxic T lymphocytes (CTLs) on HLA-A*0201 expressed on the leukemic cell membrane. PR-1-specific CTLs specifically lyse myeloid leukemia cells, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS), without lysing normal bone marrow cells. Previously, the present inventors have shown that PR-1 vaccination of HLA-A2+ patients with AML, CML, and MDS with the PR1 peptide in Montanide ISA-51 and GM-CSF induced PR-1 CTL immunity in 58% of patients and a clinical response of interest in 13 of 66 patients (20%).

B.HLA-A2

The human leukocyte antigen system (HLA) is the name for the major histocompatibility complex (MHC) in humans. This superlocus contains a large number of genes related to the function of the human immune system. This group of genes is located on chromosome 6 and encodes cell surface antigen-presenting proteins and many other genes. The proteins encoded by certain genes are also called antigens, a result of their historical discovery as factors in organ transplantation. The major HLA antigens are essential for immune function. Different classes have different functions.

HLA class I antigens (A, B & C) present peptides (including viral peptides, if present) from inside the cell. These peptides are produced from digested proteins that break down in lysosomes. The peptides are generally small polymers, about 9 amino acids in length. Foreign antigens attract killer T cells (also known as CD8 ⁺ cells) that destroy the cell. HLA class II antigens (DR, DP & DQ) present antigens to T lymphocytes from outside the cell. These specific antigens stimulate T helper cells to multiply, and these T helper cells, in turn, stimulate antibody-producing B cells, while self-antigens are suppressed by suppressor T cells.

HLA-A2 (A2) is a human leukocyte antigen serotype within the HLA-A "A" serotype group. This serotype identifies the gene products of many HLA-A*02 alleles, including HLA-A*0201, *0202, *0203, *0206, and *0207. A*02 is common globally, while A*0201 is found at high frequencies in northern Asia and North America. A2 is the most diverse serotype, showing diversity in East Africa and Southwest Asia. While A*0201 is found at high frequencies in northern Asia, its diversity is limited to A*0201, with less common Asian variants A*0203 and A*0206.

Serotype is determined by antibody recognition of the ^α2 subset of the HLA-A α chain. For A2, the α "A" chain is encoded by the HLA-A*02 allele group, and the β chain is encoded by the B2M locus. A2 and A*02 are nearly synonymous. A2 is more common in northern Asia and North America than elsewhere and is part of several long haplotypes.

III. Antibodies

The present invention relates to the production and use of antibodies that bind to PR1 in the presence of HLA-A2. Antibodies are capable of "specific binding" to a specific target or a range of antigenically related targets. As used herein, an antibody is considered to be capable of "specific binding" to an antigen if the antigen can be distinguished from antigenically different molecules based on binding to the antibody variable region. Such interactions are in contrast to nonspecific binding, which involves classes of compounds without regard to their chemical structure (e.g., binding of proteins to nitrocellulose, etc.). In particular, the antibodies of the present invention can exhibit "highly specific binding," such that they are unable or substantially unable to bind to even closely related molecules.

Monoclonal antibodies can be readily prepared using well-known techniques, such as those illustrated in U.S. Patent No. 4,196,265, which is incorporated herein by reference. Typically, the technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the invention) in a manner sufficient to provide an immune response. Rodents (e.g., mice and rats) are preferred animals. Splenocytes from the immunized animal are then fused with immortal myeloma cells. Successful fusions are then screened for the production of suitable antibodies.

In one embodiment, the antibody molecule will comprise fragments (e.g., F(ab'), F(ab')2) produced, for example, by proteolytic cleavage of a mAb, or single-chain immunoglobulins, such as can be produced by recombinant means. Such antibody derivatives are monovalent. In one embodiment, these fragments can be combined with each other or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Notably, these chimeric molecules may comprise substituents capable of binding to different epitopes of the same molecule, or they may be capable of binding to both an activated protein C epitope and an "inactivated protein C" epitope.

When an antibody or fragment thereof is intended for therapeutic purposes, it may be desirable to "humanize" it to attenuate any immune response. Such humanized antibodies can be studied in vitro or in vivo. Humanized antibodies can be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding but non-immunogenic portion (i.e., a chimeric antibody). PCT application PCT/U.S.86/02269, EP application 184,187, EP application 171,496, EP application 173,494, PCT application WO 86/01533, EP application 125,023, Sun et al. (1987), Wood et al. (1985), and Shaw et al. (1988); all references are incorporated herein by reference. Morrison (1985) provides a general review of "humanized" chimeric antibodies; it is also incorporated herein by reference. Alternatively, "humanized" antibodies can be produced by CDR or CEA replacement. Jones et al. (1986), Verhoeyan et al. (1988), Beidler et al. (1988); all references are incorporated herein by reference.

A. Variants

The following is a discussion based on altering the amino acids of a protein to produce a modified protein. When making these alterations, the hydropathic index of the amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is believed that the relative hydropathic character of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local average hydrophilicity of a protein (as governed by the hydrophilicity of its neighboring amino acids) is correlated with the biological properties of the protein. As described in detail in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic nonionic amino acids: serine (+3.0), asparagine (+0.2), glutamine (+0.2); The amino acids were cysteine (-1.0), methionine (-1.3), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0). The amino acids were tyrosine (-2.3), cysteine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0). The amino acids were tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It will be appreciated that an amino acid can be substituted for another amino acid of similar hydrophilicity and produce a biologically or immunologically modified protein. In such alterations, amino acid substitutions whose hydrophilicity values are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the above characteristics into account are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

The present invention can also employ the use of peptide mimetics to prepare polypeptides that possess many of the natural properties of antibodies but with altered and/or improved characteristics (see, e.g., Johnson, 1993). The basic principle behind the use of mimetics is that the peptide backbone of a protein primarily orients the amino acid side chains in such a way as to facilitate molecular interactions, such as those of an antibody with an antigen. These principles can be used in conjunction with the above principles to engineer second-generation molecules that possess many of the natural properties of an antibody but with altered and/or improved characteristics.

It is contemplated that the present invention may also employ sequence variants, such as insertion variants or deletion variants. Deletion variants lack one or more residues of the native protein. Insertion mutants generally involve the addition of material to a non-terminal point of the polypeptide. It will also be understood that insertion sequence variants may comprise an N-terminal or C-terminal amino acid and still be essentially as represented by one of the sequences disclosed herein, so long as the sequence meets the criteria described above, including maintenance of biological activity.

The present invention also contemplates isotype modification. As discussed below, antibody 8F4 is identified as IgG2a-κ. By modifying the Fc region to have different isotypes, different functions can be achieved. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, switching to the A class can improve tissue distribution, and switching to the M class can improve potency.

Modified antibodies can be prepared by any technique known to those skilled in the art, including expression by standard molecular biology techniques or chemical synthesis of polypeptides. Methods for recombinant expression are also discussed elsewhere in this document.

B. Single-chain antibody

Single chain variable fragment (scFv) is the fusion that the variable region of the heavy chain and light chain of immunoglobulin are connected together by short (usually serine, glycine) joint.Although the constant region has been removed and a linker peptide has been introduced, the chimeric molecule retains the specificity of the original immunoglobulin. The image on the right shows how this modification retains specificity without changing usually. Historically, these molecules were produced to help to be very convenient for phage display of expressing the antigen-binding domain as a single peptide. Alternatively, scFv can be directly produced by the subclone heavy chain and light chain derived from a hybridoma. Single chain variable fragment lacks the constant Fc region present in the complete antibody molecule, and therefore lacks the common binding site (e.g., protein A/G) for purifying antibodies. Because protein L interacts with the kappa light chain variable region, these fragments can often use protein L to carry out purification/immobilization.

Flexible linkers are generally composed of helix-promoting and turn amino acid residues (e.g., alanine, serine, and glycine). However, other residues may also play a role. Tang et al. (1996) used phage display as a means to rapidly select custom linkers for single-chain antibodies (scFvs) from a library of protein linkers. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approximately 5×10 ⁶ different members) was displayed on filamentous phage and subjected to affinity screening for haptens. The population of selected variants showed a significant increase in binding activity while retaining considerable sequence diversity. Screening of 1054 individual variants subsequently yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the conserved proline in the linker, the two residues after the V _H C-terminus, and the abundance of arginine and proline at other positions as the only common features of the selected range.

The recombinant antibodies of the present invention may also comprise sequences or portions that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers bound to the J chain. Another multimerization domain is the Ga14 dimerization domain. In other embodiments, the chains may be modified with substances such as biotin/avidin, allowing for the combination of two antibodies.

In a different embodiment, single-chain antibodies can be produced by connecting the receptor light chain and heavy chain using a non-peptide linker or chemical unit. Generally, the light chain and heavy chain are produced in different cells, purified, and then linked together in an appropriate manner (i.e., the N-terminal of the heavy chain is connected to the C-terminal of the light chain by a suitable chemical bridge).

Cross-linking reagents are used to form molecular bridges that bind the functional groups of two different molecules, such as stabilizers and coagulants. However, it is expected that dimers or multimers of the same analog or heteropolymeric complexes composed of different analogs can be produced. To link two different compounds in a stepwise manner, heterobifunctional cross-linkers can be used to eliminate unwanted homopolymer formation. Table 1 describes several cross-linking agents.

Table 1 - Heterobifunctional crosslinkers

Exemplary heterobifunctional cross-linkers contain two reactive groups: one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine-reactive group, the cross-linker can react with lysine residues of one protein (e.g., a selected antibody or fragment), and through the thiol-reactive group, the cross-linker, already bound to the first protein, can react with cysteine residues (free sulfhydryl groups) of another protein (e.g., a selected substance).

Preferably, a cross-linking agent with reasonable stability in blood is used. Various types of disulfide-containing linkers are known to be successfully used to conjugate targeting agents and therapeutic/prophylactic agents. Linkers containing sterically hindered disulfide bonds have been shown to produce greater stability in vivo, thereby preventing the release of the targeting peptide before it reaches the site of action. Therefore, these linkers are a group of connecting agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and methyl groups. It is believed that the steric hindrance of the disulfide bond functions to protect the bond from attack by thiolate anions (e.g., glutathione) that may be present in tissues and blood, thereby helping to prevent the conjugate from uncoupling before the linked substance is delivered to the target site.

SMPT crosslinking reagents, like many other known crosslinking reagents, are capable of crosslinking functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of crosslinker includes heterobifunctional photoreactive phenylazides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamino)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimide group reacts with primary amine groups, and the phenylazide (after photolysis) reacts non-selectively with any amino acid residue.

In addition to sterically hindered crosslinkers, non-sterically hindered crosslinkers may also be used in accordance with the present invention. Other useful crosslinkers that are not believed to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of these crosslinkers is well understood in the art. Another embodiment involves the use of flexible linkers.

U.S. Patent No. 4,680,338 describes bifunctional linkers that can be used to produce conjugates of ligands with amine-containing polymers and/or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. The linkers are particularly useful for allowing a substance of interest to be directly bonded to the linker, and cleavage results in release of the active agent. Particular uses include adding free amino groups or free sulfhydryl groups to proteins (e.g., antibodies or drugs).

U.S. Patent No. 5,856,456 provides peptide linkers for connecting polypeptide components to prepare fusion proteins (e.g., single-chain antibodies). The linker is up to about 50 amino acids in length and contains at least one occurrence of a charged amino acid (preferably arginine or lysine). It is followed by a proline and is characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers that can be used in a variety of immunodiagnostic and separation techniques.

C. Purification

In certain embodiments, the antibodies of the present invention may be purified. As used herein, the term "purified" is intended to refer to a composition that is isolatable from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. Thus, a purified protein also refers to a protein that has been liberated from its naturally occurring environment. When the term "substantially purified" is used, the designation refers to a composition in which the protein or peptide forms the major component of the composition, e.g., the composition comprises about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein.

Protein purification techniques are well known to those skilled in the art. These techniques include, at one level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After the polypeptide is separated from other proteins, the polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to uniformity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or by thermal denaturation followed by centrifugation; gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; and combinations of these and other techniques.

When purifying the antibodies of the present invention, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. Affinity columns that bind to the polypeptide tag portion may be used to purify the polypeptide from other cellular components. As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or that certain steps can be omitted, and still produce a suitable method for preparing a substantially purified protein or peptide.

Typically, complete antibodies are fractionated using reagents that are bound to the Fc portion of the antibody (i.e., protein A). Alternatively, antigens can be used to purify and select appropriate antibodies simultaneously. These methods often utilize selectors that are bound to supports (e.g., columns, filters, or beads). The antibody is bound to the support, contaminants are removed (e.g., washed off), and the antibody is released by applying conditions (salt, heat, etc.).

In accordance with the present disclosure, various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art. These include, for example, determining the specific activity of the active fraction, or analyzing and assessing the amount of polypeptide in the fraction by SDS/PAGE. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and calculate the purity therefrom. Of course, the actual unit used to express the amount of activity will depend on the specific assay technique selected after purification and whether the expressed protein or peptide exhibits detectable activity.

It is known that under different conditions of SDS/PAGE, the migration of polypeptides can vary, sometimes significantly (Capaldi et al., 1977). Therefore, it should be understood that the apparent molecular weight of a purified expression product or partially purified expression product may vary under different electrophoretic conditions.

D. Conjugation of Antibodies to Therapeutic or Diagnostic Agents

In one embodiment, the antibodies of the present invention can be linked to a variety of agents for disease diagnosis and disease treatment. Linkage can be performed using a variety of well-known chemical reactions and reagents, some of which are described elsewhere in this document.

1. Diagnostic reagents

Many diagnostic/imaging agents are known in the art, as are methods for their attachment to proteins (including antibodies) (see, e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509, each of which is incorporated herein by reference). The imaging moieties used may be paramagnetic ions, radioisotopes, fluorescent dyes, NMR detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, for example, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III) may be mentioned, with gadolinium being particularly preferred. Ions that can be used in other contexts (e.g., X-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioisotopes for therapeutic and/or diagnostic applications, mention may be made of astatine ²¹¹ , carbon ¹⁴ , chromium ⁵¹ , chlorine ³⁶ , cobalt ⁵⁷ , cobalt ⁵⁸ , copper ⁶⁷ , Eu ¹⁵² , gallium ⁶⁷ , hydrogen ³ , iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , iron ⁵⁹ , phosphorus ³² , rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , selenium ⁷⁵ , sulfur ³⁵ , technetium ^99m and/or yttrium ^{90. 125} I is often preferred for use in certain embodiments, and technetium ^99m and/or indium ¹¹¹ are also often preferred due to their low energy and suitability for long-range detection. The radiolabeled receptors of the present invention can be produced according to methods known in the art. For example, the receptor can be iodinated by contact with sodium iodide and/or potassium iodide and a chemical oxidant, such as sodium hypochlorite or an enzymatic oxidant, such as lactoperoxidase. The TcR according to the present invention can be labeled with technetium ^-99m by a ligand exchange process, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to the column. Alternatively, a direct labeling technique can be used, for example, by incubating pertechnetate, a reducing agent (such as _SNCl2 ), a buffer solution (such as sodium potassium phthalate solution), and the antibody. Intermediate functional groups commonly used for binding radioisotopes are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA), which are present as metal ions in the antibody.

Fluorescent labels contemplated for use as conjugates are Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine and/or Texas Red.

2. Therapeutic agents

A wide variety of therapeutic agents can be linked to the antibodies of the invention. For example, the radioisotopes discussed above, while useful in diagnostic settings, can also be used as therapeutic agents. Chemotherapeutic agents can also be conjugated to the antibody and include cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate.

Another class of therapeutic agents are toxins. Cholera toxin, botulinum toxin, pertussis toxin, ricin A and B chains, and other natural and synthetic toxins are contemplated.

Cytokines and lymphokines are another class of therapeutic agents that can be coupled to the TcRs of the invention and include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, TNFα, GM-CSF, INFα, IFNβ and IFNγ.

In other embodiments, anti-inflammatory agents are contemplated as therapeutic agents that can be conjugated to the antibody. Anti-inflammatory agents include NSAIDs, steroids, rapamycin, infliximab, and denileukin (ontak). Immunosuppressants include FK-506 and cyclosporin A.

TLR agonists can be linked to antibodies, for example, through the Fc portion of the molecule. Agonists of TLRs are compounds that stimulate or "turn on" the immune system. The natural agonists for TLR9 are components of DNA commonly found in bacteria and viruses. The natural agonists for TLR7 and TLR8 are RNA patterns found in viruses. Upon recognizing their natural DNA and RNA agonists, TLR7, 8, and 9 each trigger a different cascade of protective immune responses. TLR agonists include oligodeoxynucleotides, hyaluronic acid fragments, imiquimod, lavandin C, lipid A, loroxibine, LPS, monophosphoryl lipda A, myristyl ether, resiquimod, S. typhimurium flagellin, HKLM, PAM3CSK4, and polyinosinic-polycytidylic acid (polyI:C).

IV. Nucleic Acids and Expression

A. Antibody-encoding nucleic acid

In one aspect of the present invention, nucleic acids encoding multiple parts of antibody heavy and light chains, variable domains and constant domains are provided. The nucleic acid segments can be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA. When it is desired to be incorporated into an expression vector, the nucleic acid may also include natural introns or introns derived from another gene, as well as other non-coding regions (e.g., regulatory regions) and coding regions (e.g., joints). The term "cDNA" as used herein is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. Compared to genomic DNA or DNA polymerized from a non-processed or partially processed RNA template of a genome, the advantage of using cDNA is that cDNA primarily comprises the coding sequence of the corresponding protein.

The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide and generally refers to a polypeptide that is produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule. Recombinant vectors and isolated nucleic acid segments may variously contain their own antibody coding regions, coding regions with selected changes or modifications in the basic coding regions, or they may encode a larger polypeptide comprising non-antibody regions.

As used herein, "nucleic acid" includes single-stranded and double-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids, and nucleic acid analogs. It is contemplated that nucleic acids within the scope of the present invention may have a length of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 460, about 475, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 575, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 675, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870 about 75, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more nucleotide residues.

It is contemplated that the antibody may be encoded by any nucleic acid sequence encoding the appropriate amino acid sequence, such as those in SEQ ID NOs: 3, 60, 5, 8, 9, 10 (heavy chain CDRs 1, 2, and 3; light chain CDRs 1, 2, and 3), and SEQ ID NOs: 11 or 27, which comprise heavy chain CDRs and framework regions 1, 2, and 3, flanked upstream by heavy chain CDRs 1, 2, and 3, respectively; and SEQ ID NO: 23, which comprises light chain CDRs and framework regions 1, 2, and 3, flanked upstream by light chain CDRs 1, 2, and 3, respectively. The design and generation of nucleic acids encoding the desired amino acid sequence is well known to those skilled in the art using a standardized codon table (Table 2). In some specific embodiments, the codons selected to encode each amino acid may be modified to optimize expression of the nucleic acid in the desired host cell. As used herein, the term "functionally equivalent codon" refers to codons encoding the same amino acid, such as the six codons for arginine or serine, and also refers to codons encoding biologically equivalent amino acids. Codon preferences for host cells of different species are well known in the art. Codon preferences for humans are well known to those skilled in the art (Wada et al., 1990). Codon preferences for other organisms are also well known to those skilled in the art (Wada et al., 1990, which is incorporated herein by reference in its entirety).

Table 2 - Codon table

B. Nucleic Acid Expression

Prokaryotic and/or eukaryotic systems can be used to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides. The present invention contemplates the use of such expression systems to produce antibodies that bind to PR-1/HLA-A2. One powerful expression technology utilizes the insect-cell/baculovirus system. Such insect-cell/baculovirus systems can produce high-level protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Patents 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and are commercially available, for example, from the company under the designation 2.0 and from the company under the designation BacPack ^™ Baculovirus Expression System.

In addition, there are many other expression systems that are commercially available and can be widely used. An example of these systems is the Complete Control inducible mammalian expression system (which includes a synthetic ecdysone inducible receptor) or its pET expression system (a kind of Escherichia coli expression system). Another example of an inducible expression system can be derived from it with T-Rex ^™ (tetracycline regulated expression) system (a kind of inducible mammalian expression system using a full-length CMV promoter). A yeast expression system called Pichia methanolica expression system is also provided, which is designed to produce recombinant proteins at high levels in the methylotrophic yeast Pichia methanolica. Those skilled in the art know how to express vectors (e.g., expression constructs) to produce nucleic acid sequences or their associated polypeptides, proteins, or peptides.

1. Viral Vectors and Delivery

There are many ways to introduce expression vectors into cells. Viruses provide powerful tools for expressing protein products encoded by nucleic acids. Therefore, in certain embodiments of the present invention, expression vectors include viruses or modified vectors derived from viral genomes. Certain viruses can enter cells through receptor-mediated endocytosis to integrate into the host cell genome and stably and effectively express viral genes, making them attractive candidates for transferring foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The viruses first used as gene vectors are DNA viruses, including papovaviruses (simian virus 40, bovine papillomavirus and polyomavirus) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).

Adenoviral vectors. A special method for delivering nucleic acids involves the use of adenoviral expression vectors. Although adenoviral vectors are known to have a low ability to integrate into genomic DNA, this characteristic is offset by the high efficiency of gene transfer provided by these vectors. "Adenoviral expression vector" is meant to include those constructs that contain sufficient adenoviral sequences to (a) support construct packaging and (b) ultimately express tissue-specific or cell-specific constructs cloned therein. Knowledge of the genetic organization or adenovirus (a 36 kb linear double-stranded DNA virus) allows large pieces of adenoviral DNA to be replaced with up to 7 kb of foreign sequence (Grunhaus and Horwitz, 1992).

AAV vector. Adenovirus-assisted transfection can be used to introduce nucleic acid into cells. Increased transfection efficiency has been reported in cell systems using adenovirus coupling systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for the vaccine of the present invention (Muzyczka, 1992). AAV has infectivity to a wide host range (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details on the production and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

Retroviral vectors. Retroviruses hold promise as gene delivery vehicles in vaccines because they can integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a broad spectrum of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

In order to construct a retroviral vector, nucleic acid (for example, nucleic acid encoding a target antigen) is inserted into the viral genome in place of certain viral sequences to produce a replication-defective virus. In order to produce virions, a packaging cell line (Mann et al., 1983) comprising gag, pol and env genes but not containing LTR and packaging components was constructed. When a recombinant plasmid comprising cDNA and retroviral LTR and packaging sequences is introduced into a special cell line (for example, by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium comprising the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviruses can infect a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentivirus vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses: HIV-1, HIV-2, and simian immunodeficiency viruses: SIV. Lentivirus vectors are produced by multiple attenuation of HIV virulence genes, for example, genes env, vif, vpr, vpu, and nef are deleted, making the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, U.S. Patent No. 5,994,136 (which is incorporated herein by reference) describes a recombinant lentivirus capable of infecting non-dividing cells, wherein suitable host cells are transfected with two or more vectors (i.e., gag, pol and env and rev and tat) carrying packaging functions. The recombinant virus can be targeted by connecting the envelope protein to an antibody or a special ligand for targeting a receptor of a specific cell type. By inserting the sequence of interest (including regulatory regions) and another gene encoding a receptor ligand on a specific target cell into the viral vector, for example, the vector is now target-specific.

Other viral vectors. Other viral vectors can be used as vaccine constructs in the present invention. Vectors derived from viruses (e.g., vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus) can be used. They provide multiple attractive features for a variety of mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990). Lentiviruses can also be developed as vaccine vectors (Vanden Driessche et al., 2002).

Use modified viruses for delivery. The nucleic acid to be delivered can be housed within an infectious virus that has been engineered to express a specific binding ligand. Thus, the viral particles will specifically bind to the target cell's associated receptors and deliver the contents to the cell. Based on chemical modification of retroviruses by chemically adding lactose residues to the viral envelope, a new approach designed to allow specific targeting of retroviral vectors has been developed. This modification allows for specific infection of hepatocytes via the sialoglycoprotein receptor.

Another approach to targeting recombinant retroviruses has been designed using biotinylated antibodies directed against retroviral envelope proteins and against specific cell receptors. The antibodies were coupled via a biotin component using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated that a variety of human cells with those surface antigens were infected in vitro with ecotropic viruses (Roux et al., 1989).

2. Non-viral nucleic acid delivery

Suitable non-viral methods for nucleic acid delivery to achieve expression of the compositions of the present invention are contemplated to include virtually any method as described herein or known to those of ordinary skill in the art by which nucleic acids (e.g., DNA) can be introduced into organelles, cells, tissues, or organisms. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microparticle bombardment (PCT Application No. WO 94/09699 and 95/06128; U.S. Patents 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each of which is incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985). By applying techniques such as these, organelles, cells, tissues or organisms can be stably or transiently transformed.

V. Antibodies for Diagnosis of Cancer or Hyperplastic or Dysplastic Disorders

In one embodiment of the invention, methods are provided for diagnosing cancer, such as leukemia (e.g., AML, CML, MDS), and myelodysplasia disorders. Myelodysplasias (MDS) refer to a group of disorders in which the bone marrow does not function normally and produces insufficient numbers of normal blood cells. MDS affects the production of any and occasionally all types of blood cells, including red blood cells, platelets, and white blood cells (cytopenias). Approximately 50% of pediatric myelodysplasias may be one of five types of MDS: refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia. The remaining 50% typically present with isolated or combined cytopenias, such as anemia, leukopenia, and/or thrombocytopenia (low platelet count). Although chronic, MDS progresses to acute myeloid leukemia (AML) in approximately 30% of patients.

Solid tumor cancers are also contemplated according to the diagnosis of the present invention. Such cancers are lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma and bladder cancer. Other hyperplastic, neoplastic and dysplastic diseases (including benign hyperproliferative diseases) are also within the scope of the diagnostic methods described herein.

A. Administration of Diagnostic Reagents

Administration of diagnostic agents is well known in the art and will vary depending on the diagnosis to be achieved. For example, when a discrete tumor mass is to be imaged, local or regional administration (e.g., in the tumor vasculature, local lymphatic system, or local artery or vein) may be utilized. Alternatively, the diagnostic agent may be provided regionally or systemically. When a known specific tumor mass has been identified, or when metastasis is suspected, this may be the approach of choice for imaging an entire limb or organism.

B. Injectable Compositions and Formulations

One method for delivering the drug according to the present invention is systemic. However, the pharmaceutical compositions disclosed herein may alternatively be administered parenterally, intravenously, intradermally, intramuscularly, transdermally, or even intraperitoneally, as described in U.S. Patent No. 5,543,158, U.S. Patent No. 5,641,515, and U.S. Patent No. 5,399,363, each of which is specifically incorporated herein by reference in its entirety.

Drug injection can be performed by syringe or any other method for injecting solutions, as long as the drug can be injected through the specific gauge of needle required. A new needle-free injection system has been described (U.S. Patent No. 5,846,233) that has a nozzle defining an ampoule chamber for holding the solution and an energy device for propelling the solution from the nozzle to the delivery site. A syringe system has also been described for gene therapy that allows for precise multiple injections of predetermined amounts of solution at any depth (U.S. Patent No. 5,846,225).

The active compound can be prepared as a solution of a free base or a pharmaceutically acceptable salt by appropriately mixing it with a surfactant (e.g., hydroxypropylcellulose) in water. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal conditions of storage and use, these preparations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in their entirety) for the temporary preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be mobile to a certain extent, and the degree is easy to exist for injectability. It must be stable under manufacturing and storage conditions and must be preserved against the contamination of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), a suitable mixture thereof, and/or a vegetable oil. Suitable fluidity can be maintained, for example, by the use of a coating (e.g., phosphatidylcholine), in the case of dispersions, by maintaining the desired particle size, and by the use of a surfactant. Microbial action can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferred to include an isotonic agent, such as a sugar or sodium chloride. The absorption of the injectable composition can be prolonged by using an absorption delaying agent (e.g., aluminum monostearate and gelatin) in the composition.

For parenteral administration of aqueous solutions, for example, the solution may be appropriately buffered and first made isotonic with enough saline or glucose. These special aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, according to the present disclosure, employable sterile aqueous media will be known to those skilled in the art. For example, 1 dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermic perfusion fluid (hypodermolysis fluid) or injected into the infusion site of the suggestion (see, for example, "Remington's Pharmaceutical Sciences" 15th edition, pages 1035-1038 and 1570-1580). Some variations in dosage will inevitably occur depending on the condition of the subject being treated. In any case, the person in charge of administration will determine the appropriate dosage for the individual subject. Moreover, for human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

Sterile injectable solutions are prepared by the following method: the active compound is incorporated in the desired amount in an appropriate solvent with a variety of the other ingredients listed above, as needed, and then filtered sterile. Generally, dispersions are prepared by incorporating a variety of sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and, as needed, other ingredients from those listed above. Where sterile powders are used to prepare sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

The compositions disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins) and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). After preparation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The preparation is easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, etc.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of these media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or agents being compatible with the active ingredient, their use in therapeutic compositions is also contemplated. Supplementary active ingredients may also be incorporated into the composition.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an allergic reaction or similar troublesome reaction when administered to a human. The preparation of aqueous compositions comprising a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared.

VI. Treatment Methods

A. Cancer and Proliferative/Dysplastic/Neoplastic Disorders

The antibodies of the present invention can be used in methods for treating proliferative/dysplastic/neoplastic diseases/conditions including cancer. Types of diseases/conditions expected to be treated with the peptides of the present invention include, but are not limited to, leukemias (e.g., AML, MDS, and CML) and myelodysplasia. Other types of cancer may include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, preneoplastic lesions in the lung, colon cancer, melanoma, bladder cancer, and any other neoplastic disease.

In order to kill cells, inhibit cell growth, inhibit metastasis, reduce tumor/tissue size, tumor cell load, or reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present invention, the hyperplastic/neoplastic/cancer cells are generally contacted with a therapeutic compound (e.g., a polypeptide or an expression construct encoding an antibody of the present invention) that is typically dispersed in a pharmaceutically acceptable buffer or carrier (see discussion of diagnostic agents above). The route of administration varies naturally with the location and nature of the lesion and includes, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulations. For neoplastic diseases and conditions, any of the administration formulations and routes discussed for the treatment or diagnosis of cancer may also be employed. Ex vivo embodiments are contemplated in which tumor cells are treated/transduced externally in the patient's body (specifically or as part of a larger cell population).

For discrete, solid, accessible tumors, intratumoral injection or injection into the tumor vasculature is specifically contemplated. Local administration, regional administration, or systemic administration may also be appropriate. For tumors > 4 cm, the volume to be administered will be about 4 ml to 10 ml; while for tumors < 4 cm, a volume of about 1 ml to 3 ml will be used. Multiple injections delivered in a single dose will comprise a volume of about 0.1 ml to about 0.5 ml. The viral particles may be advantageously contacted by injecting multiple injections into the tumor at intervals of about 1 cm.

In the case of surgical intervention, the present invention can be used at the time of surgery and/or thereafter to treat residual or metastatic disease. For example, a formulation comprising the antibody can be injected or perfused into the resected tumor bed. Perfusion can be continued after resection, for example, by leaving an implanted catheter at the surgical site. Periodic postoperative treatment is also contemplated.

Continuous administration may also be used where appropriate, for example, when a tumor is removed and the tumor bed is treated to eliminate residual microscopic disease. Delivery is preferably by syringe or catheter. After initiation of treatment, such continuous perfusion may be performed for a period of about 1 to 2 hours to about 2 to 6 hours, to about 6 to 12 hours, to about 12 to 24 hours, to about 1 to 2 days, to about 1 to 2 days, or longer. Generally, the dosage of the therapeutic composition administered by continuous perfusion is the same as that administered by single or multiple injections, adjusted for the time period over which perfusion occurs. It is also contemplated that perfusion of the extremities may be used to administer the therapeutic composition of the present invention, particularly for the treatment of melanomas and sarcomas.

Treatment regimens are equally variable and often depend on tumor type, tumor location, disease progression, and the health and age of the patient. Obviously, certain types of tumors will require more aggressive treatments, while at the same time, some patients cannot tolerate more onerous regimens. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic agents.

In certain embodiments, the tumor being treated may be, at least initially, unresectable. Treatment may increase the resectability of the tumor by shrinking the margins or by eliminating certain particularly aggressive portions. Following treatment, resection may be performed. Additional treatment after resection will be used to eliminate microscopic residual disease at the tumor site.

A typical course of treatment for a primary tumor or a resected tumor bed includes multiple doses. Typically, primary tumor treatment involves six doses administered over a two-week period. The two-week regimen can be repeated one, two, three, four, five, six, or more times. During the course of treatment, the need to complete the planned dosing can be reassessed.

B. Combination therapy

It can also be proved that the use of a conjugate therapy comprising a second anticancer agent is advantageous." anticancer " agent can have a negative impact on the cancer of the object, such as by killing cancer cells, inducing cancer cell apoptosis, reducing cancer cell growth rate, reducing the incidence or quantity of metastasis, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumor or cancer cell, promoting the immune response for cancer cell or tumor, preventing or inhibiting cancer progression or increasing the life span of the cancer subject to achieve. Anticancer agent includes biological preparation (biotherapy), chemotherapeutic agent and radiotherapeutic agent. More generally, these other compositions are provided according to the therapy of the present invention with the combined amount of effectively killing cells or inhibiting cell proliferation. The method can include contacting cells with the two agents simultaneously. This can be achieved by contacting cells with a single composition or pharmaceutical preparation comprising the two agents, or by contacting cells with two different compositions or preparations simultaneously.

Alternatively, antibody therapy can be carried out before or after other drug treatments, and is spaced apart by a few minutes to a few weeks. In the embodiment in which other agents and antibodies are applied to cells separately, it is generally ensured that there is no large time period between each delivery so that medicament and expression construct can still play a favorable combined effect to cells. In such a case, it is considered that cells can be contacted with these two forms within about 12 to 24 hours and more preferably within about 6 to 12 hours of each other. However, in some cases, it can be expected that the treatment period will be significantly extended, wherein between each administration, a few days (2, 3, 4, 5, 6 or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) are passed.

Various combinations can be employed; for example, the antibody therapy (with or without a conjugated therapeutic agent) is "A," and the second anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Taking into account the toxicity of the antibody therapy (if any), a general scheme of administering a specific second therapy is performed after administering the therapeutic agent of the present invention to the patient. It is expected that the treatment cycle can be repeated as necessary. It is also expected that a variety of standard therapies and surgical interventions can be used in combination with the cancer therapy.

1. Chemotherapy

Cancer therapy also includes and is based on the multiple conjoint therapy of chemo and radiotherapy.Combined chemotherapy includes for example cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, adriamycin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agent, paclitaxel, gemcitabine, navelbine, farnesyl protein transferase inhibitor, anti-platinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, temozolomide (Temazolomide, the aqueous form of DTIC) or any analogue or derivative variant mentioned above.The combination of chemotherapy and biological therapy is referred to as biochemotherapy.The present invention contemplates any chemotherapeutic agent known in the art for the treatment or prevention of cancer.

2. Radiation therapy

Other factors that cause DNA damage and have been widely used include the direct delivery of commonly known gamma-rays, X-rays and/or radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, have also been considered. It is likely that all of these factors produce extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosomal assembly and maintenance. The dosage range of X-rays is: daily dose is 50 roentgens to 200 roentgens in an extended period of time (3 to 4 weeks), to a single dose of 2000 roentgens to 6000 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the intake of neoplastic cells.

As used herein, the terms "contacting" and "exposing" as applied to cells describe the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or placing them in direct proximity to a target cell. To achieve cell killing or stasis, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent cell division.

3. Immunotherapy

In general, immunotherapy relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies that are specific to some markers on the surface of tumor cells. Antibodies can serve as effectors for treatment alone, or they can recruit other cells to actually achieve cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and only serve as targeting agents. Alternatively, the effector can be a lymphocyte that carries surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of treatment modalities, i.e., the suppression or reduction of direct cytotoxic activity and Fortilin, will provide therapeutic benefits in cancer treatment.

Immunotherapy can also be used as part of a combination therapy. The general approach for combination therapy is discussed below. In one aspect of immunotherapy, tumor cells must have some markers responsible for targeting (i.e., not present on most other cells). There are many tumor markers and any of them can be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is anticancer effect and immune stimulation. There are also immunostimulatory molecules, including: cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, gamma-IFN); chemokines (e.g., MIP-1, MCP-1, IL-8) and growth factors (e.g., FLT3 ligands). Immunostimulatory molecules have been shown to enhance antitumor effects as protein forms or by gene delivery in combination with tumor suppressors (e.g., mda-7) (Ju et al., 2000).

As mentioned previously, examples of immunotherapies currently under investigation or use are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum), dinitrochlorobenzene, and aromatic compounds) (U.S. Patent 5,801,005; U.S. Patent 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy (e.g., interferon and; IL-1; GM-CSF and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent 5,830,880 and U.S. Patent 5,846,945); and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has anti-tumor activity and has been approved for the treatment of malignant tumors (Dillman, 1999). The combination therapy of Herceptin and chemotherapy for cancer is more effective than monotherapy. Therefore, it is expected that one or more anti-cancer therapies can be used together with the tumor-associated HLA-restricted peptide therapy described herein.

Adoptive immunotherapy. In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes of a patient are isolated in vitro, activated with lymphokines (e.g., IL-2) or transduced with tumor necrosis genes, and re-administered (Rosenberg et al., 1988; 1989). To achieve this therapy, an immunologically effective amount of activated lymphocytes is administered to an animal or human patient in combination with an adjuvanted antigenic peptide composition as described herein. The activated lymphocytes are most preferably the patient's own cells, which have been previously isolated from a blood or tumor sample and activated (or "expanded") in vitro. This form of immunotherapy has produced regression of several cases of melanoma and renal cancer, but the percentage of responders is small compared to the number of patients who did not respond.

Passive immunotherapy. There are several different approaches to passive immunotherapy for cancer. They can be broadly categorized as follows: injection of antibodies alone, injection of antibodies conjugated to toxins or chemotherapeutic agents, injection of antibodies conjugated to radioisotopes, injection of anti-idiotypic antibodies, and finally, depletion of tumor cells from the bone marrow.

Preferably, human monoclonal antibodies are adopted in passive immunotherapy because they produce little or no side effects in patients. However, their application is limited to its scarcity to some extent and has only been administered intralesionally so far. Human monoclonal antibodies to ganglioside antigens have been administered intralesionally to patients with recurrent melanoma of the skin (Irie & Morton, 1986). After daily or weekly intralesional injections, six out of ten patients observed degeneration. In another study, moderate success (Irie et al., 1989) was achieved by intralesional injections of two human monoclonal antibodies. Possible therapeutic antibodies include anti-TNF, anti-CD25, anti-CD3, anti-CD20, CTLA-4-IG and anti-CD28.

Advantageously, more than one monoclonal antibody directed against two different antigens may be administered, or even antibodies with multiple antigen specificities may be administered. Treatment may also include administering lymphokines or other immunopotentiators as described by Baiorin et al. (1988). The development of human monoclonal antibodies is described in more detail elsewhere in this specification.

4. Gene therapy

In another embodiment, the second treatment is gene therapy, in which the therapeutic polynucleotide is administered before, after, or simultaneously with the administration of the tumor-associated HLA-restricted peptide. The delivery of a vector encoding the tumor-associated HLA-restricted peptide in combination with a second vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on the target tissue. Alternatively, a single vector encoding both genes can be used. The present invention encompasses a variety of proteins, some of which are described below. Various genes targeted for certain forms of gene therapy combined with the present invention are known to those of ordinary skill in the art and may include any gene involved in cancer.

Cell proliferation inducers. Proteins that induce cell proliferation are further divided into multiple categories based on their function. All of these proteins have in common their ability to regulate cell proliferation. For example, the sis oncogene, a form of PDGF, is a secreted growth factor. Oncogenes rarely originate from genes encoding growth factors, and sis is currently the only known naturally occurring oncogene growth factor. In one embodiment of the present invention, it is contemplated that antisense mRNA directed against a specific cell proliferation inducer may be used to prevent the expression of the cell proliferation inducer.

The proteins FMS, ErbA, ErbB, and neu are growth factor receptors. Mutations in these receptors lead to loss of regulatory function. For example, point mutations affecting the transmembrane domain of the Neu receptor protein generate the neu oncogene. The erbA oncogene is derived from the intracellular receptor for thyroid hormone. Modified oncogenic ErbA receptors are thought to compete with endogenous thyroid hormone receptors, leading to uncontrolled growth.

The largest class of oncogenes includes signal transduction proteins (e.g., Src, Abl, and Ras). Protein Src is a cytoplasmic protein-tyrosine kinase, and in some cases its conversion from a proto-oncogene to an oncogene is produced by a mutation at tyrosine residue 527. In contrast, in one example, the conversion of the GTPase protein ras from a proto-oncogene to an oncogene is produced by a mutation from valine at amino acid 12 in the sequence to glycine, which reduces ras GTPase activity. Proteins Jun, Fos, and Myc are proteins that act as transcription factors directly on nuclear function.

Cell proliferation inhibitors. Tumor suppressor oncogenes function to inhibit excessive cell proliferation. Inactivation of these genes disrupts their inhibitory activity, leading to unregulated proliferation. The most common tumor suppressors are Rb, p53, p21, and p16. Other genes that can be used according to the present invention include APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, C-CAM, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusion, and 21/p27 fusion.

Regulator of programmed cell death. Apoptosis or programmed cell death is an essential process for normal embryonic development, maintenance of adult tissue homeostasis and inhibition of carcinogenesis (Kerr et al., 1972). The Bcl-2 protein family and ICE-like proteases have been shown to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein found in association with follicular lymphoma plays a prominent role in controlling apoptosis and enhancing cell survival in response to various apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsu.jimoto et al., 1985; Tsu.jimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein is now considered to be a member of a family of related proteins that can be classified as death agonists or death antagonists.

Following the discovery of this protein, Bcl-2 was shown to inhibit cell death triggered by a variety of stimuli. Furthermore, it is now apparent that there exists a family of Bcl-2 cell death regulatory proteins that share common structural and sequence homology. These various family members have been shown to have functions similar to those of Bcl-2 (e.g., _BclxL , _BclW , _BclS , Mcl-1, A1, Bfl-1), or to function in opposition to Bcl-2 and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

5. Surgery

About 60% of cancer patients undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other therapies (e.g., the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or alternative therapies).

Curative surgery includes excision, wherein physical removal, cutting away and/or destroying all or part of cancerous tissue. Tumor resection refers to physical removal of at least a portion of a tumor. Except tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs surgery). It is also contemplated that the present invention can be used in combination with the removal of superficial cancer, procarcinoma or the normal tissue of the accompanying amount.

After excision of some or all of the cancer cells, tissues or tumors, a cavity can be formed in vivo. Treatment can be achieved by perfusion, direct injection or by additional anticancer therapy for local application in this region. This treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also use different dosages.

C. Autoimmune disease

The present invention also contemplates the use of the antibodies of the present invention to treat autoimmune diseases. PR-1 is derived from myeloid autoproteins. Proteinase 3 (Pr3), which contains PR1, is the target of the autoimmune attack of Wegener's granulomatosis. Myeloperoxidase (MPO) is a target antigen in small vessel vasculitis (Franssen et al., 1996; Brouwer et al., 1994; Molldrem et al., 1996), as evidenced by the presence of both T cell and antibody immunity in patients with these diseases. Wegener's granulomatosis is associated with the production of cytoplasmic anti-neutrophil cytoplasmic antibodies (cAMCA) specific for Pr3 (Molldrem et al., 1997), while microscopic polyangiitis and Churg-Strauss syndrome are associated with perinuclear ANCA (pANCA) specific for MPO (Molldrem et al., 1999; Savage et al., 1999). Therefore, inhibiting immune cell recognition of PR1 can treat autoimmune diseases.

Thus, the antibodies of the invention are administered to subjects with autoimmune diseases to neutralize the effects of other autoantibodies (e.g., pANCA to proteinase 3). Alternatively, the antibodies can be engineered to be "bispecific," i.e., to have immunospecificity for two antigens, one of which is PR1/HLA-A2 and the other is a dendritic cell surface antigen (e.g., DEC-205, LOX-1, RAGE), thereby blocking dendritic cell function in antigen presentation.

1. Vasculitis

Vasculitis is a process caused by inflammation of the blood vessel walls and leads to a variety of conditions. There is no accepted classification system for vasculitis, but it can be classified as large vessel vasculitis, medium vessel vasculitis, or small vessel vasculitis by the size or type of blood vessels involved. Small vessel vasculitis is defined as vasculitis that affects blood vessels smaller than arteries (i.e., arterioles, venules, and capillaries); however, small vessel vasculitis can also involve medium-sized arteries. Anti-neutrophil cytoplasmic antibody (AMCA)-associated vasculitis is the most common cause of small vessel vasculitis and includes microscopic polyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome, and certain types of drug-induced vasculitis.

Wegener's granulomatosis. Wegener's granulomatosis is a rare disease that causes inflammation of blood vessels in the upper respiratory tract (nose, sinuses, ears), lungs and kidneys. It can also affect many other areas of the body, where arthritis (inflammation of the joints) occurs in almost half of all cases. It can also affect the eyes and skin. Although the cause is unknown, Wegener's granulomatosis is considered an autoimmune disease and is often classified as one of the rheumatic diseases. Destructive lesions occur in the upper and lower respiratory tract and kidneys. In the kidneys, these lesions cause glomerulonephritis, which can lead to hematuria (blood in the urine) and kidney failure. It most often occurs between the ages of 30 and 50, and men are usually twice as likely to be afflicted as women. It is rare in children, but is seen in infants as young as 3 months old. Kidney disease can progress rapidly, with kidney failure occurring within a few months of initial diagnosis. If left untreated, more than 90% of all Wegener's granulomatosis patients will develop kidney failure and die.

Early symptoms may include fatigue, malaise, fever, and discomfort around the nose and sinuses. Upper respiratory infections (such as sinusitis or ear infections) often precede the diagnosis of Wegener's granulomatosis. Other upper respiratory symptoms include nose bleeds, pain, and sores at the nasal opening. Persistent fever for no apparent reason (fever of unknown origin - FUO) may be the initial symptom. Night sweats may accompany the fever. Loss of appetite and weight loss are common. Skin lesions are common, but there is no characteristic lesion associated with the disease. Kidney disease is necessary to make a definitive diagnosis of Wegener's granulomatosis. The urine may contain blood, which often first appears as red or smoky urine. Although there may be no symptoms, the diagnosis is easily made through laboratory studies. Eye problems occur in a large number of patients and can range from mild conjunctivitis to severe inflammation of the eye and the tissues around the eye. Additional symptoms include weakness, loss of appetite, weight loss, bloody discharge from the nose, sinus pain, sinusitis, lesions in and around the nose, cough, coughing up blood, bloody sputum, shallow breathing, wheezing, chest pain, blood in the urine, rash, and joint pain.

Diagnosis is made by biopsy of abnormal tissue, which may include open lung biopsy, upper airway biopsy, nasal mucosal biopsy, bronchoscopy and transtracheal biopsy, kidney biopsy, urinalysis, chest x-ray, bone marrow aspirate, and blood tests for autoantibodies. Treatment includes corticosteroids, cyclophosphamide, methotrexate, or azathioprine, which can produce long-term remissions in more than 90% of affected people.

Churg-Strauss syndrome. Churg-Strauss syndrome (CSS), also known as allergic granulomatosis, is a form of systemic vasculitis. CSS is similar to polyarteritis nodosa, but the abundance of eosinophils distinguishes it from that disease. Most CSS patients are middle-aged with a history of new or increasing severity of asthma, one of the main features of CSS. Symptoms of asthma can begin long before the onset of vasculitis. Other early symptoms include nasal polyps and allergic rhinitis. The disease often turns into eosinophilia, with counts as high as 60%. The next stage of the disease is overt vasculitis, which can involve the skin, lungs, nerves, kidneys, and other organs. Peripheral nerve involvement can be particularly debilitating and include pain, numbness, or tingling in the extremities (neuropathy/mononeuritis multiplex). Before the advent of therapy, CSS was often a fatal disease. Most patients died from the widespread, uncontrolled disease.

Although the cause of CSS is unknown, it appears to be multifactorial. Although there may be a genetic factor, CSS is only rarely seen in two members of the same family. Therefore, environmental factors and infections are more likely to be the cause, but there is no clear evidence for this. Diagnosis is made by a specific combination of symptoms and signs, the type of organ involved, and certain abnormal blood tests (particularly eosinophilia). In addition to a detailed patient history and physical examination, blood tests, chest X-rays, and other types of imaging studies, nerve conduction tests and tissue biopsies (lung, skin, or nerve) may be performed to aid in the diagnosis. In order to be classified as a CSS patient, the patient should have at least 4 of the following 6 criteria: 1) wheezing; 2) eosinophilia [>10% in the differential WBC count]; 3) mononeuropathy; 4) transient pulmonary infiltrates on chest X-ray; 5) lateral nasal sinus abnormalities; and 6) tissue biopsy containing blood vessels with extravascular eosinophils.

CSS is usually responsive to prednisone. Initially, high-dose oral prednisone was used, but after about the first month, this high-dose of prednisone gradually decreased in the following month. In addition to prednisone, other immunosuppressive drugs such as azathioprine, cellcept, methotrexate or cyclophosphamide can also be used. High-dose intravenous steroids can be used for those patients with serious illnesses, or for those patients who do not respond to other treatments. By suitable therapy, symptoms begin to subside quickly, and cardiac function and renal function gradually improve, and the pain caused by peripheral nerve involvement is improved. According to the continuation of patient response and disease, therapy can be sustainable for 1 to 2 years.

2. Crohn’s disease

Symptoms of Crohn's disease include intestinal inflammation and the development of intestinal strictures and fistulas; these symptoms are often accompanied by neuropathy. Anti-inflammatory drugs (e.g., 5-aminosalicylic acid (e.g., mesalamine) or corticosteroids) are commonly prescribed but are not always effective (reviewed in V.A. Botoman et al., 1998). Immunosuppression with cyclosporine is sometimes beneficial for patients who are resistant or intolerant to corticosteroids (Brynskov et al., 1989).

However, 90% of patients ultimately require surgical correction; 50% undergo colectomy (Leiper et al., 1998; Makowiec et al., 1998). Postoperative recurrence rates are high, with 50% requiring reoperation within 5 years (Leiper et al., 1998; Besnard et al., 1998).

One hypothesis for the etiology of Crohn's disease is that intestinal mucosal barrier failure, which can be caused by genetic susceptibility and environmental factors (e.g., smoking), exposes the immune system to antigens from the intestinal lumen (including bacterial and food antigens) (e.g., Soderholm et al., 1999; Hollander et al., 1986; Hollander, 1992). Another hypothesis is that persistent intestinal infection with pathogens (e.g., Mycobacterium paratuberculosis, Listeria monocytogenes, abnormal Escherichia coli, or paramyxoviruses) stimulates the immune response; or alternatively, symptoms are caused by a dysregulated immune response to ubiquitous antigens (e.g., normal intestinal microflora and its metabolites and toxins) (Sartor, 1997). The presence of IgA and IgG anti-Sacccharomyces cerevisiae antibodies (ASCA) in serum was found to be highly diagnostic for pediatric Crohn's disease (Ruemmele et al., 1998; Hoffenberg et al., 1999).

In Crohn's disease, the dysregulated immune response tends to be cell-mediated immunopathology (Murch, 1998). However, immunosuppressive drugs (e.g., cyclosporine, tacrolimus, and mesalamine) have been used to treat corticosteroid-resistant cases of Crohn's disease with some success (Brynskov et al., 1989; Fellerman et al., 1998).

Recent efforts to develop diagnostic and therapeutic tools for Crohn's disease have focused on the central role of cytokines (Schreiber, 1998; van Hogezand & Verspaget, 1998). Cytokines are small secreted proteins or factors (5 kD to 20 kD) that have specific effects on cell-cell interactions, intercellular communication, or the behavior of other cells. Cytokines are produced by lymphocytes, especially T _H 1 and T _H 2 lymphocytes, monocytes, intestinal macrophages, granulocytes, epithelial cells, and fibroblasts (reviewed in Rogler & Andus, 1998; Galley & Webster, 1996). Some cytokines are proinflammatory (e.g., TNF-α, IL-1 (α and β), IL-6, IL-8, IL-12, or leukemia inhibitory factor (LIF)); others are anti-inflammatory (e.g., IL-1 receptor antagonists, IL-4, IL-10, IL-11, and TGF-β). However, their roles may overlap and be functionally redundant in certain inflammatory conditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α and IL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, and IL-8 are overproduced locally by mucosal cells (Funakoshi et al., 1998). These cytokines can have far-ranging effects on physiological systems, including bone development, hematopoiesis, and liver, thyroid, and neuropsychiatric function. In addition, an imbalance in the IL-1β/IL-1ra ratio favoring proinflammatory IL-1β has been observed in Crohn's patients (Rogler & Andus, 1998; Saiki et al., 1998; Dionne et al., 1998; but see S. Kuboyama, 1998). One study showed that cytokine profiles in stool samples can serve as a useful diagnostic tool for Crohn's disease (Saiki et al., 1998).

Treatments proposed for Crohn's disease include the use of various cytokine antagonists (e.g., IL-1 ra), inhibitors (e.g., IL-1β converting enzyme and antioxidants), and anti-cytokine antibodies (Rogler and Andus, 1998; van Hogezand & Verspaget, 1998; Reimund et al., 1998; N. Lugering et al., 1998; McAlindon et al., 1998). In particular, monoclonal antibodies against TNF-α have been tried in Crohn's disease treatment with some success (Targan et al., 1997; Stack et al., 1997; van Dullemen et al., 1995). These compounds may be used in combination therapy with the compounds of the present invention.

Another approach to treating Crohn's disease focuses on at least partially eradicating the bacterial flora that can trigger an inflammatory response and replacing it with a non-pathogenic flora. For example, U.S. Patent No. 5,599,795 discloses a method for preventing and treating Crohn's disease in human patients. The method involves sterilizing the intestine with at least one antibiotic and at least one antifungal agent to kill the existing flora and replace them with a selection of well-characterized bacteria taken from a normal human. Borody teaches a method for treating Crohn's disease by at least partially removing the existing intestinal microbial flora by lavage and replacing it with a new bacterial flora introduced by a fecal inoculum from a disease-screened human donor or by a new bacterial flora introduced by a composition comprising Bacteroides and Escherichia coli species (U.S. Patent No. 5,443,826). However, the cause of Crohn's disease that can be diagnosed and/or treated is unknown.

3. Rheumatoid arthritis

The exact etiology of RA is still unknown, but it is clear that it has an autoimmune aspect. The first signs of arthritis appear in the synovial lining, where synovial fibroblasts proliferate and attach to the articular surfaces of the joint margins (Lipsky, 1998). Subsequently, macrophages, T cells and other inflammatory cells are recruited into the joints, where they produce a large amount of mediators, including the following cytokines: interleukin-1 (IL-1), which contributes to the chronic sequelae of bone and cartilage destruction, and tumor necrosis factor (TNF-α), which plays a role in inflammation (Dinarello, 1998; Burger & Dayer, 1995; van den Berg, 2001). The IL-1 concentration in the plasma of RA patients is significantly higher than that in healthy individuals, and it is noteworthy that plasma IL-1 levels are correlated with RA disease activity (Eastgate et al., 1988). Moreover, synovial fluid levels of IL-1 are associated with a variety of radiographic and histological features of RA (Kahle et al., 1992; Rooney et al., 1990).

In normal joints, the effects of these and other proinflammatory cytokines are balanced by a variety of anti-inflammatory cytokines and regulatory factors (Burger & Dayer, 1995). The significance of this cytokine balance is illustrated in adolescent RA patients who have increased fever cycles throughout the day (Prieur et al., 1987). After each peak of fever, factors that block the effects of IL-1 are found in serum and urine. This factor was isolated, cloned and identified as IL-1 receptor antagonist (IL-1ra) (a member of the IL-1 gene family) (Hannum et al., 1990). IL-1ra, as its name indicates, is a natural receptor antagonist that competes with IL-1 for binding to the type I IL-1 receptor and thus blocks the effects of IL-1 (Arend et al., 1998). A 10-fold to 100-fold excess of IL-1ra may be required to effectively block IL-1; however, synoviocytes isolated from RA patients do not appear to produce enough IL-1ra to counteract the effects of IL-1 (Firestein et al., 1994; Fujikawa et al., 1995).

4. Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by the deposition of autoantibodies and immune complexes in tissues, leading to tissue damage (Kotzin, 1996). Compared to autoimmune diseases such as MS and type 1 diabetes, SLE potentially directly involves multiple organ systems, and its clinical manifestations are diverse and variable (reviewed by Kotzin & O'Dell, 1995). For example, some patients may demonstrate primarily rash and joint pain, show spontaneous remission, and require very little medication. At the other end of the spectrum are patients who demonstrate severe and progressive kidney involvement, requiring treatment with high doses of steroids and cytotoxic drugs such as cyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE and the primary diagnostic test available is elevated serum levels of IgG antibodies to nuclear components such as double-stranded DNA (dsDNA), single-stranded DNA (ss-DNA), and chromatin. Of these autoantibodies, IgG anti-dsDNA antibodies play a major role in the development of lupus glomerulonephritis (GN) (Hahn & Tsao, 1993; Ohnishi et al., 1994). Glomerulonephritis is a serious condition in which the capillary walls of the glomeruli, the blood-purifying vessels of the kidney, thicken due to accretion on the epithelial side of the glomerular basement membrane. The disease is often chronic and progressive and can lead to eventual renal failure.

The mechanism of induction of autoantigens in these autoimmune diseases remains unclear. Because there is no known cause of SLE that can be diagnosed and/or treated, treatment is directed toward suppressing the immune response, such as with macrolide antibiotics, rather than targeting the underlying cause (e.g., U.S. Patent No. 4,843,092).

5. Juvenile rheumatoid arthritis

Juvenile rheumatoid arthritis (JRA), the term for the most common form of childhood arthritis, is applied to a family of diseases characterized by chronic inflammation and synovial hypertrophy. The term overlaps with, but is not entirely synonymous with, the family of diseases known in Europe as juvenile chronic arthritis and/or juvenile idiopathic arthritis.

Jarvis (1998) and others (Arend, 2001) have proposed that the pathogenesis of rheumatoid diseases in adults and children involves a complex interplay between innate and adaptive immunity. This complexity lies at the heart of the difficulty in unraveling the pathogenesis of the disease.

Both the innate and adaptive immune systems utilize a variety of cell types, a vast array of cell surface and secreted proteins, and an interconnected network of positive and negative feedback (Lo et al., 1999). Furthermore, when considered separate, the innate and adaptive wings of the immune system functionally intersect (Fearon & Locksley, 1996), and the pathological events that occur at these intersections may be highly relevant to our understanding of the pathogenesis of adult and pediatric forms of chronic arthritis (Warrington, et al., 2001).

Polyarticular JRA is a distinct clinical subtype characterized by inflammation and synovial proliferation in multiple joints (four or more), including the small joints of the hands (Jarvis, 2002). Because of its multi-joint involvement and its ability to progress rapidly over time, this JRA subtype can be severe. Although clinically distinct, polyarticular JRA is not uniform, and patients vary in their clinical presentation of the disease, age of onset, prognosis, and response to treatment. These differences most likely reflect the varying spectrum of immune and inflammatory attacks that can occur in this disease (Jarvis, 1998).

6. Sjögren's syndrome

Primary Sjögren's syndrome (SS) is a chronic, slowly progressive systemic autoimmune disease that primarily affects middle-aged women (female to male ratio 9:1), but can be seen in all ages, including children (Jonsson et al., 2002). It is characterized by lymphocytic infiltration and destruction of exocrine glands by mononuclear cells including CD4+, CD8+ lymphocytes and B cells (Jonsson et al., 2002). In addition, extraglandular (systemic) manifestations are seen in one-third of patients (Jonsson et al., 2001).

Glandular lymphocytic infiltration is a progressive feature (Jonsson et al., 1993), which can replace large parts of the organ when it expands. Interestingly, in some patients, the glandular infiltrates are very similar to ectopic lymphoid microstructures in the salivary glands (called ectopic germinal centers) (Salomonsson et al., 2002; Xanthou & Polihronis, 2001). In SS, ectopic GCs are defined as aggregates of proliferating T cells and B cells with a network of follicular dendritic cells and activated endothelial cells. These GC-like structures formed in target tissues also depict the functional properties of producing autoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson &, Jonsson, 2003).

In other systemic autoimmune diseases (such as RA), factors that are key to ectopic GC have been identified. Rheumatoid synovial tissue with GC was shown to produce chemokines CXCL13, CCL21, and lymphotoxin (LT)-β (detected on follicular center and mantle area B cells). Multivariate regression analysis of these analytes identified CXCL13 and LT-β as separate cytokines that predict GC in rheumatoid synovitis (Weyand & Goronzy, 2003). Recently, salivary gland CXCL13 and CXCR5 have been shown to play an essential role in the inflammatory process by recruiting B cells and T cells, thus contributing to lymphoid regeneration and ectopic GC formation in SS (Salomonsson & Larsson, 2002).

7. Psoriasis

Psoriasis is a chronic skin disease of scaling and inflammation that affects 2% to 2.6% of the U.S. population, or 5.8 to 7.5 million people. Although the disease occurs in all age groups, it primarily affects adults. It occurs with about the same frequency in men and women. Psoriasis occurs when skin cells rapidly emerge from their source beneath the skin's surface and accumulate on the surface before they have a chance to mature. Usually this movement (also known as turnover) takes about a month, but in psoriasis, this can occur in just a few days. In its typical form, psoriasis causes patches of thick, red (inflamed) skin covered with silvery scales. These patches (which are sometimes called plaques) are usually itchy or painful. It most commonly occurs on the elbows, knees, other parts of the legs, scalp, lower back, face, palms, and soles of the feet, but it can also occur on the skin anywhere on the body. The disease can also affect the fingernails, toenails, genitals, and the soft tissue inside the mouth. Although skin breakdown around affected joints is common, about 1 million people with psoriasis experience joint inflammation that produces the symptoms of arthritis. This condition is called psoriatic arthritis.

Psoriasis is a skin disorder driven by the immune system, particularly involving T cells. In psoriasis, T cells misbehave and become so overactive that they trigger another immune response, leading to inflammation and rapid turnover of skin cells. In about a third of cases, there is a family history of psoriasis. Researchers have studied several families affected by psoriasis and identified genes associated with the disease. People with psoriasis may notice that their skin sometimes worsens and then improves. Conditions that can cause flare-ups include infection, stress, and changes in climate that dry out the skin. In addition, certain medications prescribed for high blood pressure, including lithium and beta-blockers, can trigger flare-ups or exacerbations of the disease.

8. Multiple sclerosis

Multiple sclerosis (MS) is still a serious health problem that only tortures hundreds of thousands of people in the United States and tortures millions of people in the world every year. It is one of the most common diseases of the central nervous system (brain and spinal cord). MS is an inflammatory disease relevant to demyelination or myelin loss. Myelin (the fatty substance that isolates nerves) acts as an insulating layer so that nerves can transmit impulses from one point to another. In MS, myelin loss is accompanied by the destruction of the ability of nerve conduction electrical impulses to and from the brain, and this has produced a plurality of symptoms of MS, such as vision, muscle coordination, strength, sensation, speaking and swallowing, bladder control, libido and cognitive ability are impaired. The plaque or lesion where myelin is lost shows as a hardened scar-like area. These scars occur at different times and in different areas of the brain and spinal cord, and are therefore the term "multiple" sclerosis, literally meaning a plurality of scars.

Currently, no single laboratory test, symptom, or physical finding provides a definitive diagnosis of MS. Further complicating matters, the symptoms of MS can be easily confused with a wide variety of other diseases, such as acute disseminated encephalomyelitis, Lyme disease, HIV-associated myelopathy, HTLV-1-associated myelopathy, neurosyphilis, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, polyarteritis nodosa, Sjögren's syndrome, Behçet's disease, sarcoidosis, paraneoplastic syndromes, subacute combined degeneration of the spinal cord, subacute myelo-optic neuropathy, adrenomyoneuropathy, spinocerebellar syndrome, hereditary spastic paraparesis/primary lateral sclerosis, stroke, tumors, arteriovenous malformations, arachnoid cysts, Arnold-Chiari malformation, and cervical spondylosis. Therefore, the diagnosis of MS must be made through a process of demonstrating findings consistent with MS and excluding other causes.

In general, the diagnosis of MS relies on two criteria. First, there must be two attacks at least one month apart. An attack (also called an exacerbation, flare-up, or relapse) is the sudden onset or worsening of MS symptoms that lasts for at least 24 hours. Second, there must be more than one area of central nervous system myelin damage. The sheath damage must occur at more than one time point and not be caused by any other disease that can cause demyelination or similar neurological symptoms. MRI (magnetic resonance imaging) is currently the preferred method for imaging the brain to detect the presence of plaques or scars caused by MS.

However, the diagnosis of MS cannot be made based solely on MRI. Other diseases can cause lesions in the brain that are similar to those caused by MS. In addition, the appearance of brain lesions by MRI can be quite different from one patient to another, even resembling brain or spinal cord tumors in some aspects. Moreover, a normal MRI scan does not rule out the diagnosis of MS, as a small number of patients with confirmed MS do not show any lesions in the brain on MRI. These individuals often have spinal cord lesions or lesions that are not detectable by MRI. Therefore, it is crucial that a thorough clinical examination also includes a medical history and functional testing. This should cover mental, emotional and language function, movement and coordination, vision, balance, and the five senses. When symptoms first begin, the person's gender, place of birth, family history, and age are also important considerations. In some cases, additional tests may be needed, including evoked potentials (electrodiagnostic studies that can reveal delays in central nervous system conduction times), cerebrospinal fluid (looking for the presence of clonally expanded immunoglobulin genes, also known as oligoclonal bands), and blood (to rule out other causes).

D. Combination therapy

Combination therapies for the immune disorders listed above are also contemplated. These therapies include standard therapies used in conjunction with the treatment methods of the present invention, such as anti-inflammatory agents and immunosuppressants. These standard therapies will be able to negatively impact the immune cells that cause the disease in the subject or alleviate the symptoms of these diseases. The process may involve contacting the cell or subject with two agents simultaneously. This may be accomplished with a single composition or pharmaceutical formulation comprising both agents, or with two different compositions or formulations simultaneously. Alternatively, the antibody therapy may be administered before or after treatment with the other agent, with intervals ranging from a few minutes to a few weeks.

Various combinations can be employed; for example, an antibody therapy (with or without a conjugated therapeutic agent) is "A," and a second immune disease therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Taking into account the toxicity of the antibody treatment (if any), a general scheme of administering a specific second therapy is performed after administering the therapeutic agent of the present invention to the patient. It is expected that the treatment cycle can be repeated as necessary. It is also expected that a variety of standard therapies and surgical interventions can be used in combination with the therapy.

VII. Examples

The following examples are included to demonstrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present invention and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes can be made to the disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present invention.

Example 1: Method

Antibody Production. To generate antibodies against the combined PR1/HLA-A*0201 epitope, the present inventors immunized BALB/c mice with recombinant PR1/HLA-A*0201 monomers via subcutaneous (SQ) and intraperitoneal (IP) routes, three times at two-week intervals. Splenocytes were isolated from the immunized animals and B cells were fused with HGPRT-negative immortalized myeloma cells using polyethylene glycol (PEG). Hybridoma cells were then selected using pp65/HLA-A*0201 and PR1/HLA-A*0201 monomers and plated in 96-well plates for single-cell cloning.

Antibody Screening and Characterization. Monoclonal cell lines (~20,000) were screened with PR1/HLA-A*0201 monomers by ELISA to identify positive antibody-secreting hybridomas. The 8F4 hybridoma was identified for its specificity for PR1/HLA-A*0201 by ELISA and characterized using isotype-specific antibodies and immunoglobulin light chain antibodies.

Antibody Cloning, Sequence Analysis, and Binding Studies. The 8F4 heavy chain was cloned from hybridoma cDNA, and the primary sequence was obtained. Epitope mapping was performed by binding an altered PR1 peptide containing Ala substitutions at each position from P1 to P9 to the HLA-A*0201 heavy chain and the β-2 microglobulin fold. The binding affinity of 8F4 for PR1/HLA-A*0201 was determined by surface plasmon resonance on a Biacore instrument using immobilized 8F4 and increasing concentrations of soluble PR1/HLA-A*0201. FACS analysis and confocal imaging were used to investigate the binding of 8F4 to normal and abnormal cells.

Antibody activity. To determine whether 8F4 binding to AML triggers cell lysis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were performed. AML cells from patient material that showed sensitivity to 8F4 CDC-mediated lysis were incubated in the presence or absence of 8F4 or an isotype control and then transferred into irradiated (200 cGy) immunodeficient HLA-A2 transgenic NOD/SCID mice. Animals were sacrificed at two weeks and spleen cells and bone marrow were analyzed by FACS.

Total RNA isolation. Qiagen RNA easy kit with Minerut columns was used. RNA loading buffer was prepared as follows: 1 μl of ethidium bromide (EtBr) (10 mg/ml) was added to 100 μl of 10× DNA loading dye in 1× TAE, RNAse-free H ₂ O, and 1% agarose (in the kit). The instructions read "hybridoma cells or frozen vials of 1 × 10 ⁶ to 5 × 10 ⁶ viable cells. If active culture is available, pellet 1 × 10 ⁶ to 5 × 10 ⁶ viable cells in a 15 ml conical tube in step 4 and proceed to step 5. If only frozen cells are available, thaw one vial of hybridoma cells at 37°C, remove from the water bath immediately after thawing, and gently mix. Wipe the vial with 70% ethanol and carefully unscrew the cap to avoid contact with the threads. Transfer the contents of the vial to a 15 ml conical tube containing 15 ml complete medium. Centrifuge at 100 × g (~1,000 rpm for a low-speed Sorvall centrifuge) for 5 minutes. During the spin, add b-mercaptoethanol to a small aliquot of RLT buffer. Carefully remove all medium from the cells with a 10 ml pipette. Lyse the cell pellet in buffer RLT using a Qiagen precipitator and follow the Qiagen protocol for RNA isolation. Depending on the starting cell amount, use 2X Elute RNA from the Minerut column with 15 μl of RNAse-free H ₂ O (1 x 13 μl if eluting from a 6-well column). The RNA should remain on the ICE throughout the following procedure. Pour a 1% agarose microgel containing 1 μg/ml EtBr and quantify the RNA during the 15 minute curing time. Using the same RNAse-free _{H 2} O as above as a blank, quantify 2 μl of RNA using a spectrophotometer. Calculate the RNA concentration: (A ₂₆₀ )(40) - μg/ml. The A 260/280 ratio should be > 1.6. Check the quality of the RNA by running 1 μg on a 1% agarose microgel in a total volume of 10 μl of 1X RNA loading buffer. Run ~1 inch into the gel. Analyze the gel on a photo documentation system. A high-quality RNA banding pattern is characterized by distinct 28s and 18s ribosomal RNA bands with an ideal ratio of 2:1 intensities. A 1:1 ratio is also acceptable; however, the absence of bands or a smear at the bottom of the gel indicates that the RNA is denatured and should not be used.

Isolation and sequence analysis of rearranged Ig variable region (V) genes from hybridomas. In order to obtain DNA sequences by V heavy chain (VH) and V light chain (VL) genes, rapid cloning (RACE) PCR of cDNA ends in combination with human heavy chain constant region primers or light chain constant region primers was used. 5' RACE cDNA amplification was performed using BD Smart™ RACE cDNA amplification kit (BD Bioscience) and according to the instructions provided. PFU ultra (Stratagene), Universla primer A mixture (UPM) and gene specific primers (GSP) of human IgG H & L constant region were used.

The clone of 5 ' RACE PCR product and DNA sequencing use TOPO cloning kit (Invitrogen) and gel extraction kit (Qiagen).For IgG L, separate 8 colonies and be used for a small amount of preparation and pass through EcoRI digestion screening.With M13rev and T7 primer, six positive clones are checked order.For IgG, separate 8 colonies and be used for a small amount of preparation and pass through EcoRI digestion screening.With M13rev and T7 primer, six positive clones are checked order.

Example 2: Results

Antibody Production. To generate antibodies against the combined PR1/HLA-A*0201 epitope, the present inventors immunized BALB/c mice with recombinant PR1/HLA-A*0201 monomers via subcutaneous (SQ) and intraperitoneal (IP) routes, three times at two-week intervals. Splenocytes were isolated from the immunized animals and B cells were fused with HGPRT-negative immortalized myeloma cells using polyethylene glycol (PEG). Hybridoma cells were then selected using pp65/HLA-A*0201 and PR1/HLA-A*0201 monomers and plated in 96-well plates for single-cell cloning.

Antibody Screening and Characterization. Monoclonal cell lines were screened using PR1/HLA-A*0201 monomers by ELISA to identify positive antibody-secreting hybridomas. Nearly 2,000 hybridomas were screened, and one, designated 8F4, was identified by ELISA as specific for PR1/HLA-A*0201. Characterization of the 8F4 hybridoma using isotype-specific antibodies and immunoglobulin light chain antibodies revealed secretion of a single IgG2a-κ PR1/HLA-A*0201-specific antibody.

Antibody Binding Evaluation. Epitope mapping was performed by binding altered PR1 peptides containing Ala substitutions at each position from P1 to P9 to the HLA-A*0201 heavy chain and the B2 microglobulin fold. P1 was found to be the most critical for 8F4 binding, although alterations at all amino acid positions abolished binding ( FIG1 ). The binding affinity of 8F4 for PR1/HLA-A*0201 was determined by surface plasmon resonance on a Biacore instrument using immobilized 8F4 and increasing concentrations of soluble PR1/HLA-A*0201, as shown in FIG2 . The _KD of 8F4 was 9.9 nM, compared to a _KD of 162 nM for the commercially available BB7.2 murine monoclonal antibody that recognizes a different allele-specific site on HLA-A*0201. Using confocal microscopy, a direct fluorescent conjugate of 8F4 bound only to PR1 peptide-pulsed T2 cells (which express HLA-A*0201), but not to irrelevant pp65-pulsed T2 cells or unpulsed T2 cells. In summary, 8F4 specificity and high 8F4 binding affinity for the combined PR1/HLA-A*0201 were confirmed. Using both FACS analysis and confocal imaging (again using 8F4, FITC-conjugated BB7.2 anti-HLA-A*0201 antibody, and DAPI), 8F4 was shown to bind to circulating immature cells (blasts) from HLA-A2+ patients with AML, but not to PBMCs from HLA-A2+ healthy donors, nor to HLA-A2-negative AML blasts (Figures 3 and 5).

Cloning and sequencing of mouse 8F4 variable region genes. Mouse 8F4 hybridoma cells were grown in RPMI-1640 medium (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS; HyClone) and 1 mM sodium pyruvate at 37°C in _a 7.5% CO2 incubator. Total RNA was extracted from approximately ¹⁰⁷ hybridoma cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the supplier's protocol. Oligo dT-primed cDNA was synthesized using the SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA) according to the supplier's protocol. 8F4 heavy and light chain variable region cDNAs were amplified by polymerase chain reaction (PCR) using Phusion DNA polymerase (New England Biolabs, Beverly, MA) using 3' primers that anneal to the mouse gamma-2a and kappa chain constant regions, respectively, and the universal primer A mix or nested universal primer A provided in the SMARTer RACE cDNA amplification kit as the 5' primer. For PCR amplification of the heavy chain variable region (VH), the 3' primer had the sequence 5'-GCCAGTGGATAGACCGATGG-3' (SEQ ID NO: 46). For PCR amplification of the light chain variable region (VL), the 3' primer had the sequence 5'-GATGGATACAGTTGGTGCAGC-3' (SEQ ID NO: 47). The amplified VH and VL cDNAs were cloned into the pCR4Blunt-TOPO vector (Invitrogen) for sequencing. DNA sequencing of the variable regions was performed using Tocore (Menlo Park, CA). Several heavy and light chain clones were sequenced and unique sequences homologous to representative mouse heavy and light chain variable regions were identified. The consensus cDNA sequences and deduced amino acid sequences of 8F4 VH and VL are shown in Figures 1 and 2, respectively. No unusual features were noted in the mature 8F4 VH and VL amino acid sequences.

Construction of chimeric 8F4 IgG1/κ antibody. A gene encoding 8F4 VH was generated as an exon containing splice donor signals and appropriate flanking restriction enzyme sites by PCR using 8F4 VH cDNA as a template, 5'-GCAACTAGTACCACCATGAACTTCGGGCTCAGC-3' (SEQ ID NO: 48; SpeI site underlined) as a 5' primer, and 5'-CGAAAGCTTGAAGTTAGGACTCACCTGCAGAGAGAGTGACCAG AG-3' (SEQ ID NO: 49; HindIII site underlined) as a 3' primer. Similarly, a gene encoding 8F4 VL was generated by PCR using 8F4 VL cDNA as a template, 5'-GCAGCTAGCACCACCATGGAGTCACAGATTCAG-3' (SEQ ID NO: 50; NheI site underlined) as a 5' primer, and 5'-CGAGAATTCTTTGGATTCTACTTACGTTTGATTTCCAGCTTGG TG-3' (SEQ ID NO: 51; EcoRI site underlined) as a 3' primer. The splice donor signals for the 8F4 VL and VL exons were derived from mouse germline JH3 and Jκ1 sequences, respectively. The PCR amplified fragment was gel purified using the NucleoSpin Extraction II kit (Macherey-Nagel, Bethlehem, PA) and cloned into the pCR4 Blunt-TOPO vector (Invitrogen) for sequence confirmation. The correct V segments were digested with SpeI and HindIII (for VH) or NheI and EcoRI (for VL), gel purified, and cloned into a mammalian expression vector carrying human γ-1 and κ constant regions for the production of chimeric 8F4 IgG1/κ antibodies. The schematic structure of the resulting expression vector pCh8F4 is shown in Figure 11.

Generation of humanized 8F4 VH and VL genes. The humanized 8F4 VH and VL amino acid sequences were designed as follows. First, a three-dimensional molecular model of the 8F4 variable region was constructed using JNBiosciences' proprietary algorithm. This molecular model was then used to identify framework amino acid residues important for forming the CDR structure. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to 8F4 VH and VL were selected, respectively. Finally, the CDR sequences, along with framework amino acid residues important for maintaining the CDR structure, were transplanted from 8F4 VH and VL into the correspondingly selected human framework sequences.

The GenBank database was searched for human VH sequences homologous to the 8F4 VH framework, and the VH sequence encoded by the human U96282 cDNA (U96282VH) (GenBank Accession No.; Rassenti and Kipps, J. Exp. Med. 185:1435, 1997) was selected as the acceptor for humanization. The CDR sequences of 8F4 VH were first transferred to the corresponding positions of U96282 VH. No human framework amino acid substitutions were predicted to be necessary to maintain the CDR structure. The amino acid sequence of the resulting humanized VH (Hu8F4 VH) is shown in Figure 12 along with the 8F4 and U96282 VH sequences.

Based on a homology search with the 8F4 VL framework sequence, the human Vκ region encoded by the AY043146 cDNA (AY043146VL) (GenBank accession number; Ghiotto et al., submitted to GenBank on June 29, 2001) was selected as the recipient for humanization. First, the CDR sequences of 8F4 VL were transferred to the corresponding positions of AY043146 VL. Then, at framework position 70 (a site where three-dimensional modeling of the 8F4 variable region indicated contact with the CDRs), the mouse 8F4 VL amino acid residue was replaced with the corresponding human residue. The amino acid sequence of the resulting humanized VL (Hu8F4VL1) is shown in Figure 13 along with the 8F4 and AY043146 VL sequences.

Although Val at position 70 in the mouse 8F4 VL is located at a framework position important for CDR formation, detailed analysis of the 8F4 variable region molecular model indicated the possibility that the amino acid residue at position 70 in Hu8F4 VL1 could be replaced with the corresponding human residue Asp in AY043146 VL without loss of antibody binding affinity. To further reduce the potential immunogenicity of the humanized 8F4 antibody, a second humanized VL (Hu8F4 VL2) was designed in which Val at position 70 in Hu8F4 VL1 was replaced with Asp. The amino acid sequence of Hu8F4 VL2 is shown in Figure 13.

The gene encoding Hu8F4VH was designed as an exon containing a signal peptide, splice donor signal, and appropriate restriction enzyme sites for subsequent cloning into a mammalian expression vector. The splice donor signal in the Hu8F4VH exon is derived from the human germline JH3 sequence. The signal peptide sequence in the humanized Hu8F4VH exon is derived from the corresponding mouse 8F4VH sequence.

The genes encoding Hu8F4 VL1 and VL2, respectively, were designed as exons containing a signal peptide, splice donor signal, and appropriate restriction enzyme sites for subsequent cloning into a mammalian expression vector. The splice donor signals for the Hu8F4 VL1 and VL2 exons were derived from the human germline Jκ4 sequence. The signal peptide sequences in the humanized Hu8F4 VL1 and VL2 exons were derived from the corresponding mouse 8F4 VL sequence.

Hu8F4 VH, VL1, and VL2 genes were constructed by GenScript USA (Piscataway, NJ) under a confidentiality non-disclosure agreement. After digestion with SpeI and HindIII (for VH) or NheI and EcoRI (for VL), the Hu8F4 VH, VL1, and VL2 genes were subcloned into corresponding sites of a mammalian expression vector for production of a human IgG1/κ format. The resulting expression vector, pHu8F4-1, expresses a humanized 8F4 IgG1/κ antibody (Hu8F4-1) comprising Hu8F4 VH and VL1. Similarly, pHu8F4-2 expresses a humanized 8F4 IgG1/κ antibody (Hu8F4-2) comprising Hu8F4 VH and VL2. The schematic structures of pHu8F4-1 and pHu8F4-2 are shown in Figure 11. The nucleotide sequences of Hu8F4 VH, VL1 and VL2 genes, together with the deduced amino acid sequences, are shown in SEQ ID NOs: 22/23, 24/25 and 26/27, respectively.

The Hu8F4 VH and VL2 genes were also cloned into another mammalian expression vector for the production of a variant human IgG1/κ format, designated IgG1-AA. The IgG1-AA format carries two amino acid substitutions in the γ-1 heavy chain, a Leu to Ala substitution at position 234 and a Leu to Ala substitution at position 235 (Eu numbering; Kabat et al., 1991), resulting in greatly reduced binding to Fcγ receptors (U.S. Patent No. 6,491,916). The schematic structure of the resulting plasmid, pHu8F4-2-AA, is shown in FIG11 .

Generation of Stable Transfectants of NS0 Producing Chimeric and Humanized 8F4 IgG1/κ Antibodies. To generate cell lines stably producing Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA IgG1/κ antibodies, expression vectors pCh8F4, pHu8F4-1, pHu8F4-2, and pHu8F4-2-AA, respectively, were introduced into the chromosome of the mouse myeloma cell line NS0 (European Association for Animal Cell Culture, Salisbury, Wiltshire, UK). NS0 cells were grown in DME medium containing 10% FBS at 37°C in a 7.5% _CO2 incubator. Stable transfection into NS0 was performed by electroporation as described by Bebbington et al. (Bio/Technology 10:169-175, 1992). Prior to transfection, each expression vector was linearized using FspI. Approximately ¹⁰ cells were transfected with 20 μg of linearized plasmid, suspended in DME medium containing 10% FBS, and plated into several 96-well plates. After 48 hours, selection medium (DME medium containing 10% FBS, HT medium supplement (Sigma, St. Louis, MO), 0.25 mg/ml xanthine, and 1 μg/ml mycophenolic acid) was applied. Approximately 10 days after the start of selection, the culture supernatant was assayed for antibody production.

The expression of chimeric and humanized 8F4IgG1/κ antibodies was measured by sandwich ELISA. In a typical experiment, goat anti-human IgG Fcγ chain specific polyclonal antibody (Sigma) was coated with 1/2,000 dilution of 100 μl/well PBS at 4°C overnight, washed with wash buffer (PBS containing 0.05% Tween 20), and blocked with 300 μl/well blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20) for 0.5 hour at room temperature. After washing with wash buffer, 100 μl/well samples appropriately diluted in ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20) were applied to the ELISA plate. Appropriate humanized IgG1/κ antibodies were used as standards. After incubating the ELISA plate for 1 hour at room temperature and washing with wash buffer, bound antibodies were detected using 100 μl/well of a 1/2,000 dilution of HRP-conjugated goat anti-human kappa chain polyclonal antibody (SouthernBiotech). After incubating at room temperature for 0.5 hour and washing with wash buffer, color development was performed by adding 100 μl/well of ABTS substrate (bioWORLD, Dublin, OH). Color development was stopped by adding 100 μl/well of 2% oxalic acid. Absorbance was read at 405 nm. Stable transfectants of NS0 producing high levels of Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA antibodies (NS0-Ch8F4 1-G8, NS0-Hu8F4-1 1-D2, NS0-Hu8F4-2 1-F5, and NS0-Hu8F4-2-AA 1D3, respectively) were adapted for growth in serum-free medium using Hybridoma-SFM (Invitrogen). NS0-Ch8F4 1-G8, NS0-Hu8F4-1 1-D2, NS0-Hu8F4-2 1-F5, and NS0-Hu8F4-2-AA 1D3 were negative for the presence of mycoplasma using a PCR Mycoplasma Detection Set (Takara Bio USA, Madison, WI).

The authenticity of the heavy and light chains produced in NS0-Ch8F4 1-G8, NS0-Hu8F4-1 1-D2, NS0-Hu8F4-2 1-F5, and NS0-Hu8F4-2-AA 1D3 was confirmed by cDNA sequencing. Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and oligo-dT-primed cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the supplier's protocol. The coding region of the gamma-1 heavy chain was amplified by PCR using CMV2 and JNT098 as primers ( Figure 11 ) and Phusion DNA polymerase. The PCR fragment was gel purified and sequenced using CMV2, JNT082, JNT097, and JNT098 as primers, as shown in SEQ ID NOs: 28 and 30-32. Similarly, the coding region of the kappa light chain was amplified using CMV2 and JNT026 (SEQ ID NOs: 28 and 29). Gel-purified DNA fragments were sequenced using CMV2 and JNT026 as primers. For each of the Ch8F4 heavy chain, Ch8F4 light chain, Hu8F4-1 heavy chain, Hu8F4-1 light chain, Hu8F4-2 heavy chain, Hu8F4-2 light chain, Hu8F4-2-AA heavy chain, and Hu8F4-2-AA light chain, the resulting nucleotide sequences of the coding regions perfectly matched the corresponding sequences in pCh8F4, pHu8F4-1, pHu8F4-2, or pHu8F4-2-AA vectors (SEQ ID NOs: 33/34, 35/36, 37/38, 39/40, 41/42, and 43/44).

Purification of 8F4-4, Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA Antibodies. Hybridoma 8F4-4 (provided by Dr. Molldrem) was cultured in RPMI medium (Hyclone) containing 10% FBS and adapted for growth in Hybridoma-SFM. 8F4-4, NS0-Ch8F41-G8, NS0-Hu8F4-11-D2, NS0-Hu8F4-21-F5, and NS0-Hu8F4-2-AA1D3 cells were grown in Hybridoma-SFM in roller bottles to a density of approximately 10 ^cells /ml, 1/10 volume of 60 mg/ml ultrafiltered soy hydrolyzate (Irvine Scientific, Santa Ana, CA) dissolved in SFM4 MAb medium (Hyclone) was added, and further grown until cell viability became less than 50%. After centrifugation and filtration, the culture supernatant is loaded onto a protein A agarose column (HiTrap MABSeIect SuRe, GE Healthcare, Piscataway, NJ). After washing the column with PBS, the antibody is eluted with 0.1M glycine-HCl (pH 3.0). After neutralization with 1M Tris-HCl (pH 8), the buffer solution through which the antibody is eluted is changed to PBS by dialysis. Antibody concentration (1 mg/ml=1.4 OD) is determined by measuring the absorbance at 280 nm. The purification and yield of each batch of 8F4-4, Ch8F4, Hu8F4-1, Hu8F4-2 and Hu8F4-2-AA are summarized in Table 3.

Table 3

Multiple purification batches and yields of 8F4-4, Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA.

Purified 8F4-4, Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA were characterized by SDS-PAGE according to standard procedures. Analysis under reducing conditions showed that each antibody was composed of a heavy chain with a molecular weight of approximately 50 kDa and a light chain with a molecular weight of approximately 25 kDa ( FIG. 14 ). The purity of each antibody appeared to be greater than 95%.

Characterization of Ch8F4 and Hu8F4 Antibodies. Antigen binding of Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA was examined by ELISA using a complex of the PR1 peptide (VLQELNVTV (SEQ ID NO: 45)) and HLA-A2 (PR1/HLA-A2). ELISA plates were first coated with 100 μl/well of 5 μg/ml streptavidin in PBS (Jackson ImmunoResearch, West Grove, PA). After washing the wells with wash buffer (PBS containing 0.05% Tween 20) and blocking with blocking buffer, 50 μl/well of 2 μg/ml biotinylated PR1/HLA-A2, provided by Dr. Molldrem, was added. After incubation at room temperature for 30 minutes, the ELISA plates were washed with wash buffer. Ch8F4, Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA antibodies were added, starting at 1 μg/ml and serially diluted 3-fold in ELISA buffer for binding to PR1/HLA-A2. After incubating the ELISA plate at room temperature for 1 hour and washing with wash buffer, bound antibodies were detected using 100 μl/well of a 1/2,000 dilution of HRP-conjugated goat anti-human kappa chain polyclonal antibody. After incubation at room temperature for 30 minutes and washing with wash buffer, color development was performed by adding 100 μl/well of ABTS substrate. Color development was stopped by adding 100 μl/well of 2% oxalic acid. Absorbance was read at 405 nm. Data are shown in Figure 15. _EC50 values calculated using GraphPad Prism (GraphPad Software, San Diego, CA) were 0.054 μg/ml for Ch8F4, 0.050 μg/ml for Hu8F4-1, 0.07 μg/ml for Hu8F4-2, and 0.07 μg/ml for Hu8F4-2-AA. This result indicates that Hu8F4-1, Hu8F4-2, and Hu8F4-2-AA all retain the antigen-binding affinity of the mouse 8F4 antibody.

Antibody effect on target cells. In order to determine whether 8F4 is combined with AML to trigger cell lysis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were performed. 8F4 is to HLA-A2+AML, but not to the CDC-mediated lysis of HLA-A2 negative AML or HLA-A2+ healthy donor control PMBC, which is shown to be antibody dose-dependent (Fig. 4). AML cells from patients' materials showing sensitivity to 8F4CDC-mediated lysis were incubated in the presence or absence of 8F4 or isotype controls and then transferred to irradiated (200cGy) immunodeficient HLA-A2 transgenic NOD/SCID mice. Animals were sacrificed at two weeks and spleen cells and bone marrow were analyzed by FACS. At autopsy, AML was identified only in animals treated with IgG2a isotype controls and not in animals treated with 8F4 (Fig. 6). Compared with mice treated with isotypes, there was no obvious toxicity in mice receiving 8F4 alone. In summary, these data support the following conclusions: the 8F4 monoclonal antibody: (1) binds specifically to the combined PR1/HLA-A*0201 epitope with high affinity; (2) binds specifically to HLA-A*0201 molecules occupied by PR1 peptides on the surface of human cells (including myeloid leukemias) and can be used to identify such molecules; (3) causes specific lysis of HLA-A2+ AML in the presence of complement; and (4) can prevent AML engraftment in an immunodeficient mouse model.

Prevention of tumor implantation. After injection of AML cells and 8F4, AML infiltrates in experimental mouse tissues were measured and shown in Figures 8A-B. No AML cells were detected in the bone marrow and peripheral blood of the 8F4-treated mice without transfer control and experiment. Mice receiving AML cells mixed with isotype-matched control antibodies (iso) showed AML1 and AML5 implantation two or four weeks after AML transfer. Flow cytometric analysis of AML implantation was performed using an extended graph, including mouse cell-specific markers (mCD45), 3 to 6 human markers (CD45, CD13, CD33, CD34, CD38, HLA-DR), and Live/DeadFixable Aqua (Invitrogen). All figures show live mCD45 cells.

8F4 induces transient neutrophil reduction in HLA-A2 transgenic NOD/SCID. 8F4 or control Ab was injected into HLA-A2Tg NOD/SCID mice that showed endogenous PR1 presentation. Bone marrow cells were harvested and stained with mAbs against mouse antigens. Granulocytopenia was evident in the scatter plot of the bone marrow (Fig. 9A; left panel). Gr-1lo immature neutrophils were present in the bone marrow of 8F4-treated mice, but Gr-1hi mature neutrophils were greatly reduced (Fig. 9A; middle panel). In addition, monocytes (SSClo CD11b+; FIG.9A; right lower gate of the right panel) were reduced in 8F4-treated animals. Intravenous injection of 8F4 induced a transient reduction in the absolute number of circulating mature granulocytes, macrophages, and monocytes in HLA-A2Tg NOD/SCID mice (Fig. 9B). Three weeks after treatment, all populations were still present. No significant pathological changes were evident in the liver, lung, spleen, kidney, heart, and brain of HLA-A2 Tg NOD/SCID mice 7 days after injection of 200 μg (10 mg/kg) 8F4 ( FIG. 9C ).

8F4 induces transient leukopenia in established human hematopoietic cells. Peripheral blood was taken from mice to monitor cord blood engraftment, and 8F4 was injected into the mice 9 to 12 weeks after transfer. The mice were then sacrificed and the blood, spleen, and bone marrow were analyzed for human cell engraftment ( FIG. 10B ). As can be seen, antibody injection transiently reduced the % engraftment of the transferred cells ( FIG. 10A ).

Binding specificity, affinity and activity of humanized 8F4 antibody against human AML. To characterize the binding specificity of Hu8F4, the present inventors performed a FACS-based assay to show that Hu8F4 only binds to PR1-pulsed T2 cells ( FIG. 16A ), but not to pp65-pulsed T2 cells. To characterize the binding affinity of Hu8F4, the present inventors compared the binding of two forms of the humanized antibody, Hu8F4-1 (Hu1) and Hu8F4-2 (Hu2), to mouse 8F4 and an isotype control (rhIgG1) using ELISA. The present inventors used plates coated with recombinant PR1/HLA-A2 monomers to capture the antibodies and used anti-human antibodies in a colorimetric assay to determine the binding fraction by optical density (OD). As shown in FIG. 16B , Hu1 and Hu2 showed K _values of 7.7 nM and 7.8 nM, respectively, which is similar to mouse 8F4 ( _K = 9.9 nM). Thus, both humanized antibodies had identical ligand specificity and binding affinity compared to the parent mouse antibody.This data established biochemical justification for the use of the Hu8F4 antibody in additional experiments to determine the spectrum of activity in preclinical animal models.

To address the potential mechanism of Hu8F4 action, the present inventors treated PR1 pulsed T2 target cells with Hu8F4 or isotype control antibody (IgG) in the presence of rabbit complement and determined complement-mediated lysis using standard assays. As shown in Figure 16C, neither Hu8F4 nor chimeric Ch8F4 (human Fc from IgG1 and mouse F(ab) ₂ from 8F4) mediated complement-dependent cytotoxicity (CDC). Therefore, unlike mouse 8F4 (IgG2), Hu8F4 does not lyse target cells by complement fixation. The present inventors are conducting additional studies to determine whether Hu8F4 mediates ADCC, direct apoptosis, or inhibition of mitosis and proliferation in ongoing experiments.

Next, the inventors used Hu8F4 to treat established primary human AML xenografts in NSG mice. Mice were first implanted with AML for 2 weeks and then treated with Hu8F4, Ch8F4, or isotype IgG1 3x/week at 10 mg/1 kg antibody for 2 weeks. BM and peripheral blood chimerism were analyzed after treatment. As shown in Figure 17, three separate experiments with three different AML samples eliminated or significantly inhibited their growth by Hu8F4 and Ch8F4 compared to isotype controls. Therefore, these data confirm that Hu8F4 has high biological activity for primary human AML from patients with treatment-refractory relapsed disease, and that the mechanism of action of Hu8F4 is independent of complement because NSG mice lack expression of key complement proteins.

Biological safety data of 8F4 in HLA-A2 transgenic immunocompetent (B6) and immunodeficient (NOD/scid) mice. The inventors established three mouse models for preclinical studies of Hu8F4: HLA-A2 transgenic B6 immunocompetent mice, HLA-A2 transgenic NOD/scid mice, and NSG mice (lacking the IL-2 common γ chain). To determine potential toxicity, the inventors first showed that PR1 is expressed on HLA-A2 on 5% and 6% of hematopoietic stem cells and granulocytes in HLA-A2 transgenic animals, respectively. Next, the inventors showed that a single IV administration of high-dose 8F4 (10 mg/kg) induced transient and completely reversible cytopenias in both HLA-A2 transgenic mice (Figures 18 to 19). NSG mice were transplanted with CD34-selected human umbilical cord blood to establish long-term stable human chimerism, which was then treated with single and multiple doses of Hu8F4 to determine the effect of the mAb against PR1/HLA-A2+ human hematopoietic stem cells.

H8F4 (anti-PR1/HLA-A2 mAb) delays tumor growth and prolongs survival of triple-negative breast cancer xenografts. In addition to the above work on leukemia, the inventors have also shown that PR1 9-mer peptides derived from the hematopoietic-restricted serine proteases neutrophil elastase (NE) and proteinase 3 (P3) can also be cross-presented on HLA-A2 of various non-hematopoietic tumors that do not express endogenous P3 or NE, including melanoma, non-small cell lung cancer, and breast cancer (Alatrash et al., 2012). The triple-negative breast cancer cell line MB-MDA-231 (also known as 231 cells) does not express P3 and NE but expresses HLA-A2. However, these cells take in soluble P3 and NE and cross-present PR1, subsequently making 231 cells susceptible to 8F4-mediated cleavage. Importantly, PR1/HLA-A2 is expressed on breast cancer cells from patient tissue biopsies (Alatrash et al., 2012), including triple-negative breast cancer (TNBC) patients. Therefore, PR1/HLA-A2 may be a target antigen on breast cancer, and the inventors inferred that 8F4 may have biological activity against HLA-A2+ breast cancer.

To test this hypothesis, the inventors studied the effect of h8F4 in (a) primary tumors and (b) metastatic tumor xenograft models of NSG mice. In the primary tumor model, 231TNBC cells were injected into the mammary fat pad of NSG mice, followed by a single dose of h8F4, isotype control antibody or PBS. 231 cells were transfected with the ffluc gene so that tumor growth could be monitored over time using bioluminescent imaging (BLI). H & E staining of biopsies of the tumor site 1 to 2 days after transplantation showed that neutrophils and macrophages (which are cells that naturally express P3 and NE) infiltrated the tumor (Figure 20A). As shown in Figure 20B, tumor growth in mice receiving h8F4 was delayed compared to mice receiving isotype control or PBS. In addition, h8F4 prolonged survival (p < 0.01) compared to control mice.

In the second model, 231 cells (2×10 ⁵ ) modified by the ffluc gene were injected into the tail vein of NSG mice and received 10 mg/kg of h8F4 or isotype control antibody on day 7, 3x/week. In untreated NSG mice, 231 cells injected by IV rapidly metastasized to the lungs (determined by BLI) and subsequently metastasized to other tissues, including the spleen, GI tract, and liver. As shown in Figure 20C, h8F4 significantly delayed the metastatic tumor growth of 231 cells compared to isotype-treated mice and significantly increased survival (p=0.0006). These results indicate that 231 cells in vivo take up P3 and NE from tumor-associated neutrophils and macrophages, and PR1 cross-presents on HLA-A2, which inhibits TNBC cell growth by h8F4 treatment. Thus, h8F4 has biological activity against TNBC, and our results strongly suggest that h8F4 mAb has the potential to be a therapeutic mAb for the treatment of non-hematopoietic HLA-A2+ tumors, including breast cancer.

Example 3: Method

Patient tissues, cells and cell cultures. Patient breast cancer frozen tissue blocks were purchased from Origene. Patients and healthy donor samples were collected after obtaining informed consent to participate in a study approved by the Institutional Review Board of MD Anderson Cancer Center (Houston, TX). MDA-MB-231, MCF-7, MDAMB-453 and T47D breast cancer cell lines and SW-620 (colorectal adenocarcinoma), OVCAR-3 (ovarian adenocarcinoma), MIA PaCa-2 (pancreatic cancer), Jurkat (acute T cell leukemia), T2 (B cell/T cell hybridoma), HL-60 (acute promyelocytic leukemia) and U-937 (histiocytic leukemia) cell lines were obtained from the American Type Culture Collection. The MCF-HER-18 cell line was provided by M.-C.Hung (MD Anderson Cancer Center). Mel526, Mel 624, MT 2019, and MT 2333 melanoma cell lines were provided by L. Radvanyi (MD Anderson Cancer Center). Cell lines were authenticated by DNA fingerprinting at MD Anderson Cancer Center within 6 months of experimental use.

Breast cancer cells were grown in 2.5 mM l-glutamine DMEM (HyClone) supplemented with 10% FBS (Gemini Bio-Products) and 100 U/ml penicillin/100 mg/ml streptomycin (Cellgro). G418 (Lonza) (0.5 mg/ml) was added to MCF-7-HER18 cell cultures as a selection agent. RPMI 1640 (HyClone) containing 25 mM HEPES and l-glutamine was used to replace the DMEM used for leukemia cell line cultures. All cell lines were cultured at 37°C in 5% CO2. PBMCs and polymorphonuclear neutrophils (PMNs) from healthy donors and patients were enriched using standard Histopaque 1077 and 1119 (Sigma-Aldrich) gradient centrifugation, respectively.

RT-PCR. mRNA was extracted from cell lines and laser capture microdissection (LCM) samples using the RNA Stat 60 kit (TelTest). cDNA synthesis was performed using the Gene Amp RNA kit (PerkinElmer). The following primers were used: P3, forward primer 59-GACCCCACCATGGCTCAC-39 [SEQ ID NO: 52] and reverse primer 59-ATGGGAAGGACAGACAGGAG-39 [SEQ ID NO: 53]; mammaglobin-1, forward primer 59-AGCACTGCTACGCAGGCTCT-39 [SEQ ID NO: 54] and reverse primer 59-ATAAGAAAGAGAAGGTGTGG-39 [SEQ ID NO: 55]; actin, forward primer 59-CCAGAGCAAGAGAGCTATCC-39 [SEQ ID NO: 56] and reverse primer 59-CTGTGGTGGTGAAGCTGTAG-39 [SEQ ID NO: 57] and GAPDH, forward primer 59-TAGACGGGAAGCTCACTGGC-39 [SEQ ID NO: The reverse primer was 59-AGGTCCACCACCCTGTTGCT-39 [SEQ ID NO: 58] and reverse primer 59-AGGTCCACCACCCTGTTGCT-39 [SEQ ID NO: 59]. After denaturation at 95°C for 5 minutes, the sample was amplified for 35 cycles using an iCycler (Bio-Rad). The sample was run on a 1.5% agarose gel. The bands were imaged using a GelDoc 2000 (Bio-Rad) and analyzed using QuantityOne software (Bio-Rad).

Western blot. Whole cell lysates (WCL) were generated by suspending the cell pellet in a lysis buffer (10 mM/L HEPES [pH 7.9], 10 mM/L KCl, 0.1 mM/L EGTA, 0.1 mM/L EDTA, and 1 mM/L DTT) containing protease inhibitors and subsequently subjected to a freeze-thaw cycle for 15 minutes. WCL were separated by electrophoresis on a 10% SDS gel under reducing conditions, transferred to a polyvinylidene difluoride membrane, blocked with 5% milk, and stained with anti-NE (Santa Cruz Biotechnology), anti-P3 (NeoMarkers), anti-tubulin (Sigma-Aldrich), or anti-GAPDH (Sigma-Aldrich) Ab. Chemiluminescence was captured on Kodak film.

Ag cross-presentation. To determine protein uptake, cells were pulsed in reduced serum medium (0.5% FBS) containing 10 mg/ml P3, NE (both from Athens Research & Technology), EndoGrade OVA (Hyglos), or irradiated (7500 cGy) PMN or PBMC in a 1:1 ratio (breast cancer: irradiated cells). The cells were then permeabilized (BD Biosciences) and stained with anti-P3 (clone MCPR3-2; Thermo Scientific) or anti-NE (Santa Cruz Biotechnology) directly conjugated to Alexa-488 or 647 and analyzed by flow cytometry. To determine cross-presentation, cells were surface stained with fluorescently conjugated 8F4 as previously described (Sergeeva et al., 2011). Anti-P3, anti-NE, and anti-PR1/HLA-A2 (8F4) Abs were directly conjugated using Alexa-488 or 647 kits (Inbitrogen). Aqua live/dead staining (Invitrogen) was used to evaluate viability. For all flow cytometry experiments, light scattering was used to establish preliminary gates, followed by aqua live/dead staining. To inhibit cross-presentation, cells were incubated with the endoplasmic reticulum (ER) to Golgi apparatus antegrade inhibitor brefeldin A (Sigma-Aldrich) or the proteasome inhibitor lactocysteine (Sigma-Aldrich) (Francois et al., 2009; Kovacsovics-Bankowski and Rock 1995; and Mukai et al., 2009).

Use the Leica Microsystems SP2 SE confocal microscope (Leica) with 310/25 air objective lens, 363/1.4 oil objective lens to carry out confocal imaging to illustrate the intracellular P3 location and use Leica LCS software (version 2.61) to analyze.Use lysosomal associated membrane protein-2 (LAMP-2) that FITC is puted together; eBioscience) lysosome and late endosome are dyed (Kuronita etc., 2002).Use Cytomation CyAn flow cytometer (Dako) to carry out flow cytometry.Use FlowJo software (Tree Star) analysis data.

Immunohistochemistry. Cryopreserved breast and melanoma tumor tissues (Origene) were formalin-fixed and paraffin-embedded for immunohistochemistry. Prior to staining, tissue sections were deparaffinized in xylene, rehydrated, and quenched for endogenous peroxidase activity. Sections were blocked with 10% normal horse serum and then incubated with either a primary WGM2 anti-P3 mAb clone (1:10) (Abcam) or anti-NE (Santa Cruz Biotechnology) for 30 minutes at room temperature. Melanoma slides were co-stained with an anti-microphthalmia-associated transcription factor (MITF) Ab (Thermo Scientific). Slides were then washed and incubated with a secondary anti-mouse IgG-biotin Ab (1:200) (Vector Laboratories) followed by avidin-biotin peroxidase (1:100) (Vector Laboratories). Staining was visualized using the chromagen 3,39-diaminobenzidine (Dako). All slides were counterstained with hematoxylin. Normal tonsil tissue stained for PMNs was used as a positive control. Negative controls were stained as described above after removal of the primary Ab.

Peptide-specific CTL lines. As previously described, PR1-specific CTLs were amplified by stimulating PBMCs from healthy HLA-A2 donors with PR1 peptide in vitro (Molldrem et al., 2000 and Molldrem et al., 1999). Briefly, T2 cells were washed with serum-free PRMI1640 culture medium and incubated with 20 mg/ml of PR1 peptide at 37°C for 90 minutes. Peptide-loaded T2 cells were irradiated with 7500 cGy, washed, and cultured with freshly isolated PBMCs in a 1:1 ratio in PRMI 1640 culture medium supplemented with 10% human AB serum. Cultures were restimulated with peptide-pulsed T2 cells on days 7, 14, and 21, and 20 IU/ml human rIL-2 (BioSource International) was added one day thereafter.

Cell-mediated cytotoxicity assay. As previously described, specific lysis was determined using a standard cytotoxicity assay (Molldrem et al., 1996 and Molldrem et al., 1997). Briefly, 1000 target cells (1.0 3 10 ⁵ cells/ml) in 10 ml were stained with calcein-AM (Invitrogen) at 37 ° C for 90 minutes, washed three times with RPMI1640, and then incubated with 10 ml peptide-specific CTL at a changed E:T ratio. After a 4-hour incubation period at 37 ° C in 5% CO ₂ , 5 ml of trypan blue was added to each well and fluorescence was measured using an automated CytoFluor II plate reader (PerSeptive Biosystems). Specific cytotoxicity percentages were calculated as follows: ([fluorescent _target + _effector fluorescence _{culture medium} ]/[fluorescent _target fluorescence _{culture medium} alone]) × 100.

Complement-mediated cytotoxicity assay. In order to determine whether cross-presentation increases the susceptibility of breast cancer to 8F4, complement-mediated cytotoxicity assays were performed as previously described (Sergeeva et al., 2011 and Prang et al., 2005). MDA-MB-231 cells were cultured for 24 hours in a culture medium containing NE (10 mg/ml) or P3 (10 mg/ml). Cells were incubated with calcein-AM (Invitrogen), washed three times and resuspended in serum-free PRMI 1640. One million cells were mixed with increasing doses of 8F4Ab (0.624, 1.25, 2.5, 5 and 10 mg/ml) or isotype Ab (negative single-shot) (final concentration of 10 mg/ml) and incubated at 37°C for 10 minutes. Standard rabbit complement (5 ml; Cedarlane Laboratories) was then added and the cells were incubated for 60 minutes at 37°C. Supernatant from BB7.2 hybridoma (anti-HLA-A2 source) and digitonin (Promega) were used as positive controls. Fluorescence was measured and specific killing was calculated as described above.

LCM and RNA extraction from breast tumor tissue. LCM was performed to isolate breast cancer cells from breast tumor tissue biopsies using an Arcturus PixCell laser capture microscope (Life Technologies, Applied Biosystems) with an IR diode laser. Tissues were sectioned (5 mm thick), placed on unloaded glass slides, and fixed in 75% ethanol and diethylpyrocarbonate water. Nuclear staining was performed using hematoxylin after tissue hydration. After dehydration in graded alcohols, the samples were stored in xylene until ready for LM. H&E staining was used to identify areas for microdissection. Tissue was pulsed with a laser beam adjusted between 30 and 70 mW to maintain a 10 mm diameter. Approximately 5,000 breast cancer cells were captured in an Arcturus Capsure HSLCM collector (Life Technologies, Applied Biosystems). Total RNA was extracted and purified using the Arcturus PicoPure RNA Isolation Kit (Life Technologies, Applied Biosystems). RNA integrity and quantity were determined using a Nano Drop ND-1000 spectrophotometer (Thermo Scientific). RNA was amplified using two rounds of T7-based amplification using the Arcturus RiboAmp RNA Amplification Kit. This produced 2.5 mg of amplified RNA. cDNA was synthesized from 1 mg of amplified RNA using the Roche Transcription First Strand cDNA Synthesis Kit (Roche Applied Science) according to the manufacturer's instructions.

Staining of PR1-CTL in breast cancer patients. PBMCs from patients were stained with the following fluorescent Abs: CD8 allophycocyanin-H7 (BD Biosciences), CD3PE Cy7 (BD Biosciences), CD4pacific orange (Invitrogen), PE-conjugated PR1/HLA-A2-dextramer (Immudex), and the following pacific blue-conjugated germline Abs: CD14 (BD Biosciences), CD16 (BD Biosciences), and CD19 (BioLegend). Dead cells were excluded using aqua live/dead staining (Invitrogen). Samples were fixed with 4% paraformaldehyde. Data were acquired on a Canto flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). The frequency of PR1-CTL was determined as the percentage of live cells (germline-, ^CD4- , CD3+, CD8+, and PR1-dextramer ⁺ ).

Confocal imaging of patient tissue. Tissue sections were fixed and cryopreserved with cold acetone. Breast cancer tissue was stained with mouse anti-cytokeratin-7 (CK7) Ab (Abcam) conjugated to Alexa-488, a breast cancer marker, and 8F4 Ab conjugated to Alexa-647 (Sergeeva et al., 2011). To determine whether PR1/HLA-A2 expression is achieved by breast cancer cells and not by infiltrating leukocytes, serial breast cancer tissue sections were also stained with mouse anti-CD45 Ab conjugated to Alexa-647 (Invitrogen) as a leukocyte marker. Human tonsil tissue sections (Origene) were used as a positive staining control for CD45. For melanoma, tissue sections were fixed with cold acetone, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 15 minutes, and blocked with 5% normal goat serum (Jackson ImmunoResearch Laboratories). The sections were then incubated with mouse anti-MITF (Thermo Scientific), a melanoma marker, for 1 hour, washed with PBS, and then incubated with goat anti-mouse IgG conjugated to Alexa-488 (Jackson ImmunoResearch Laboratories). The slides were then washed, blocked with 5% normal mouse serum (Jackson ImmunoResearch Laboratories), and incubated with 8F4Ab conjugated to Alexa-647. ProLong Gold antifade reagent (Invitrogen) containing DAPI was added. Confocal imaging was performed using a Leica Microsystems SP2SE confocal microscope with a 310/25 air objective and a 363/1.4 oil objective. Imaging analysis was performed using LeicaLCS software (version 2.61).

Example 4: Results

Solid tumors take up NE and P3. To determine whether NE and P3 uptake is a ubiquitous phenomenon, the inventors co-cultured various solid tumor cell lines with 10 mg/ml NE or P3 and then used flow cytometry to assess intracellular uptake. The inventors showed that not all tumor types take up NE and P3, and that the extent of uptake varied between tumor types (Figure 21). Furthermore, NE uptake appeared to plateau over time and was much lower than P3 uptake, suggesting different uptake mechanisms and suggesting receptor-mediated processes that may be involved in NE uptake.

There is no P3 in breast cancer. Because the present inventors have shown before, there is no NE in breast cancer and it is taken in by breast cancer cells (Mittendorf etc., 2012) and is differentiated from P3 taking in by endogenous expression differentiation, so we analyzed breast cancer cell line and primary tumor tissue for P3 expression on mRNA and protein level. PCR shows that breast cancer cell line MDA-MB-231, MCF-7, MCF-7-HER18 (HER18) and MDAMB-453 all lack P3mRNA (Figure 22 A). Similarly, breast cancer cells (Figure 22 B, Table 4) extracted from three different breast tumors also lack P3mRNA. Immunoblotting from the WCL of cell line has determined that there is no P3 protein (Figure 22 C) in breast cancer cells. The immunohistochemical staining of primary breast cancer has detected P3 in breast cancer tissue, but P3 is limited to the inflammatory component in breast tumor, rather than the inflammatory component (Figure 22 D) in breast tumor cells. These data are consistent with previous reports showing P3 in breast cancer (Desmedt et al., 2006), but our data suggest that the source of P3 is inflammatory cells within the tumor rather than within breast cancer cells.

Table 4 - Pathological characteristics of breast and melanoma tumor tissues used for LCM and confocal microscopy

ER, estrogen receptor; HER2, HER2/neu; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; N/D, not determined; PR, progesterone receptor; TNM, tumor/node/metastasis classification of malignant tumors.

Breast cancer cells take in P3. Because we show that breast cancer cells do not endogenously express P3, the present inventors assume that P3 can be taken in by breast cancer cells, as previously shown for NE (Mittendorf etc., 2012). HLA-A2 positive cell lines MDA-MB-231, MCF-7 and HER18 were co-cultured with 10mg P3 for 1, 4 and 24 hours, then the intracellular uptake (Figure 23 A) of P3 was used for flow cytometry analysis. The present inventors detected the time-dependent increase of P3 intake in all three cell lines. They also proved that the dose-dependent intake of P3 did not seem to occur platform, showed the non-receptor mediated process (Figure 23 B) of P3 intake. In order to further characterize P3, take in, because it is relevant to the Ag cross presentation that occurs in different cell compartments (Cresswell etc., 2005), so the present inventors carried out laser confocal microscopy, and demonstrated that after taking in, P3 is positioned in lysosome, as shown in the P3 co-staining (Figure 23 C) that is carried out with LAMP-2. Uptake into lysosomal compartments occurs at an early time point (1 to 4 hours) and may be the initial step in Ag degradation as it is processed for cross-presentation on HLA class I molecules (Basha et al., 2008).

Because different cellular pathways are involved in the uptake and processing of soluble and cell-associated proteins, which can determine whether they are cross-presented (Burgdorf et al., 2006), and because neutrophils have been reported in tumor tissues (including breast cancer) (Queen et al., 2005 and Jensen et al., 2009), the present inventors evaluated whether breast cancer cells take up soluble P3 and cell-associated P3 differently. To examine this, MDA-MB-231 cells were co-cultured with soluble P3 (10 mg/ml) or with irradiated PMNs or PBMCs at a 1:1 ratio for 4 hours (Figure 24A; data not shown). The data demonstrate that breast cancer cells can take up both soluble PE and cell-associated P3. In fact, uptake by cell-associated P3 appears to be more efficient than uptake by soluble proteins (median fluorescence intensity [MFI] = 12,292 vs. 1,356; p, 0.05), which may be due to the association of P3 with other proteins that can promote uptake.

Cross-presentation of P3 and NE by breast cancer cells. Because the present inventors have shown that NE is also taken up by breast cancer (Mittendorf et al., 2012) and because PR1 is derived from the neutrophil azurophil granule proteases NE and P3, they investigated whether NE and P3 are cross-presented by breast cancer cells after ingestion. HLA-A2+ MDA-MB-231 cells were co-cultured with soluble P3 or NE at increasing time points and subsequently analyzed for PR1/HLA-A2 expression using mouse anti-PR1/HLA-A2 Ab 8F4 (Sergeeva et al., 2011). These data show that breast cancer cells can cross-present PR1 from both NE and P3. Significant PR1 cross-presentation was primarily seen at 24 hours (Figure 24B), with PR1/HLA-A2 on the breast cancer cell surface increasing 2.5-fold and 3.0-fold, respectively, after incubation with NE and P3 compared to unpulsed cells. There was no significant increase in HLA-A2 expression on the cell surface (data not shown).

In addition, to study the intracellular mechanisms involved in NE and P3 cross-presentation, the present inventors studied whether proteases and ER/Golgi apparatus are involved in NE and P3 cross-presentation, as previously shown for other Ags (Francois et al., 2009, Kovacsovics-Bankowski et al., 1995 and Mukai et al., 2009). Our data show that both ER/Golgi apparatus and proteases are involved in NE and P3 cross-presentation, because after co-culture with NE or P3, cells were incubated with brefeldin A, which inhibits ER to Golgi anterograde transport, and with the proteasome inhibitor lactocysteine, which reduced PR1/HLA-A2 expression in MDA-MB-231 breast cancer cells (Figures 24C, 24D). This is similar to the results of the present inventors who previously demonstrated that proteasomes and ER/Golgi apparatus participate in NE and P3 cross-presentation through APC (Alatrash et al., 2012).

PR1 cross presentation makes breast cancer susceptible to PR1 targeted therapy. Because PR1 peptide vaccine (Rezvani et al., 2008), PR1-CTL (Rezvani et al., 2007 and Ma et al., 2010) and anti-PR1/HLA-A2Ab (8F4) (Sergeeva et al., 2011) have been effectively targeted in leukemia, the present inventors studied whether PR1/HLA-A2 expression on breast cancer cells after cross presentation makes these cells susceptible to PR1-CTL and 8F4Ab killing. In standard calcein-AM cytotoxicity assays, HLA-A2+MDA-MB-231 cells were cultured in a culture medium comprising 10mg/mlNE or P3 for 24 hours, and then incubated with PR1-CTL amplified by healthy donors for 4 hours (Molldrem et al., 1996; Jiang et al., 1996) (Figure 24E). The data demonstrate that cross-presentation of NE and P3 increases the susceptibility of MDA-MB-231 cells to PR1-CTL killing after NE or P3 pulses compared to unpulsed MDA-MB-231 cells. Similarly, using 8F4Ab ( FIG. 24F ) ( Sergeeva et al., 2011 ) in a complement-dependent cytotoxicity assay, the present inventors observed dose-dependent killing of MDA-MB-231 cells after cross-presentation of NE or P3 compared to unpulsed cells. It was noted that maximal killing occurred at the highest dose of 8F4Ab (10 mg/ml).

Detection of PR1/HLA-A2 and PR1-CTL in Breast Cancer Patients. Because the present inventors have shown that cultured breast cancer cell lines and tumor tissues lack endogenous NE and P3, and because they have observed in vitro evidence of cross-presentation of NE and P3 by breast cancer cells and subsequent susceptibility to PR1-targeted therapy, the present inventors investigated whether PR1 could be detected in primary breast cancer patient tissues and whether PR1-CTL could be detected in peripheral blood from breast cancer patients. Confocal laser microscopy of two HLA-A2-positive breast cancer tissues demonstrated 8F4 in both tumor tissues (Figure 25A). 8F4 staining was absent in HLA-A2-negative tissues (data not shown). Furthermore, to verify that PR1/HLA-A2 is expressed by breast cancer cells rather than infiltrating leukocytes, the present inventors stained serial breast cancer tissue sections with the leukocyte marker CD45. The present inventors showed the absence of CD45 staining in breast cancer tissue areas co-stained with 8F4 and CK7, further confirming that PR1/HLA-A2 is expressed by breast cancer cells rather than adjacent inflammatory cells ( FIG. 25B ).

To determine whether PR1-CTLs can be detected in breast cancer patients, we used 11 peripheral blood samples from patients with early breast cancer for PR1/HLA-A2 dextramer staining (Figure 25C). The median frequency of PR1-CTLs in these HLA-A2 ₊ patients was 0.05% (range, 0.02% to 0.2%) of CD8 ₊ T cells, which is slightly higher than the frequency of PR1-CTLs in healthy donors (1/15,000 to 1/350,000 CD8 ₊ T cells) (Molldrem et al., 1997). In summary, these in vivo data suggest that serine proteases NE and P3 present in the tumor microenvironment can be taken up and cross-presented by breast cancer cells, which may contribute to the adaptive immune response against epitopes PR1 derived from NE and P3.

PR1/HLA-A2 and PR1-CTL in melanoma patients. Because melanoma tissue has also been shown to have inflammatory cells that can be the source of NE and P3 (Jensen et al., 2012), and because melanoma is known to be susceptible to immunotherapy (Dudley et al., 2002 and Schwartzentruber et al., 2011), the present inventors next investigated whether cross-presentation of NE and P3 could also be detected in melanoma. To determine whether PR1-CTL was also detected in melanoma, the present inventors stained PBMCs from melanoma patients with PR1/HLA-A2 dextramer and detected PR1-CTL in all seven patients, with a median frequency of 0.014% (range 0.0053% to 0.019%) of CD8 ₊ T cells (Figure 25C), similar to that seen in blood from normal donors. The present inventors also detected PR1/HLA-A2 expression in one HLA-A2 ₊ (Melanoma 1) melanoma tissue but not in the HLA-A2 ₂ (Melanoma 2) melanoma tissue ( FIG. 25D ).

Melanoma cross-presentation of NE and P3 increases susceptibility to PR1-CTL. To determine whether melanoma expresses NE and P3, the present inventors stained melanoma tissue obtained from patients for NE and P3 and showed the absence of NE and P3 (Figures 26A, 26B). The present inventors also analyzed NE and P3 expression in four melanoma cell lines, MEL526, MEL624, MT2019, and MT2333. Western blot analysis showed the absence of NE and P3 in the melanoma cell lines (Figure 26C). Similar to breast cancer, the present inventors demonstrated that the HLA-A2 ₊ Mel 526 cell line took up and cross-presented NE and P3 (Figures 26D, 26E). Because 8F4 Ab binds to HLA-A2 molecules that constitute a significant portion of the conformational PR1/HLA-A2 epitope (Sergeeva et al., 2011 and Porgador et al., 1997), Mel 526 cells did show staining with 8F4 before co-culture with NE or P3 (data not shown). However, after co-culture with NE or P3, staining with 8F4 increased, while HLA-A2 surface staining did not increase (data not shown), indicating an increase in PR1/HLA-A2 expression on the cell surface. Furthermore, cross-presentation of NE and P3 increased the susceptibility of HLA-A2 ₊ Mel 526 cells to PR1-CTL killing, and maximal killing was observed at the highest E:T ratio ( FIG. 26F ).

******************

In light of the present disclosure, all compositions and/or methods disclosed and claimed herein can be realized and performed without undue experimentation. Although the compositions and methods of the present invention have been described with respect to some preferred embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the compositions and/or methods and methods described herein may be varied without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically related and physiologically related agents may replace the agents described herein and achieve the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

VIII. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims (21)

1. A humanized antibody that, when bound to an HLA-A2 receptor, binds to VLQELNVTV (SEQ ID NO: 45), said antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19 or 20. 2. The antibody according to claim 1, wherein the antibody comprises the following heavy chain and light chain: the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain constant region consisting of the human γ-1 heavy chain constant region, and the light chain comprises a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19 and a light chain constant region consisting of the human κ light chain constant region. 3. The antibody according to claim 1, wherein the antibody comprises the following heavy chain and light chain: the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16 and a heavy chain constant region consisting of the human γ-1 heavy chain constant region, and the light chain comprises a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 20 and a light chain constant region consisting of the human κ light chain constant region. 4. The antibody according to claim 2 or 3, wherein the human γ-1 heavy chain constant region is the constant region in the amino acid sequence shown in SEQ ID NO: 38, and the human κ light chain constant region is the constant region in the amino acid sequence shown in SEQ ID NO: 42. 5. The antibody according to claim 1, wherein the antibody is fused with a non-antibody peptide or polypeptide segment. 6. The antibody of claim 1, wherein the antibody is linked to a diagnostic reagent or the antibody is linked to a therapeutic reagent. 7. The antibody according to claim 6, wherein the diagnostic reagent is a dye, a radioactive isotope, a chemiluminescent molecule, a paramagnetic ion, or a spin trapping reagent. 8. The antibody according to claim 6, wherein the diagnostic reagent is a fluorophore. 9. The antibody according to claim 6, wherein the diagnostic reagent is a chromophore. 10. The antibody according to claim 6, wherein the therapeutic agent is a cytokine, a chemotherapeutic agent, a radiotherapy agent, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule, or an immune co-stimulatory molecule. 11. An expression vector comprising a nucleic acid encoding the variable region of the heavy chain of the antibody as described in any one of claims 1 to 3 and a nucleic acid encoding the variable region of the light chain of the antibody as described in any one of claims 1 to 3. 12. Host cells, selected from (a) and (b) below: (a) Host cells transfected with an expression vector containing the following nucleic acids: nucleic acids containing a sequence encoding the sequence of the antibody heavy chain variable region of claim 1 and nucleic acids containing a sequence encoding the sequence of the antibody light chain variable region of claim 1; and (b) Host cells transfected with the following expression vectors: an expression vector containing a nucleic acid encoding a sequence of the antibody heavy chain variable region of claim 1 and an expression vector containing a nucleic acid encoding a sequence of the antibody light chain variable region of claim 1. 13. A method for generating a humanized antibody, comprising culturing the host cell of claim 12 under conditions supporting the expression of the humanized antibody, wherein the humanized antibody binds to VLQELNVTV (SEQ ID NO: 45) when bound by an HLA-A2 receptor. 14. The humanized antibody produced by the method of claim 13, which binds to VLQELNVTV (SEQ ID NO: 45) when bound to the HLA-A2 receptor. 15. The antibody of claim 14, wherein the antibody is linked to a diagnostic reagent or the antibody is linked to a therapeutic reagent. 16. Use of the antibody according to any one of claims 1-10 and 14-15 in the manufacture of a medicament for treating a person suffering from cancer. 17. The use according to claim 16, wherein the cancer is a solid tumor. 18. The use according to claim 17, wherein the solid tumor is a head and neck tumor, a brain tumor, an esophageal tumor, a breast tumor, a lung tumor, a liver tumor, a spleen tumor, a stomach tumor, a small intestine tumor, a large intestine tumor, a rectal tumor, an ovarian tumor, a uterine tumor, a cervical tumor, a prostate tumor, a testicular tumor, or a skin tumor. 19. The use according to claim 16, wherein the cancer is a leukemia. 20. The use according to claim 19, wherein the blood cancer is leukemia or lymphoma. 21. The use according to claim 16, wherein the cancer is a recurrent cancer or a metastatic cancer.