These antibodies appear to be functionally equivalent and cross-react with the beta 2 - chain found on human, sheep, pig, rabbit and dog leukocytes but not with the beta-2 chain found on murine and rat leukocytes. Summary of the Invention. The present invention is directed to a method for producing humanized monoclonal antibodies by utilizing a process of comparative model building. In this method computer data bases are searched to locate homologous human protein sequences that correspond to specified regions of the non-human derived usually murine antibody, and a series of models is formulated, tested and modified to produce a model of a humanized antibody which is then constructed by recombinant DNA technology.
In a preferred embodiment, a humanized monoclonal antibody corresponding to the murine anti-CD18 antibody The variable V region sequences from both the heavy H and light L chains were determined from cDNA amplified by PCR , and spliced onto human constant C regions, resulting in a chimeric The chimeric Ab was expressed in tissue culture Ag8.
The results from competition and inhibition assays showed that the chimeric Ab was as effective as the murine The deduced murine VH and VL protein sequences were compared to the protein sequence data base, and two human Ig protein sequences were selected to be used as templates. The present inventors modeled a murine Based on the Construction of the humanized These were then attached onto vectors containing genes for appropriate C regions to form humanized Ab IgG1, kappa.
The humanized proteins were again expressed in Ag8. About a dozen of the humanized Brief Description of the Drawings.
Figure 1 illustrates an amino acid comparison of the murine All amino acids which are identical to the phase IV sequence are shaded. In addition, sequences which are different than the previous phase sequence are shown in bold. Figure 2 illustrates an amino acid residue comparison of the murine FIGURE 3 illustrates five pairs of complementary oligonucleotides corresponding to the variable regions of the light chain.
FIGURE 4 illustrates five pairs of complementary oligonucleotides corresponding to the variable region of the heavy chain. Fluorescein isothiocyanate FITC labelled antibody was incubated with cells at the concentrations indictated on the abscissa and the amount of antibody bound is indicated by relative fluorescence intensity on the ordinate.
In the absence of competing antibody, FITC - m The dashed line shows the fluorescent intensity of binding by FITC-murine FIGURE 8 illustrates the results of a chemiluminescence binding assay of murine closed square , chimeric open square and humanized closed diamond The anticancer antibody L6 open diamond , which does not bind to HL60 cells, was used as a control.
FIGURE 11 illustrates a series of restriction maps for plasmids utilized in the production of the variable heavy chain plasmid pN gamma 1. Description of Preferred Embodiments. The present invention is directed to a method of producing humanized monoclonal antibodies mAbs by utilizing a process of comparative model building and rational design.
In a preferred embodiment this method is utilized to produce a humanized molecule of the anti-CD18 murine monoclonal antibody The mouse mAb It would be advantageous to modify mAb Therefore, in one embodiment, the present invention was directed to "humanize" the For the studies described in the present invention, murine, chimeric and humanized antibodies were purified from solution by protein A chromatography on IPA - Fast Flow Immobilized rProteinA Repligen, Cambridge, MA using the manufacturer's recommended protocol.
In the present invention recombinant methods are utilized to produce humanized monoclonal antibodies that contain complementarity determining regions CDRs analogous to the originally derived monoclonal antibody, and which have homologous human heavy and light chain framework regions.
The resulting antibodies demonstrate the binding affinity and specificity of the original antibody yet are completely humanized monoclonal antibodies. As used herein the term "humanized" and its various grammatical forms as it relates to antibodies is defined to mean that the amino acid residues of the antibody in the heavy and light chains are replaced with amino acid residues corresponding to homologous human protein regions without altering the binding activity of the antibody.
For the humanized Some variation of individual amino acids in the antigen binding and framework regions are contemplated by this invention and are within the scope of this invention when such variations do not interfere or inhibit the binding to antigen, such as the Ile for Glu substitution at position of the light chain.
As used herein the term "canonical loop conformation" refers to a small repertoire of main chain conformations for five of the six loops all except H3. The particular conformation adopted is determined by only a limited number of residues within the loop or the framework.
As used herein the term "framework residues" means residues which are located outside the structurally defined CDR loops. These residues can be part of the hypervariable regions for the antibody. As used herein the term "monoclonal antibody" refers to all recombinant antibodies derived from an initial single cell and includes murine monoclonal antibodies, chimeric antibodies and humanized antibodies.
In the present invention a procedure of comparative model building was utilized to construct the appropriately designed humanized antibody. As a preferred embodiment, the modeling of the murine The Brookhaven Protein Database Bernstein et al. If the variable light chain and heavy chain templates which fulfill these criteria are from different antibodies, these structures are combined by superposition of the set of structural invariants at the VL- VH domain interface Novotny et al.
USA 82 : This provides the "structural template" for model building of murine The CDR loops and their known structural framework determinants of murine Chothia et al.
The structurally defined CDR loops consist on average of shorter sequence segments than the hypervariable regions defined by Kabat Kabat et al Sequences of Proteins of Immunological Interest. The non-canonical H3 loop region within the All non-conserved amino acid side chains in similar positions are replaced using interactive computer graphics.
The model then consists of a combination of backbone fragments of different antibodies with replaced side chains. New York: Plenum Press pp. The most homologous human variable region sequences are found by searching the sequence data base for the most homologous human sequences for the variable light and the variable heavy chains of the The structural template for murine Furthermore the percent homology is chosen to be similar to that found for comparison of the structural template with the murine sequence.
The CDR loops and known structural determinants are then grafted onto the human template Jones et al. The CDR loop regions and structural determinants in the "human template" sequence are replaced by the analogous sequences from the murine antibody, as determined above. This provides the Phase I h A Phase I model of humanized These models now consist of the murine binding site and murine framework murine The of murine and Phase I h The models of the binding site regions were compared residue by residue from the N-terminus to the C-terminus.
By this comparison, all framework residues and residues within the framework - CDR junctions which can interact with the murine CDR loops and may therefore be important for the structural integrity of the murine binding site were identified. These residues typically include all the known structural determinants for the specified canonical CDR loop conformations Chothia et al. These residues were then "re-mutated" to the murine residues, forming the Phase II h The murine This construct represents the Phase II h In Phase III, further improvements of the structural model of h Side chain conformations of the antigen binding site loops and the framework - CDR junctions are also further refined using an iterative conformational search protocol Bruccoleri RE and Karplus M.
The refined model structure may be called Phase III h The binding site features of the construct were analyzed in detail in order to classify the antibody structure, for example, as a "groove-type" or "cavity-type" or "flat" antibody.
This allows one, in the absence of detailed structural knowledge of the antibody-antigen complex, to postulate which parts of the CDR surface or residues at the CDR-framework junctions are unlikely to be involved in antigen binding. In the Phase III and earlier models, these positions may be occupied by murine residues which can now be changed to human residues. You can explore trial locations from around the US and connect directly with a trial coordinator. You can print an overview of this trial to take in to your next appointment.
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Or call us today and we'll help answer any questions that you may have. Being exposed to radiation or certain chemicals. Tests that examine the blood, bone marrow, and urine are used to diagnose multiple myeloma and other plasma cell neoplasms. The following tests and procedures may be used: Physical exam and health history : An exam of the body to check general signs of health, including checking for signs of disease, such as lumps or anything else that seems unusual.
Blood and urine immunoglobulin studies : A procedure in which a blood or urine sample is checked to measure the amounts of certain antibodies immunoglobulins. For multiple myeloma, betamicroglobulin , M protein, free light chains, and other proteins made by the myeloma cells are measured. A higher-than-normal amount of these substances can be a sign of disease. Bone marrow aspiration and biopsy : The removal of bone marrow, blood, and a small piece of bone by inserting a hollow needle into the hipbone or breastbone.
A pathologist views the bone marrow, blood, and bone under a microscope to look for abnormal cells. The following tests may be done on the sample of tissue removed during the bone marrow aspiration and biopsy: Cytogenetic analysis : A laboratory test in which the chromosomes of cells in a sample of bone marrow are counted and checked for any changes, such as broken, missing, rearranged, or extra chromosomes.
Changes in certain chromosomes may be a sign of cancer. Cytogenetic analysis is used to help diagnose cancer, plan treatment, or find out how well treatment is working. FISH fluorescence in situ hybridization : A laboratory test used to look at and count genes or chromosomes in cells and tissues. When these dyed pieces of DNA attach to certain genes or areas of chromosomes in the sample, they light up when viewed under a fluorescent microscope. The FISH test is used to help diagnose cancer and help plan treatment.
Flow cytometry : A laboratory test that measures the number of cells in a sample, the percentage of live cells in a sample, and certain characteristics of the cells, such as size, shape, and the presence of tumor or other markers on the cell surface. The cells from a sample of a patient's bone marrow are stained with a fluorescent dye, placed in a fluid, and then passed one at a time through a beam of light.
The test results are based on how the cells that were stained with the fluorescent dye react to the beam of light. This test is used to help diagnose and manage certain types of cancers, such as leukemia and lymphoma. Skeletal bone survey : In a skeletal bone survey, x-rays of all the bones in the body are taken.
The x-rays are used to find areas where the bone is damaged. An x-ray is a type of energy beam that can go through the body and onto film, making a picture of areas inside the body. Complete blood count CBC with differential : A procedure in which a sample of blood is drawn and checked for the following: The number of red blood cells and platelets.
The number and type of white blood cells. The amount of hemoglobin the protein that carries oxygen in the red blood cells.
The portion of the blood sample made up of red blood cells. Blood chemistry studies : A procedure in which a blood sample is checked to measure the amounts of certain substances, such as calcium or albumin , released into the blood by organs and tissues in the body.
An unusual higher or lower than normal amount of a substance can be a sign of disease. Twenty-four-hour urine test : A test in which urine is collected for 24 hours to measure the amounts of certain substances. An unusual higher or lower than normal amount of a substance can be a sign of disease in the organ or tissue that makes it. A higher than normal amount of protein may be a sign of multiple myeloma. MRI magnetic resonance imaging : A procedure that uses a magnet, radio waves , and a computer to make a series of detailed pictures of areas inside the body.
This procedure is also called nuclear magnetic resonance imaging NMRI. An MRI of the spine and pelvis may be used to find areas where the bone is damaged. PET scan positron emission tomography scan : A procedure to find malignant tumor cells in the body. A small amount of radioactive glucose sugar is injected into a vein. The PET scanner rotates around the body and makes a picture of where glucose is being used in the body.
Malignant tumor cells show up brighter in the picture because they are more active and take up more glucose than normal cells do. CT scan CAT scan : A procedure that makes a series of detailed pictures of areas inside the body, such as the spine, taken from different angles.
The pictures are made by a computer linked to an x-ray machine. A dye may be injected into a vein or swallowed to help the organs or tissues show up more clearly. This procedure is also called computed tomography, computerized tomography, or computerized axial tomography.
The combined scans give more detailed pictures of areas inside the body, such as the spine, than either scan gives by itself. Certain factors affect prognosis chance of recovery and treatment options. The prognosis depends on the following: The type of plasma cell neoplasm. The stage of the disease. Whether a certain immunoglobulin antibody is present. Whether there are certain genetic changes. Whether the kidney is damaged. Whether the cancer responds to initial treatment or recurs comes back.
Treatment options depend on the following: The type of plasma cell neoplasm. The age and general health of the patient. Whether there are signs, symptoms, or health problems, such as kidney failure or infection, related to the disease.
Stages of Plasma Cell Neoplasms There are no standard staging systems for monoclonal gammopathy of undetermined significance MGUS , macroglobulinemia, and plasmacytoma. After multiple myeloma has been diagnosed, tests are done to find out the amount of cancer in the body.
The following tests and procedures may be used in the staging process: Skeletal bone survey : In a skeletal bone survey, x-rays of all the bones in the body are taken. MRI magnetic resonance imaging : A procedure that uses a magnet, radio waves , and a computer to make a series of detailed pictures of areas inside the body, such as the bone marrow.
Bone densitometry : A procedure that uses a special type of x-ray to measure bone density. The stage of multiple myeloma is based on the levels of betamicroglobulin and albumin in the blood. The following stages are used for multiple myeloma: Stage I multiple myeloma In stage I multiple myeloma , the blood levels are as follows: betamicroglobulin level is lower than 3.
Refractory Plasma Cell Neoplasms Plasma cell neoplasms are called refractory when the number of plasma cells keeps going up even though treatment is given. Treatment Option Overview There are different types of treatment for patients with plasma cell neoplasms. Eight types of treatment are used: Chemotherapy Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells , either by killing the cells or by stopping them from dividing.
Other drug therapy Corticosteroids are steroids that have antitumor effects in multiple myeloma. Targeted therapy Targeted therapy is a treatment that uses drugs or other substances to identify and attack specific cancer cells without harming normal cells. High-dose chemotherapy with stem cell transplant High doses of chemotherapy are given to kill cancer cells. Biologic therapy Biologic therapy is a treatment that uses the patient's immune system to fight cancer.
International Mylom Print Share. Mylom Mylom may be available in the countries listed below. Further information Always consult your healthcare provider to ensure the information displayed on this page applies to your personal circumstances.
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