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  Pharmaceutical Patents  

 

Title:  Anti-CD20 antibodies and fusion proteins thereof and methods of use
United States Patent: 
7,435,803
Issued: 
October 14, 2008

Inventors: 
Hansen; Hans (Picayune, MS), Qu; Zhengxing (Warren, NJ), Goldenberg; David M. (Mendham, NJ)
Assignee:
 Immunomedics, Inc. (Morris Plains, NJ)
Appl. No.: 
11/534,103
Filed: 
September 21, 2006


 

Outsourcing Guide


Abstract

The present invention provides humanized, chimeric and human anti-CD20 antibodies and CD 20 antibody fusion proteins that bind to a human B cell marker, referred to as CD20, which is useful for the treatment and diagnosis of B-cell disorders, such as B-cell malignancies and autoimmune diseases, and methods of treatment and diagnosis.

Description of the Invention

SUMMARY OF THE INVENTION

Accordingly, the present invention provides humanized, chimeric and human anti-CD20 antibodies that bind to a human B cell marker, referred to as CD20, which is useful for the treatment and diagnosis of B-cell disorders, such as B-cell malignancies and autoimmune diseases.

The present invention further provides methods of treatment of mammalian subjects, such as humans or domestic animals, with one or more humanized, chimeric and human CD20 antibodies, alone, as an antibody fusion protein, as a therapeutic conjugate alone or as part of an antibody fusion protein, in combination, or as a multimodal therapy, with other antibodies, other therapeutic agents or immunomodulators or as an immunoconjugate linked to at least one therapeutic agent, therapeutic radionuclide or immunomodulator. These humanized, chimeric and human CD20 antibodies can also be used as a diagnostic imaging agent alone, in combination with other diagnostic imaging agents, and/or in conjunction with therapeutic applications.

The present invention additionally is directed to anti-CD20 mAbs or fragments thereof that contain specific murine CDRs or a combination of murine CDRs from more than one murine or chimeric anti-CD20 MAb that have specificity for CD20. These mAbs can be humanized, chimeric or human anti-CD20 mAbs. The present invention is further directed to light and/or heavy chain variable regions or fragments thereof of these anti-CD20 Mabs and to light and/or heavy chains or fragments thereof that have specficity for CD20.

The present invention is also directed to antibody fusion proteins comprising at least two anti-CD20 mAbs or fragments thereof or a first MAb comprising an anti-CD20 mAbs or fragments thereof and a second MAb.

The present invention is further directed to a therapeutic or diagnostic conjugates of the anti-CD20 mAbs or fragments thereof or antibody fusion proteins of the anti-CD20 mAbs or other mAbs or fragments thereof bound to at least one therapeutic agent or at least one diagnostic agent. Antibody fusion proteins with multiple therapeutic agents of the same or different type are encompassed by the present invention.

The present invention is additionally directed to a method of using the anti-CD20 mAbs or fragments thereof or antibody fusion proteins thereof or fragments thereof for therapy, either alone, in combination with each other, as the antibody component of a therapeutic immunoconjugate with one or more therapeutic agents or each administered in combination with one or more therapeutic agents or with an immunoconjugate with one or more therapeutic agents.

The present invention further is directed to a method of using the anti-CD20 mAbs or fragments thereof or antibody fusion proteins thereof or fragments thereof as a diagnostic bound to one or more diagnostic agents.

The present invention additionally is directed to a method of pretargeting a cell in a patients suffering from a B-cell lymphoma or leukemia or an autoimmune disease using an antibody fusion protein or fragment thereof of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

As discussed above, anti-CD20 antibodies that are unconjugated or labeled with a therapeutic radionuclide, have failed to provide high rates of objective and lasting responses in patients with intermediate or aggressive forms of B-cell lymphoma. The present invention provides a humanized, a chimeric and a human anti-CD20 antibody, and antibody fusion proteins thereof, useful for treatment of mammalian subjects, humans and domestic animals, alone, as a conjugate or administered in combination with other therapeutic agents, including other naked antibodies and antibody therapeutic conjugates.

The anti-CD20 mAbs of the present invention contain specific murine CDRs or a combination of murine CDRs from more than one murine or chimeric anti-CD20 MAb that have specificity for the CD20 antigen. The anti-CD20 mAbs of the present invention are humanized, chimeric or human mAbs, light and/or heavy chains thereof or light and/or heavy chain variable regions thereof, and they contain the amino acids of the CDRs of a murine anti-CD20 MAb and retain substantially the B-cell and B-cell lymphoma and leukemia cell targeting of the murine anti-CD20 MAb. The CDRs of the light chain variable region of the anti-CD20 MAb comprises CDR1 comprising amino acids RASSSVSYIH (SEQ ID NO: 1), RASSSLSFMH (SEQ ID NO: 2) or RASSSVSYMH (SEQ ID NO: 3) CDR2 comprising amino acids ATSNLAS (SEQ ID NO: 4) and CDR3 comprising amino acids QQWTSNPPT (SEQ ID NO: 5), HQWSSNPLT (SEQ ID NO: 6)or QQSFSNPPT (SEQ ID NO: 7) and the CDRs of the heavy chain variable region of the anti-CD20 MAb comprises CDR1 comprising amino acids SYNMH (SEQ ID NO: 8) CDR2 comprising amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9) and CDR3 comprising amino acids STYYGGDWYFDV (SEQ ID NO: 10), STYYGGDWYFNV (SEQ ID NO: 11), SHYGSNYVDYFDV (SEQ ID NO: 12) or VVYYSNSYWYFDV (SEQ ID NO: 13). The humanized antibody further comprises the framework regions of the light and heavy chain constant regions of a human antibody.

In one embodiment, the humanized and chimeric MAb or fragment thereof does not contain the CDR3 of the heavy chain variable region comprising STYYGGDWYFNV (SEQ ID NO: 11). More preferably, CDR1 of the light chain variable region does not comprise RASSSLSFMH (SEQ ID NO: 2) when the CDR3 of the light chain variable region comprises HQWSSNPLT (SEQ ID NO: 6) and the CDR3 of the heavy chain variable region comprises SHYGSNYVDYFDV (SEQ ID NO: 12). In another embodiment, the CDR3 of the light chain variable region does not comprise HQWSSNPLT (SEQ ID NO: 6) when CDR1 of the light chain variable region comprises RASSSLSFMH (SEQ ID NO: 2) and when CDR3 of the heavy chain variable region comprises SHYGSNYVDYFDV (SEQ ID NO: 12). In a further embodiment, the CDR3 of the heavy chain variable region does not comprise SHYGSNYVDYFDV (SEQ ID NO: 12) when the CDR1 of the light chain variable region comprises RASSSLSFMH (SEQ ID NO: 2) and the CDR3 of the light chain variable region comprises HQWSSNPLT (SEQ ID NO: 6). In another embodiment, the CDR1 of the light chain variable region does not comprise RASSSVSYMH (SEQ ID NO: 3) when the CDR3 of the light chain variable region comprises QQSFSNPPT (SEQ ID NO: 7) and the CDR3 of the heavy chain variable region comprises VVYYSNSYWYFDV (SEQ ID NO:13).

Further, in another embodiment, the anti-CD20 monoclonal antibody (MAb) or fragment thereof does not contain CDR3 of the light chain variable region of amino acids QQSFSNPPT (SEQ ID NO: 7) when CDR1 of the light chain variable region comprises RASSSVSYMH (SEQ ID NO: 3) and the CDR3 of the heavy chain variable region comprises VVYYSNSYWYFDV (SEQ ID NO: 13). Additionally, the anti-CD20 MAb does not contain CDR3 of the heavy chain variable region with amino acids VVYYSNSYWYFDV (SEQ ID NO: 13) when the CDR1 of the light chain variable region comprises RASSSVSYMH (SEQ ID NO: 3) and the CDR3 of the light chain variable region comprises QQSFSNPPT (SEQ ID NO: 7).

In a preferred embodiment, the humanized anti-CD20 (hCD20) monoclonal antibody or antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of at least one murine anti-CD20 MAb variable region and the framework regions (FRs) of at least one human MAb variable region, wherein said humanized anti-CD20 MAb or fragment thereof retains substantially the B-cell and B-cell lymphoma and leukemia cell targeting of said murine anti-CD20 MAb. The humanized antibody's variable region may comprise a light chain variable region, a heavy chain variable region or a both light and heavy chain variable regions. The humanized antibody or fragment thereof may further comprise light and heavy chain constant regions of at least one human antibody.

The humanized anti-CD20 MAb or fragment thereof of the present invention comprises the CDRs of a murine anti-CD20 MAb and the framework (FR) regions of the light and heavy chain variable regions of a human antibody, while retaining substantially the B-cell, and B-cell lymphoma and leukemia cell targeting of the parent murine antiCD20 MAb, and wherein the CDRs of the light chain variable region of the murine antiCD20 MAb comprises CDR1 comprising amino acids RASSSVSYIH (SEQ ID NO: 1), CDR2 comprising amino acids ATSNLAS (SEQ ID NO: 4)and CDR3 comprising QQWTSNPPT (SEQ ID NO: 5) and the CDRs of the heavy chain variable region of murine anti-CD20 MAb comprises CDR1 comprising amino acids SYNMH (SEQ ID NO: 8), CDR2 comprising amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9) and CDR3 comprising amino acids STYYGGDWYFDV (SEQ ID NO: 10). But the humanized anti-CD20 MAb or fragment thereof may further contain in the FRs of the light and heavy chain variable regions of the antibody at least one amino acid from the corresponding FRs of the murine MAb. The humanized MAbs may further contain the light and heavy chain constant regions of a human antibody. Specifically, the humanized anti-CD20 MAb or fragment thereof contains at least one amino acid residue 1, 5, 27, 30, 38, 48, 67, 68, 70, 95, 115 and 116 of the murine heavy chain variable region of FIG. 4A (see Original Patent), designated as hA20VH1 or hA20VH2 and of at least one amino acid residue 4, 21, 35, 38, 45, 46, 59, 99, 104 and 106 of the murine light chain variable region FIG. 4B (see Original Patent), designated hA20Vk. One or more of the murine amino acid sequences can be maintained in the human FR regions of the light and heavy variable chains if necessary to maintain proper binding or to enhance binding to the CD20 antigen. More preferably the humanized anti-CD20 MAb or fragment thereof of the present invention comprises the hA20Vk of FIG. 4B and the hA2VH1 of FIG. 4A. Most preferably, the humanized anti-CD20 MAb or fragment thereof of the present invention comprises the hA20Vk of FIG. 4B and the hA2VH2 of FIG. 4A. This latter sequence contains more human amino acid sequences in the FRs of the VH2 chain than the VH1, and thus is more humanized.

The preferred chimeric anti-CD20 (cCD20) MAb or fragment thereof of the present invention comprises the CDRs of a murine anti-CD20 MAb and the FR regions of the light and heavy chain variable regions of the murine anti-CD20 MAb, i.e., the Fvs of the parental murine MAb, and the light and heavy chain constant regions of a human antibody, wherein the chimeric anti-CD20 MAb or fragment thereof retains substantially the B-cell, and B-cell lymphoma and leukemia cell targeting of the murine anti-CD20 MAb, wherein the CDRs of the light chain variable region of the chimeric anti-CD20 MAb comprise CDR1 comprising amino acids RASSSVSYIH (SEQ ID NO: 1), RASSSLSFMH (SEQ ID NO: 2) or RASSSVSYMH (SEQ ID NO: 3) CDR2 comprising amino acids ATSNLAS (SEQ ID NO: 4) and CDR3 comprising amino acids QQWTSNPPT (SEQ ID NO: 5), HQWSSNPLT (SEQ ID NO: 6) or QQSFSNPPT (SEQ ID NO: 7) and the CDRs of the heavy chain variable region of the chimeric anti-CD20 MAb comprise CDR1 comprising amino acids SYNMH (SEQ ID NO: 8) CDR2 comprising amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9)and CDR3 comprising STYYGGDWYFDV (SEQ ID NO: 10), STYYGGDWYFNV (SEQ ID NO: 11), SHYGSNYVDYFDV (SEQ ID NO: 12) or VVYYSNSYWYFDV (SEQ ID NO: 13) with the following provisos,

(a) wherein the CDR3 of the heavy chain variable region does not comprise STYYGGDWYFNV (SEQ ID NO: 11), when the CDR1 of the light chain variable region comprises amino acids RASSSVSYIH (SEQ ID NO: 1), CDR2 of the light chain variable region comprises amino acids ATSNLAS (SEQ ID NO: 4), CDR3 of the light chain variable region comprises amino acids QQWTSNPPT (SEQ ID NO: 5), CDR1 of the heavy chain variable region comprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chain variable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9)

(b) wherein the CDR3 of the heavy chain variable region does not comprise SHYGSNYVDYFDV (SEQ ID NO: 12), when the CDR1 of the light chain variable region comprises amino acids RASSSLSFMH (SEQ ID NO: 2), CDR2 of the light chain variable region comprises amino acids ATSNLAS (SEQ ID NO: 4), CDR3 of the light chain variable region comprises amino acids HQWSSNPLT (SEQ ID NO: 6), CDR1 of the heavy chain variable region comprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chain variable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9) and

(c) wherein the CDR3 of the heavy chain variable region does not comprise VVYYSNSYWYFDV (SEQ ID NO: 13), when the CDR1 of the light chain variable region comprises amino acids RASSSVSYMH (SEQ ID NO: 3), CDR2 of the light chain variable region comprises amino acids ATSNLAS (SEQ ID NO: 4), CDR3 of the light chain variable region comprises amino acids QQSFSNPPT (SEQ ID NO: 7), CDR1 of the heavy chain variable region comprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chain variable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9).

More preferably the chimeric anti-CD20 MAb or fragment thereof comprising the complementarity-determining regions (CDRs) of a murine anti-CD20 MAb and the framework (FR) regions of the light and heavy chain variable regions of the murine anti-CD20 MAb and further, the light and heavy chain constant regions of a human antibody, wherein the chimeric anti-CD20 MAb or fragment thereof retains substantially the B-cell, and B-cell lymphoma and leukemia cell targeting of the murine anti-CD20 MAb, wherein the CDRs of the light chain variable region of the chimeric anti-CD20 MAb comprises the CDRs shown in FIGS. 4B and 4A, respectively, designated cA20Vk and cA20VH. Most preferably, the chimeric anti-CD20 MAb or fragment thereof comprises the light and heavy chain variable regions of murine anti-CD20 MAb shown in FIGS. 4B and 4A, respectively, designated cA20Vk and cA20 VH.

The present invention also encompasses a human anti-CD20 MAb or fragment thereof comprising the light and heavy chain variable, wherein said human CD20 MAb retains substantially the B-cell, and B-cell lymphoma and leukemia cell targeting and cell binding characteristics of a murine anti-CD20 MAb, wherein the CDRs of the light chain variable region of the human anti-CD20 MAb comprises the same CDRs as set forth above for the chimeric and humanized anti-CD20 mAbs and as shown in FIGS. 4A and 4B. This human anti-CD20 MAb or fragment thereof furether comprises light and heavy chain constant regions of at least one human antibody.

The present invention is also intended to encompass antibody fusion proteins or fragments thereof comprising at least two anti-CD20 mAbs or fragments thereof, as described above. The antibody fusion protein or fragment thereof of the present invention is also intended to encompass an antibody fusion protein or fragment thereof comprising at least one first anti-CD20 MAb or fragment thereof as described above and at least one second MAb or fragment thereof, other than the antiCD20 MAb or fragment described above. More preferably this second MAb is a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof, and even an anti-CD20 MAb that is different than the anti-CD20 MAb described herein. The antibody fusion proteins of the present invention may be composed of one CD20 MAb and one or more of the second mAbs to provide specificity to different antigens, and are described in more detail below.

The humanized, chimeric and human anti-CD20 antibody may possess enhanced affinity binding with the epitope, as well as antitumor and anti-B-cell activity, as a result of CDR mutation and manipulation of the CDR and other sequences in the variable region to obtain a superior therapeutic agent for the treatment of B-cell disorders, including B-cell lymphomas and leukemias and autoimmune diseases. Modification to the binding specificity, affinity or avidity of an antibody is known and described in WO 98/44001, as affinity maturation, and this application summarizes methods of modification and is incorporated in its entirety by reference.

It may also be desirable to modify the antibodies of the present invention to improve effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist. One or more amino acid substitutions or the introduction of cysteine in the Fc region may be made, thereby improving internalization capability and/or increased complement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med. 176:1191-1195 (1991) and Shopes, B.J. Immunol. 148:2918-2022 (1992), incorporated herein by reference in their entirety. An antibody fusion protein may be prepared that has dual Fc regions with both enhanced complement lysis and ADCC capabilities.

The present invention is also directed to DNA sequences comprising a nucleic acid encoding a MAb or fragment thereof selected from the group consisting

(a) an anti-CD20 MAb or fragment thereof as described herein,

(b) an antibody fusion protein or fragment thereof comprising at least two of the anti-CD20 mAbs or fragments thereof,

(c) an antibody fusion protein or fragment thereof comprising at least one first MAb or fragment thereof comprising the anti-CD20 MAb or fragment thereof as described herein and at least one second MAb or fragment thereof, other than the antiCD20 MAb or fragment thereof, and

(d) an antibody fusion protein or fragment thereof comprising at least one first MAb or fragment thereof comprising the anti-CD20 MAb or fragment thereof and at least one second MAb or fragment thereof, wherein the second MAb is a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof.

Also encompassed by the present invention are expression vectors comprising the DNA sequences. These vectors contain the light and heavy chain constant regions and the hinge region of the human immunoglobulin, in the case of vectors for use in preparing the humanized, chimeric and human anti-CD20 mAbs or antibody fusion proteins thereof or fragments thereof. These vectors additionally contain, where required, promoters that express the mAbs in the selected host cell, immunoglobulin enhances and signal or leader sequences. Vectors that are particularly useful in the present invention are pdHL2 or GS, particularly when used to express a chimeric, humanized or human antibodies, such as gigs, where the vector codes for the heavy and light chain constant regions and hinge region of IgG1. More preferably, the light and heavy chain constant regions and hinge region are from a human EU myeloma immunoglobulin, where optionally at least one of the amino acid in the allotype positions is changed to that found in a different IgG1 allotype, and wherein optionally amino acid 253 of the heavy chain of EU based on the EU number system may be replaced with alanine. See Edelman et al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969), incorporated herein in its entirety by reference.

Host cells containing the DNA sequences encoding the anti-CD20 mAbs or fragments thereof or antibody fusion proteins or fragments thereof of the present invention or host cells containing the vectors that contain these DNA sequences are encompassed by the present invention. Particularly useful host cells are mammalian cells, more specifically lymphocytic cells, such as myeloma cells, discussed in more detail below.

Also encompassed by the present invention is the method of expressing the anti-CD20 MAb or fragment thereof or antibody fusion protein or fragment thereof comprising: (a) transfecting a mammalian cell with a DNA sequence of encoding the anti-CD20 mAbs or fragments thereof or antibody fusion proteins or fragments thereof, and (b) culturing the cell transfected with the DNA sequence that secretes the anti-CD20 or fragment thereof or antibody fusion protein or fragment thereof. Known techniques may be used that include a selection marker on the vector so that host cells that express the mAbs and the marker can be easily selected.

The present invention particularly encompasses B-lymphoma cell and leukemia cell targeting diagnostic or therapeutic conjugates comprising an antibody component comprising an anti-CD20 MAb or fragment thereof or an antibody fusion protein or fragment thereof of the present invention that binds to the B-lymphoma or leukemia cell, that is bound to at least one diagnostic or at least one therapeutic agent.

The diagnostic conjugate comprises the antibody component comprising an anti-CD20 MAb or fragment thereof or an antibody fusion protein or fragment thereof, wherein the diagnostic agent comprises at least one photoactive diagnostic agent, and more preferably wherein the label is a radioactive label with an energy between 60 and 4,000 keV or a non-radioactive label. The radioactive label is preferably a gamma-, beta-, and positron-emitting isotope and is selected from the group consisting of .sup.125I, .sup.131I, .sup.123I, .sup.124I, .sup.86Y, .sup.186Re, .sup.188Re .sup.62Cu, .sup.64Cu, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.18F, .sup.11C, .sup.13N, .sup.15O .sup.76Br and combinations thereof.

The diagnostic conjugate of the present invention also utilizes a diagnostic agent, such as a contrast agent, for example, such as manganese, iron or gadolinium.

The therapeutic conjugate of the present invention comprises an antibody component comprising an antibody fusion protein or fragment thereof, wherein each of said mAbs or fragments thereof are bound to at least one therapeutic agent. The therapeutic conjugate of preferably is selected from the group consisting of a radioactive label, an immunomodulator, a hormone, a photoactive therapeutic agent, a cytotoxic agent, which may be a drug or a toxin, and a combination thereof. The drugs useful in the present invention are those drugs that possess the pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating, antimetabolite, antibiotic, alkaloid, antiangiogenic, apoptotic agents and combinations thereof More specifically, these drugs are selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, anthracyclines, taxanes, and their analogs, and a combination thereof. The toxins encompassed by the present invention are selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

Useful therapeutic conjugates of the present invention are immunomodulators selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)), interferon, such as interferons-.alpha., -.beta., or -.gamma., and stem cell growth factor, such as designated "S1 factor". More specifically, immunomodulator, such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21 interferon-.gamma., TNF-.alpha. or a combination thereof are useful in the present invention.

Particularly useful therapeutic conjugates comprise one or more radioactive labels that have an energy between 60 and 700 keV. Such radioactive labels are selected from the group consisting of .sup.225Ac, .sup.67Ga, 90Y, .sup.111In, .sup.131I, .sup.125I, .sup.186Re, .sup.188Re, .sup.177Lu, .sup.32P, .sup.64Cu, .sup.67Cu, .sup.212Bi, .sup.213Bi, .sup.211At and combinations thereof Other useful therapeutic conjugates are photoactive therapeutic agent, such as a chromogen or dye.

Other useful therapeutic conjugates comprise oligonucleotides, especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products of B-cell malignancies, such as bcl-2.

The present invention particularly encompasses methods of treating a B-cell lymphoma or leukemia cell disease or an autoimmune disease in a subject, such as a mammal, including humans, domestic or companion pets, such as dogs and cats, comprising administering to the subject a therapeutically effective amount of an anti-CD20 MAb or a fragment thereof of the present invention, formulated in a pharmaceutically acceptable vehicle. This therapy utilizes a "naked antibody" that does not have a therapeutic agent bound to it. The administration of the "naked anti-CD20 antibody" can be supplemented by administering to the subject concurrently or sequentially a therapeutically effective amount of another "naked antibody" that binds to or is reactive with another antigen on the surface of the target cell or that has other functions, such as effector functions in the Fc portion of the MAb, that is therapeutic and which is discussed herein. Preferred additional mAbs are at least one humanized, chimeric, human or murine (in the case of non-human animals) MAb selected from the group consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, Ia, HM1.24, and HLA-R, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

Both the naked anti-CD20 therapy alone or in combination with other naked mAbs as discussed above can be further supplemented with the administration, either concurrently or sequentially, of a therapeutically effective amount of at least one therapeutic agent, formulated in a pharmaceutically acceptable vehicle. As discussed herein the therapeutic agent may comprises a cytotoxic agent, a radioactive label, an oligonucleotide, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

In another therapeutic method, both the naked anti-CD20 therapy alone or in combination with other naked mAbs, as discussed above, can be further supplemented with the administration, either concurrently or sequentially, of a therapeutically effective amount of at least one therapeutic conjugate, described herein and formulated in a pharmaceutically acceptable vehicle. The antibody component of the therapeutic conjugate comprises at least one humanized, chimeric, human or murine (for non-human subjects) MAb selected from the group consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof, formulated in a pharmaceutically acceptable vehicle. As discussed herein the therapeutic agent may comprise a cytotoxic agent, a radioactive label, an immunomodulator, a hormone, a photoactive therapeutic agent or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

As described herein the present invention particularly encompasses a method of treating a B-cell lymphoma or leukemia or an autoimmune disease in a subject comprising administering to a subject a therapeutically effective amount of an antibody fusion protein or fragment thereof comprising at least two anti-CD20 mAbs or fragments thereof of the present invention or comprising at least one anti-CD20 MAb or fragment thereof of the present invention and at least one additional MAb, preferably selected from the group consisting of mAbs reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

This therapeutic method can further be supplemented with the administration to the subject concurrently or sequentially of a therapeutically effective amount of at least one therapeutic agent, formulated in a pharmaceutically acceptable vehicle, wherein the therapeutic agent is preferably a cytotoxic agent, a radioactive label, an immunomodulator, a hormone, a photoactive therapeutic agent or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

Further, the antibody fusion proteins can be administered to a subject concurrently or sequentially a therapeutically effective amount of a therapeutic conjugate comprising at least one MAb bound to at least one therapeutic agent, wherein said MAb component of the conjugate preferably comprises at least one humanized, chimeric, human or murine (for non-human subjects) MAb selected from the group consisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR, tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combination thereof, formulated in a pharmaceutically acceptable vehicle. The antibody fusion protein itself can be conjugated to a therapeutic agent and thus provides a vehicle to attach more than one therapeutic agent to an antibody component and these therapeutic agents can be a combination of different recited agents or combinations of the same agents, such as two different therapeutic radioactive labels. Also encompassed by the present invention is a method of diagnosing a B-cell lymphoma or leukemia in a subject comprising administering to the subject, such as a mammal, including humans and domestic and companion pets, such as dogs, cats, rabbits, guinea pigs, a diagnostic conjugate comprising an anti-CD20 MAb or fragment thereof or an antibody fusion protein or fragment thereof of the present invention that binds to the lymphoma or leukemia cell, wherein the anti-CD20 MAb or fragment thereof or antibody fusion protein or fragment thereof is bound to at least one diagnostic agent, formulated in a pharmaceutically acceptable vehicle. The useful diagnostic agents are described herein.
 

Claim 1 of 26 Claims

1. A chimeric or humanized monoclonal antibody that binds to CD20, said antibody having a light chain variable region CDR1 comprising the sequence RASSSVSYIH (SEQ ID NO:1); CDR2 comprising the sequence ATSNLAS (SEQ ID NO:4); and CDR3 comprising the sequence QQWTSNPPT (SEQ ID NO:5); and said antibody having a heavy chain variable region CDR1 comprising the sequence SYNMH (SEQ ID NO:8); CDR2 comprising the sequence AIYPGNGDTSYNQKFKG (SEQ ID NO:9); and CDR3 comprising the sequence STYYGGDWYFDV (SEQ ID NO:10) or the sequence VVYYSNSYWYFDV (SEQ ID NO: 13).

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