Source: http://www.google.com/patents/US7312318?dq=6,163,776
Timestamp: 2014-03-10 08:43:03
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Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'ART-1', 'Application No. 60', 'ART-1']

Patent US7312318 - Internalizing anti-CD74 antibodies and methods of use - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention provides humanized, chimeric and human anti-CD74 antibodies, CD74 antibody fusion proteins, immunoconjugates, vaccines and bispecific that bind to CD74, the major histocompatibility complex (MHC) class-II invariant chain, Ii, which is useful for the treatment and diagnosis of B-cell...http://www.google.com/patents/US7312318?utm_source=gb-gplus-sharePatent US7312318 - Internalizing anti-CD74 antibodies and methods of useAdvanced Patent SearchPublication numberUS7312318 B2Publication typeGrantApplication numberUS 10/377,122Publication dateDec 25, 2007Filing dateMar 3, 2003Priority dateMar 1, 2002Fee statusPaidAlso published asCA2478012A1, CA2478012C, CN1649902A, CN1649902B, CN102174108A, DE60333732D1, EP1483294A2, EP1483294B1, EP2308898A1, US7772373, US7919087, US7931903, US20040115193, US20080138333, US20080166342, US20100015048, US20100266497, US20100284906, US20110195023, US20110244546, WO2003074567A2, WO2003074567A3Publication number10377122, 377122, US 7312318 B2, US 7312318B2, US-B2-7312318, US7312318 B2, US7312318B2InventorsHans J. Hansen, Shui-on Leung, Zhengxing Qu, David M. GoldenbergOriginal AssigneeImmunomedics, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (8), Non-Patent Citations (15), Referenced by (33), Classifications (34), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetInternalizing anti-CD74 antibodies and methods of useUS 7312318 B2Abstract The present invention provides humanized, chimeric and human anti-CD74 antibodies, CD74 antibody fusion proteins, immunoconjugates, vaccines and bispecific that bind to CD74, the major histocompatibility complex (MHC) class-II invariant chain, Ii, which is useful for the treatment and diagnosis of B-cell disorders, such as B-cell malignancies, other malignancies in which the cells are reactive with CD74, and autoimmune diseases, and methods of treatment and diagnosis.
1. A humanized, or chimeric anti-CD74 antibody or CD74 antigen-binding fragment thereof comprising the light chain variable region complementarity-determining region (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:19), CDR2 (TVSNRFS; SEQ ID NO:20), and CDR3 (SQSSHVPPT; SEQ ID NO:21) and the heavy chain variable region CDR sequences CDR1 (NYGVN; SEQ ID NO:22), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:23), and CDR3 (SRGKNEAWFAY; SEQ ID NO:24).
2. The anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment is a naked antibody or fragment.
3. The anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment is conjugated to at least one therapeutic or diagnostic agent.
4. The anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment comprising the complementarity-determining regions (CDRs) of a murine anti-CD74 (mLL1) antibody and the framework (FR) regions of a human antibody.
5. The humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 4, wherein said FRs of the light and heavy chain variable regions of said humanized antibody comprise at least one amino acid substituted from said corresponding FRs of said murine antibody.
6. The humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 5, wherein said at least one amino acid from said murine antibody is selected from the group consisting of amino acid residues 2, 3, 4, 46, 87 and 100 of the murine light chain variable region of the cLL1Vk sequence (SEQ ID NO:13), and amino acid residues 5, 37, 38, 46, 68, 91 and 93 of the murine heavy chain variable region of the cLL1VH sequence (SEQ ID NO:10).
7. The humanized anti-CD74 mAb or antigen-binding fragment thereof of claim 4, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region (SEQ ID NO:16) and a light chain variable region (SEQ ID NO:18).
8. The humanized anti-CD74 antibody or antigen-binding fragment thereof claim 4, wherein said antibody or antigen-binding fragment thereof further comprises a light and heavy chain constant region of a human antibody or a portion thereof.
9. The humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 4, wherein said antibody or antigen-binding fragment thereof is a humanized IgG1.
10. The anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein said antibody or antigen-binding fragment is a chimeric antibody or antigen-binding fragment.
11. The chimeric anti-CD74 antibody or antigen-binding fragment thereof of claim 10, wherein said chimeric antibody or antigen-binding fragment comprises murine LL1 antibody variable region sequences attached to human constant region sequences.
12. The chimeric anti-CD74 antibody or antigen-binding fragment thereof of claim 11, wherein said antibody or antigen-binding fragment is antibody or antigen-binding fragment.
13. The chimeric anti-CD74 antibody or antigen-binding fragment thereof of claim 12, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region (SEQ ID NO:5) and a light chain variable region (SEQ ID NO:7).
14. The chimeric anti-CD74 antibody or antigen-binding fragment thereof of claim 12, wherein said antibody or antigen-binding fragment thereof is a chimeric IgG1.
15. The chimeric, or humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 1, comprising at least one of the following properties:
(a) the binding of said antibody or antigen-binding fragment thereof to CD74 is blocked by an antibody or antigen-binding fragment thereof specific for CD74;
(b) said antibody or antigen-binding fragment thereof is internalized by Raji lymphoma cells in culture; and
(c) said antibody or antigen-binding fragment thereof induces apoptosis of Raji cells in cell culture when cross-linked with goat antisera reactive with the Fc of a murine IgG1 antibody.
16. The chimeric or humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is F(ab′)2, Fab, scFv, Fv, or a fusion construct comprising F(ab′)2, Fab, scFv, or Fv.
17. The chimeric or humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 16, wherein said fusion protein is multivalent, or multivalent and multispecific.
18. The chimeric or humanized anti-CD74 antibody or antigen-binding fragment thereof of claim 1, comprising constant regions of an IgG1 antibody, which are replaced with human constant regions of human IgG2a, IgG3, or IgG4.
19. A murine anti-CD74 monoclonal antibody (mAb) or antigen-binding fragment thereof comprising:
(a) CDRs of a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 (RSSQSLVHRNGNTYLH, SEQ ID NO:19), CDR2 (TVSNRFS, SEQ ID NO:20), and CDR3 (SQSSHVPPT, SEQ ID NO:21);
(b) CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 (NYGVN, SEQ ID NO:22), CDR2 (WINPNTGEPTFDDDFKG, (SEQ ID NO:23), and CDR3 (SRGKNEAWFAY, SEQ ID NO:24); and
(c) the framework (FR) regions of a murine anti-CD74 antibody.
20. An antibody fusion protein comprising four or more Fvs, or Fab's of the antibody or antigen-binding fragment thereof of claim 16.
21. The anti-CD 74 antibody or antigen-binding fragment thereof claim 3, wherein the therapeutic agent is an RNase.
22. The anti-CD74 antibody or antigen-binding fragment thereof of claim 21, wherein the antibody or antigen-binding fragment is chimeric.
23. The anti-CD74 antibody or antigen-binding fragment thereof of claim 21, wherein the antibody or antigen-binding fragment is humanized.
BACKGROUND OF THE INVENTION This application claims priority to U.S. Provisional Application No. 60/360,259 filed Mar. 1, 2002, which is incorporated herein by reference in its entirety by reference.
The use of targeting monoclonal antibodies conjugated to radionuclides or other cytotoxic agents offers the possibility of delivering such agents directly to the tumor site, thereby limiting the exposure of normal tissues to toxic agents (Goldenberg, Semin. Nucl. Med., 19: 332 (1989)). In recent years, the potential of antibody-based therapy and its accuracy in the localization of tumor-associated antigens have been demonstrated both in the laboratory and clinical studies (see, e.g., Thorpe, TIBTECH, 11: 42 (1993); Goldenberg, Scientific American, Science & Medicine, 1: 64 (1994); Baldwin et al., U.S. Pat. No. 4,925,922 and 4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irie et al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. No. 5,134,075 and 5,171,665). In general, the use of radio-labeled antibodies or antibody fragments against tumor-associated markers for localization of tumors has been more successful than for therapy, in part because antibody uptake by the tumor is generally low, ranging from only 0.01% to 0.001% of the total dose injected (Vaughan et al, Brit. J. Radiol., 60: 567 (1987)). Increasing the concentration of the radiolabel to increase the dosage to the tumor is counterproductive, generally, as this also increases exposure of healthy tissue to radioactivity.
Murine LL1 was developed by fusion of mouse myeloma cells with splenocytes from BALB/c mice immunized with preparations from the Raji B-lymphoma cell line (called EPB-1 in Pawlak-Byczkowska et al., Can. Res., 49: 4568 (1989)). The clinical use of mLL1, just as with most other promising murine antibodies, has been limited by the development in humans of a human anti-mouse antibody (HAMA) response. A HAMA response is generally not observed following injection of mLL1 Fab′, as evidenced in a bone marrow imaging study using re a mLL1 Fab′ labeled with 99mTc. Juweid et al., Nuc. Med. Comm. 18: 142-148 (1997). However, in some therapeutic and diagnostic uses, a full-length anti-CD74 mAb may be preferred. This use of the full-length anti-CD74 mAb can limit the diagnostic and therapeutic usefulness of such antibodies and antibody conjugates, not only because of the potential anaphylactic problem, but also as a major portion of the circulating conjugate may be complexed to and sequestered by the circulating anti-mouse antibodies. Although the use of antibody fragments of mLL1 may circumvent the problems of immunogenicity, there are circumstances in which whole IgG is more desirable and the induction of cellular immunity is intended for therapy or enhanced antibody survival time. In general, HAMA responses pose a potential obstacle to realizing the full diagnostic and therapeutic potential of murine anti-CD74 mAbs. Therefore, the development of chimeric, humanized and human anti-CD74 mAbs and fragments thereof, antibody fusion proteins thereof and fragments thereof, immunoconjugates for therapy and diagnosis, multivalent and/or multispecific mAbs, and fragments thereof and vaccine conjugates thereof would be extremely useful for therapy and diagnosis, with reduced production of human anti-mouse antibodies.
The present invention further is directed to a bispecific or multivspecific antibody comprising an antibody conjugate containing the anti-CD74 antibodies or fragments thereof or the antibody fusion proteins or fragments thereof of the present invention linked to an antibody or antibody fragment specific for a cancer marker substance, an eptitope on the surface of an infectious disease organism or a noxious substance in the blood or other body fluid.
BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the DNA and amino acid sequences of the murine LL1 heavy and light chain variable regions. FIG. 1A shows the DNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of the LL1 VH obtained by RT-PCR. FIG. 1B shows the DNA (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences of the LL1Vk obtained by 5′-RACE. Amino acid sequences encoded by the corresponding DNA sequences are given as one-letter codes below the nucleotide sequence. Numbering of the nucleotide sequence is on the right side. The amino acid residues in the CDR regions are shown in bold and underlined. Kabat's Ig molecule numbering is used for amino acid residues as shown by the numbering above the amino acid residues. The residues numbered by a letter following a particular digit indicates the insertion residues defined by Kabat numbering scheme. The insertion residues numbered with a letter have the same preceding digit. For example, residues 82A, 82B and 82C in FIG. 1A are indicated as 82A, B, and C.
FIG. 2 shows the DNA and amino acid sequences of the chimeric LL1 (cLL1) heavy and light chain variable regions expressed in Sp2/0 cells. FIG. 2A shows the DNA (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences of the cLL1 VH. FIG. 2B shows the double-stranded DNA (SEQ ID NO: 7) and amino acid (SEQ ID NO: 8) sequences of the cLL1Vk. Amino acid sequences encoded by the corresponding DNA sequences are given as one-letter codes. The amino acid residues in the CDR regions are shown in bold and underlined. The numbering of nucleotides and amino acids is same as that in FIG. 1. The restriction sites used for constructing the cLL1 are boxed and indicated.
FIG. 3 shows the alignment of the amino acid sequences of light and heavy chain variable regions of a human antibody, cLL1 and hLL1. FIG. 3A shows the VH amino acid sequence alignment of the human antibody RF-TS3 (SEQ ID NO: 9), cLL1 (SEQ ID NO: 10) and hLL1 (SEQ ID NO: 11) and FIG. 3B shows the Vk amino acid sequence alignment of the human antibody HF-21/28 (SEQ ID NO: 12), cLL1 (SEQ ID NO: 13) and hLL1 (SEQ ID NO: 14). Dots indicate the residues in cLL1 that are identical to the corresponding residues in the human antibodies. Boxed regions prepresent the CDR regions. Both N- and C-terminal residues (underlined) of cLL1 are fixed by the staging vectors used and not compared with the human antibodies. Kabat's Ig molecule number scheme is used as in FIG. 1.
FIG. 4 shows the DNA and amino acid sequences of the humanized LL1 (hLL1) heavy and light chain variable regions expressed in Sp2/0 cells. FIG. 4A shows the DNA (SEQ ID NO: 15) and amino acid (SEQ ID NO: 16) sequences of the hLL1VH and FIG. 4B shows the DNA (SEQ ID NO: 17) and amino acid (SEQ ID NO: 18)sequences of the hLL1Vk. Amino acid sequences encoded by the corresponding DNA sequences are given as one letter codes. The amino acid residues in the CDR regions are shown in bold and underlined. Kabat's Ig molecule numbering scheme is used for amino acid residues as in FIG. 1A and FIG. 1B.
FIG. 7 shows the result of a competitive binding assay in Raji cell membrane coated microwells to compare the binding affinity of hLL1 with that of cLL1. Varying concentrations of hLL1 (triangles) or cLL1 (diamonds) were mixed with a constant amount of HRP conjugated LL1 and incubated in 96-well microtitration plate coated with Raji membrane extacts at room temperature for 1 h. The membrane bound HRP-LL1 was measured. hLL1 and cLL1 competed equally well for the binding of HRP-LL1, indicating the binding specificity and affinity of mAb LL1 are preserved in the humanized LL1.
FIG. 8 shows the fate of 125I-labeled hLL1 and mLL1 bound to the surface of Raji cells. The radiolabeled hLL1 (solid line with symbols) or mLL1 (dotted line with symbols) was incubated with Raji cells and unbound Abs were removed by washing. The cells were then cultured as normal and the radiolabeled Abs associated with cells (diamond lines), secreted into medium (triangle lines) or degraded (circle lines) were measured at indicated time point. FIG. 8A shows the fate of the bound Ab followed for up to 3 days. FIG. 8B shows the result of hLL1 processing studied at early time points (less than 3 h). The data were average of two experiments.
FIG. 9 shows the cytotoxicity effect of crosslinked LL1Abs on Raji cells. 5�105 Raji cells were seeded at day 0 in 1 ml of culture medium containing (as indicated on top of the panels) 5 g/ml of mLL1, cLL1 or hLL1, or no any Ab (Nil), with 50 g/ml of -mFc or -hFc Ab, or without any crosslinker (Nil), indicated at right side of panels. The numbers of total and viable cells were counted daily for 3 days. Percentage of viable cells (squares) and the ratio of viable cells over the viable cells at time zero (diamonds) were calculated and plotted against culture time.
FIG. 10 shows the cytotoxicity effect of crosslinked hLL1 on Daudi cells. 5�105 Daudi cells were seeded at day 0 in 1 ml of culture medium containing (as indicated on top of the panels) 5 g/ml of hLL1, or hLL2 (an anti-CD22, internalizing Ab), or no any Ab (Nil), with 50 g/ml of -hFc Ab, or without (Nil), indicated at right side of panels. The numbers of total and viable cells were counted daily for 3 days. Percentage of viable cells (squares) and the ratio of viable cells over the viable cells at time zero (diamonds) were calculated and plotted against culture time.
A human antibody is an antibody obtained from transgenic mice that have been �engineered� to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See for example, McCafferty et al., Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors. In this technique, antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opiniion in Structural Biology 3:5564-571 (1993).
A diagnostic agent is a molecule or atom which is administered conjugated to an antibody moiety, i.e., antibody or antibody fragment, or subfragment, and is useful in diagnosing a disease by locating the cells containing the antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incoporated in its entirety by reference. Preferably, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose. Chelates are coupled to the peptide antigens using standard chemistries. The chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking. Other, more unusual, methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne, entitled �Antibody Conjugates�, issued Apr. 25, 1989, the disclosure of which is incorporated herein in its entirety by reference. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125I, 131I, 123I, 124I, 62Cu, 64Cu, 18F, 111In, 67Ga, 68Ga, 99mTc, 94mTc, 11C, 13N, 15O, 76Br, for radio-imaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies of the invention. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra for RAIT are encompassed by the invention.
A recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells, such as bacteria, yeast and mammalian cells, as well as an transgenic animal, that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell or cells of the host cells. Suitable mammalian host cells include myeloma cells, such as SP2/0 cells, and NSO cells, as well as Chinese Hamster Ovary (CHO) cells, hybridoma cell lines and other mammalian host cell useful for expressing antibodies. Also particularly useful to express mAbs and other fusion proteins, is a human cell line, PER.C6 disclosed in WO 0063403 A2, which produces 2 to 200-fold more recombinant protein as compared to conventional mammalian cell lines, such as CHO, COS, Vero, Hela, BHK and SP2-cell lines. Special transgenic animals with a modified immune system are particularly useful for making fully human antibodies.
General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by reference in its entirety. Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et al., Hybridoma 13:469 (1994), describe how they produced an LL2 chimera by combining DNA sequences encoding the Vκ and VH domains of LL2 monoclonal antibody, an anti-CD22 antibody, with respective human κ and IgG1 constant region domains. This publication also provides the nucleotide sequences of the LL2 light and heavy chain variable regions, Vκ and VH, respectively. Techniques for producing humanized MAbs are described, for example, by Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993), each of which is hereby incorporated by reference.
To this end, the present invention describes chimeric, humanized and human antibodies and fragments thereof that bind the CD74 antigen and can be used for diagnostic and therapeutic methods. Humanized antibodies and antibody fragments are described in Provisional U.S. Application titled �Anti-CD20 Antibodies And Fusion Proteins Thereof And Methods Of Use�, Attorney Docket No. 18733/1073, U.S. Provisional No. 60/356,132, U.S. Provisional Application No. 60/416,232 and Attorney Docket No. 18733/1155; hMN-14 antibodies, such as those disclosed in U.S. Pat. No. 5,874,540, which is a Class III anti-carcinoembryonic antigen antibody (anti-CEA antibody); Mu-9 antibodies, such as those described in U.S. application Ser. No. 10/116,116; AFP antibodies, such as those described in U.S. Provisional Application No. 60/399,707; PAM4 antibodies, such as those described in Provisional U.S. Application titled �Monoclonal Antibody cPAM4�, Attorney Docket No. 18733/1102; RS7 antibodies, such as those described in U.S. Provisional Application No. 60/360,229; and CD22 antibodies, such as those disclosed in U.S. Pat. Nos. 5,789,554 and 6,187,287 and U.S. application Ser. Nos. 09/741,843 and 09/988,013, all of which are incorporated herein by reference in their entirety.
Another method for producing the antibodies of the present invention is by production in the milk of transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690, both of which are incoporated in their entirety by reference. Two DNA constructs are prepared which contain, respectively, DNA segments encoding paired immunoglobulin heavy and light chains. The DNA segments are cloned into expression vectors which contain a promoter sequence that is preferentially expressed in mammary epithelial cells. Examples include, but are not limited to, promoters from rabbit, cow and sheep casein genes, the cow α-lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse whey acid protein gene. Preferably, the inserted fragment is flanked on its 3′ side by cognate genomic sequences from a mammary-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences. The expression cassettes are coinjected into the pronuclei of fertilized, mammalian eggs, which are then implanted into the uterus of a recipient female and allowed to gestate. After birth, the progeny are screened for the presence of both transgenes by Southern analysis. In order for the antibody to be present, both heavy and light chain genes must be expressed concurrently in the same cell. Milk from transgenic females is analyzed for the presence and functionality of the antibody or antibody fragment using standard immunological methods known in the art. The antibody can be purified from the milk using standard methods known in the art.
A fully human antibody of the present invention, i.e., human anti-CD74 MAbs or other human antibodies, such as anti-CD22, anti-CD19, anti-CD23, anti-CD20 or anti-CD21 MAbs for combination therapy with humanized, chimeric or human anti-CD74 antibodies, can be obtained from a transgenic non-human animal. See, e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997); U.S. Pat. No. 5,633,425, which are incoporated in their entirety by reference. For example, a human antibody can be recovered from a transgenic mouse possessing human immunoglobulin loci. The mouse humoral immune system is humanized by inactivating the endogenous immunoglobulin genes and introducing human immunoglobulin loci. The human immunoglobulin loci are exceedingly complex and comprise a large number of discrete segments which together occupy almost 0.2% of the human genome. To ensure that transgenic mice are capable of producing adequate repertoires of antibodies, large portions of human heavy- and light-chain loci must be introduced into the mouse genome. This is accomplished in a stepwise process beginning with the formation of yeast artificial chromosomes (YACs) containing either human heavy- or light-chain immunoglobulin loci in germine configuration. Since each insert is approximately 1 Mb in size, YAC construction requires homologous recombination of overlapping fragments of the immunoglobulin loci. The two YACs, one containing the heavy-chain loci and one containing the light-chain loci, are introduced separately into mice via fusion of YAC-containing yeast spheroblasts with mouse embryonic stem cells. Embryonic stem cell clones are then microinjected into mouse blastocysts. Resulting chimeric males are screened for their ability to transmit the YAC through their germline and are bred with mice deficient in murine antibody production. Breeding the two transgenic strains, one containing the human heavy-chain loci and the other containing the human light-chain loci, creates progeny which produce human antibodies in response to immunization.
Further recent methods for producing bispecific mAbs include engineered recombinant mAbs which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10): 1221-1225, 1997. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163, 1997. A variety of bispecific fusion proteins can be produced using molecular engineering. In one form, the bispecific fusion protein is monovalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in similar manner. Recombinant methods can be used to produce a variety of fusion proteins. For example, a fusion protein comprising a Fab fragment derived from a humanized monoclonal anti-CD74 antibody and a scFv derived from a murine anti-diDTPA can be produced. A flexible linker, such as GGGS connects the scFv to the constant region of the heavy chain of the anti-CD74 antibody. Alternatively, the scFv can be connected to the constant region of the light chain of another humanized antibody. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the Vλ and Vκ domains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing a DNA sequence encoding the VH region of an anti-CD74 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell for the expression of the bispecific fusion protein.
Antibody fragments which recognize specific epitopes can be generated by known techniques. The antibody fragments are antigen binding portions of an antibody, such as F(ab′)2, Fab′, Fab, Fv, sFv and the like. Other antibody fragments include, but are not limited to: the F(ab)′2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab′ fragments, which can be generated by reducing disulfide bridges of the F(ab)′2 fragments. Alternatively, Fab′ expression expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab′ fragments with the desired specificity. The present invention encompasses antibodies and antibody fragments.
An antibody fragment can be prepared by proteolytic hydrolysis of the full-length antibody or by expression in E. coli or another host of the DNA coding for the fragment. An antibody fragment can be obtained by pepsin or papain digestion of full length antibodies by conventional methods. For example, an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein, which patents are incorporated herein in their entireties by reference. Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10-2.10.4.
5. Anti-CD74 Antibodies
The anti-CD74 mAbs of the present invention contain specific murine CDRs that have specificity for the CD74 antigen. The anti-CD74 mAbs of the present invention are humanized, chimeric or human mAbs and they contain the amino acids of the CDRs of a murine anti-CD74 mAb, the murine LL1 mAb. The humanized anti-CD 74 monoclonal antibody (mAb) or fragment thereof comprise CDRs of a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), Further, the humanized anti-CD74 monoclonal antibody or fragment thereof comprises the heavy chain variable region of said humanized mAb that comprises CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). Further, the humanized mAb retains substantially the specificity for the CD74, i.e., the MHC class-II invariant chain, Ii, present on the surface of cells, such as B-lymphocytes, monocyte and histiocytes, as well as B-cell lymphoma and leukemia, as well as myeloma cells resulting in the rapid internalization and catabolization of these mAbs, fragments thereof or mAb conjugates.
In one embodiment, a CD74 antibodies of the present invention is a humanized anti-CD 74 monoclonal antibody (mAb) or fragment thereof comprising light and heavy chain variable regions comprising complementarity-determining regions (CDRs) of murine anti-CD74 (mLL1) and the framework (FR) regions of a human antibody, wherein the light chain variable region of the humanized mAb comprises CDRs of a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), and wherein the heavy chain variable region of the humanized mAb comprises CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). The murine CDRs of the heavy and light chain variable regions are shown in FIGS. 1A and 1B, respectively. The human FRs of the light and heavy chain variable regions may be modified to maintain specificity to CD74 by substituting at least one amino acid substituted from the corresponding FRs of the murine mAb. More specifically, one or more specific amino acids from the muine mAb identified by amino acid residue 2, 3, 4, 46, 87 and 100 of the murine light chain variable region of the cLL1 Vk sequence of FIG. 3B, and amino acid residues 5, 37, 38, 46, 68, 91 and 93 of the murine heavy chain variable region of the cLL1VH sequence of FIG. 3A may be maintained in the human FRs of the humanized anti-CD74 to maintain specificity.
In a preferred embodiment, the humanized anti-CD74 mAb, the humanized LL1 (hLL1) or fragment thereof containing a heavy chain variable region of FIG. 4A and a light chain variable region of FIG. 4B is used in the methods disclosed in the present invention. More specifically, the humanized anti-CD74 mAb or fragment thereof contains a light and heavy chain constant region of a human antibody or a portion thereof. 9. Additionally, the humanized anti-CD74 mAb or fragment thereof of any one of the humanized anti-CD74 mAbs or fragments thereof, described herein, can be a humanized IgG1.
Although humanized anti-CD74 mAbs are preferred, chimeric anti-CD74 (cCD74) mAbs or fragments thereof also are encompassed by the present invention. In one embodiment, the chimeric anti-CD74 (cCD74) monoclonal antibody, (mAb) or fragment thereof comprises a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21). In a further embodiment, the chimeric anti-CD74 monoclonal antibody or fragment thereof comprises a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). In a further embodiment the chimeric anti-CD 74 mAb comprises light and heavy chain variable regions comprising complementarity-determining regions (CDRs) of a murine anti-CD74 mAb and the framework (FR) regions of a murine anti-CD 74 mAb and the light and heavy chain constant regions of a human antibody, wherein the light chain variable region of the chimeric mAb comprises CDRs of a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), and wherein the heavy chain variable region of said chimeric mAb comprises CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). The preferred chimeric anti-CD74 mAb or fragment thereof comprises a heavy chain variable region of FIG. 2A and a light chain variable region of FIG. 2B.
Also encompassed within the present invention is a human anti-CD74 (huCD74) monoclonal antibody (mAb) or fragment thereof comprising a light chain variable region of the human anti-CD74 mAb that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21). Further, encompasses is a human anti-CD74 (huCD74) monoclonal antibody (mAb) or fragment thereof comprising a heavy chain variable region of said human mAb that comprises CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). More preferably, the present invention discloses a human anti-CD74 (huCD74) monoclonal antibody (mAb) or fragment thereof comprising the light and heavy chain variable and constant regions of a human antibody, wherein the huCD74 CDRs of the light chain variable region of the human anti-CD74 mAb comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), and wherein the heavy chain variable region of the human mAb comprises CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24).
The present invention also is directed to a murine anti-CD 74 monoclonal antibody or fragment thereof, comprising CDRs of a light chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), Further, the murine anti-CD74 monoclonal antibody or fragment thereof, comprising CDRs of a heavy chain variable region of a murine anti-CD74 mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24). More preferably, the murine anti-CD 74 monoclonal antibody or fragment thereof comprising complementarity-determining regions (CDRs) of murine anti-CD74 (mLL1) and the framework (FR) regions of a murine anti-CD74 antibody, wherein the light chain variable region of said murine mAb comprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO: 19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ ID NO: 20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ ID NO: 21), and wherein the heavy chain variable region of said murine mAb comprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO: 22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO: 23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO: 24).
Each of the human, chimeric, humanized or murine anti-CD74 mAbs or fragment thereof of the present invention possess at least one of the following properties: it binds specifically and are reactive with the antigen, CD74, its binding to CD-74 is blocked by an antibody of fragment thereof specific for or reactive with CD74; it is internalized by Raji lymphoma cells in culture; and it induces apoptosis of Raji cells in cell culture when cross-linked with goat antisera reactive with the Fc of of a murine IgG1 mAb.
6. Multispecific and Multivalent Antibodies
A preferred antibody fusion protein of the present invention contains four or more Fvs, or Fab's of the humanized, chimeric, human or murine anti-CD74 mAbs or fragments thereof described herein. Additionally, another preferred antibody fusion protein contains one or more Fvs, or Fab's of the mAbs or fragments thereof of the humanized, chimeric, human or murine anti-CD74 mAbs or fragments thereof described herein, and one or more Fvs or Fab's from antibodies specific for for another antigen that is specific for a tumor cell marker that is not a CD74 antigen, that is expressed by the CD74-expressing cells, such as, for example, a tumor marker selected from a B-cell lineage antigen, such as CD19, CD20, or CD22 for the treatment of B-cell malignancies; as well as other CD74 positive cells causing other types of malignancies, such as S100 in melanoma, etc. Further, the tumor cell marker may be a non-B-cell lineage antigen selected from the group consisting of HLA-DR, CD30, CD33, CD52 MUC1 and TAC.
An anti-CD74 multivalent antibody is also contemplated in the present invention. This multivalent target binding protein is constructed by association of a first and a second polypeptide. The first polypeptide comprises a first single chain Fv molecule covalently linked to a first immunoglobulin-like domain tht preferably is an immunoglobulin light chain variable region domain. The second polypeptide comprises a second single chain Fv molecule covalently linked to a second immunoglobulin-like domain tht preferably is an immunoglobulin heavy chain variable region domain. Each of the first and second single chain Fv molecules forms a target binding site, and the first and second immunoglobulin-like domains associate to form a third target binding site.
A single chain Fv molecule with the VL-L-VH configuration, wherein L is a linker, may associate with another single chain Fv molecule with the VH-L-VL configuration to form a bivalent dimer. In this case, the VL domain of the first scFv and the VH domain of the second-scFv molecule associate to form one target binding site, while the VH domain of the first scFv and the VL domain of the second scFv associate to form the other target binding site.
Another embodiment of the present invention is a CD74 bispecific, trivalent targeting protein comprising two heterologous polypeptide chains associated non-covalently to form three binding sites, two of which have affinity for one target and a third which has affinity for a hapten that can be made and attached to a carrier for a diagnostic and/or therapeutic agent. Preferably, the binding protein has two CD20 binding sites and one CD22 binding site. The bispecific, trivalent targeting agents have two different scFvs, one scFv contains two VH domains from one antibody connected by a short linker to the VL domain of another antibody and the second scFv contains two VL domains from the first antibody connected by a short linker to the VH domain of the other antibody. The methods for generating multivalent, multispecific agents from VH and VL domains provide that individual chains synthesized from a DNA plasmid in a host organism are composed entirely of VH domains (the VH-chain) or entirely of VL domains (the VL-chain) in such a way that any agent of multivalency and multispecificity can be produced by non-covalent association of one VH-chain with one VL-chain. For example, forming a trivalent, trispecific agent, the VH-chain will consist of the amino acid sequences of three VH-domains, each from an antibody of different specificity, joined by peptide linkers of variable lengths, and the VL-chain will consist of complementary VL-domains, joined by peptide linkers similar to those used for the VH-chain. Since the VH and VL domains of antibodies associate in an anti-parallel fashion, the preferred method in this invention has the VL domains in the VL-chain arranged in the reverse order of the VH domains in the VH-chain.
The anti-CD74 antibodies of the present invention can also be used to prepare functional bispecific single-chain antibodies (bscAb), also called diabodies, and can be produced in mammalian cells using recombinant methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995, incorporated. For example, bscAb are produced by joining two single-chain Fv fragments via a glycine-serine linker using recombinant methods. The V light-chain (VL) and V heavy-chain (VH) domains of two antibodies of interest are isolated using standard PCR methods. The VL and VH cDNA's obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fusion PCR. The first PCR step introduces the (Gly4-Ser1)3 linker (SEQ ID NO: 25), and the second step joins the VL and VH amplicons. Each single chain molecule is then cloned into a bacterial expression vector. Following amplification, one of the single-chain molecules is excised and sub-cloned into the other vector, containing the second single-chain molecule of interest. The resulting bscAb fragment is subcloned into an eukaryotic expression vector. Functional protein expression can be obtained by transfecting the vector into Chinese Hamster Ovary cells. Bispecific fusion proteins are prepared in a similar manner. Bispecific single-chain antibodies and bispecific fusion proteins are included within the scope of the present invention.
For example, a humanized, chimeric or human anti-CD74 monoclonal antibody can be used to produce antigen specific diabodies, triabodies, and tetrabodies. The monospecific diabodies, triabodies, and tetrabodies bind selectively to targeted antigens and as the number of binding sites on the molecule increases, the affinity for the target cell increases and a longer residence time is observed at the desired location. For diabodies, the two chains comprising the VH polypeptide of the humanized CD74 mAb connected to the VK polypeptide of the humanized CD74 mAb by a five amino acid residue linker are utilized. Each chain forms one half of the humanized CD74 diabody. In the case of triabodies, the three chains comprising VH polypeptide of the humanized CD74 MAb connected to the Vκ polypeptide of the humanized CD74 MAb by no linker are utilized. Each chain forms one third of the hCD74 triabody.
The ultimate use of the bispecific diabodies described herein is for pre-targeting CD74 positive tumors for subsequent specific delivery of diagnostic or therapeutic agents. These diabodies bind selectively to targeted antigens allowing for increased affinity and a longer residence time at the desired location. Moreover, non-antigen bound diabodies are cleared from the body quickly and exposure of normal tissues is minimized. The diagnostic and therapeutic agents can include isotopes, drugs, toxins, cytokines, hormones, enzymes, oligonucleotides, growth factors, conjugates, radionuclides, and metals. For example, gadolinium metal is used for magnetic resonance imaging (MRI). Examples of radionuclides are 225Ac, 18F, 68Ga, 67Ga, 90Y, 86Y, 111In, 131I, 125I, 123I, 99mTc, 94m Tc, 186Re, 188Re, 177Lu, 62Cu, 64Cu, 67Cu, 212Bi, 213Bi, 32P, 11C, 13N, 15O, 76Br, and 211At. Other radionuclides are also available as diagnostic and therapeutic agents, especially those in the energy range of 60 to 4,000 keV for diagnostic agents and in the energy range of 60-700 for the therapeutic agents.
7. Conjugated Multivalent and Multispecific Anti-CD74 Antibodies
Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD74 mAbs and other MAbs described herein, in accordance with this invention are suitable for use in therapeutic methods and diagnostic methods. Accordingly, the present invention contemplates the administration of the humanized, chimeric and human antibodies of the present invention alone as a naked antibody or administered as a multimodal therapy, temporally according to a dosing regimen, but not conjugated to, a therapeutic agent. An immunoconjugate is a conjugate comprising an antibody component comprising at least one mAb or fragment thereof or antibody fusion protein thereof of the humanized, chimeric or human CD74 mAbs desecribed in the present invention that binds to CD74, which is linked to a diagnostic or therapeutic agent.
The efficacy of the naked anti-CD74 mAbs can be enhanced by supplementing naked antibodies with one or more other naked antibodies, i.e., mAbs to specific antigens, such as CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, tenascin, HM1.24, or HLA-DR, preferably mature HLA-DR dimer, with one or more immunoconjugates of anti-CD74, or antibodies to theses recited antigens, conjugated with therapeutic agents, including drugs, toxins, immunomodulators, hormones, enzymes, therapeutic radionuclides, etc., with one or more therapeutic agents, including drugs, toxins, immunomodulators, hormones, enzymes, therapeutic radionuclides, etc., administered concurrently or sequentially or according to a prescribed dosing regimen, with the mAbs. Preferred B-cell-associated antigens include those equivalent to human CD19, CD20, CD21, CD22, CD23, CD46, CD52, CD74, CD80, and CD5 antigens. Preferred T-cell antigens include those equivalent to human CD4, CD8 and CD25 (the IL-2 receptor) antigens. An equivalent to HLA-DR antigen can be used in treatment of both B-cell and T-cell disorders. Particularly preferred B-cell antigens are those equivalent to human CD19, CD22, CD21, CD23, CD74, CD80, and HLA-DR antigens. Particularly preferred T-cell antigens are those equivalent to human CD4, CD8 and CD25 antigens. CD46 is an antigen on the surface of cancer cells that block complement-dependent lysis (CDC). Preferred malignant melanoma associated antigens are those equivalent to MART-1, TRP-1, TRP-2 and gp100. Further, preferred multiple myeloma-associated antigens are those equivalent to MUC1 and CD38.
A wide variety of diagnostic and therapeutic reagents can be advantageously conjugated to the antibodies of the invention. The therapeutic agents recited here are those agents that also are useful for administration separately with the naked antibody as described above. Therapeutic agents include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others from these and other classes of anticancer agents, and the like. Other useful cancer chemotherapeutic drugs for the preparation of immunoconjugates and antibody fusion proteins include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.
Furthermore, a radiolabeled antibody or immunoconjugate may comprise a γ-emitting radioisotope or a positron-emitter useful for diagnostic imaging. Suitable radioisotopes, particularly in the energy range of 60 to 4,000 keV, include 131I, 123I, 124I, 86Y, 62Cu, 64Cu, 111In, 67Ga, 68Ga, 99mTc, 94mTc, 18F, 11C, 13N, 15O, 75Br, and the like. See, for example, U.S. patent application entitled �Labeling Targeting Agents with Gallium-68��Inventors G. L. Griffiths and W. J. McBride, (U.S. Provisional Application No. 60/342,104), which discloses positron emitters, such as 18F, 68Ga, 94mTc. and the like, for imaging purposes and which is incoporated in its entirety by reference.
A toxin, such as Pseudomonas exotoxin, may also be complexed to or form the therapeutic agent portion of an antibody fusion protein of an anti-CD74 antibody of the present invention. Other toxins suitably employed in the preparation of such conjugates or other fusion proteins, include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA�A Cancer Journal for Clinicians 44:43 (1994). Additional toxins suitable for use in the present invention are known to those of skill in the art and are disclosed in U.S. Pat. No. 6,077,499, which is incoporated in its entirety by reference.
Any of the antibodies or antibody fusion proteins of the present invention can be conjugated with one or more therapeutic or diagnostic agents. Generally, one therapeutic or diagnostic agent is attached to each antibody or antibody fragment but more than one therapeutic agent or diagnostic agent can be attached to the same antibody or antibody fragment. The antibody fusion proteins of the present invention comprise two or more antibodies or fragments thereof and each of the antibodies that comprises this fusion protein can contain a therapeutic agent or diagnostic agent. Additionally, one or more of the antibodies of the antibody fusion protein can have more than one therapeutic of diagnostic agent attached. Further, the therapeutic agents do not need to be the same but can be different therapeutic agents. For example, one can attach a drug and a radioisotope to the same fusion protein. Particulary, an IgG can be radiolabeled with 131I and attached to a drug. The 131I can be incorporated into the tyrosine of the IgG and the drug attached to the epsilon amino group of the IgG lysines. Both therapeutic and diagnostic agents also can be attached to reduced SH groups and to the carbohydrate side chains.
A therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation. As an alternative, such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyidithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., �Modification of Antibodies by Chemical Methods,� in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, �Production and Characterization of Synthetic Peptide-Derived Antibodies,� in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). Alternatively, the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same peptide that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different peptide.
The Fc region is absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full-length antibody or antibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of which are incoporated in their entirety by reference. The engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
The present invention contemplates the use of naked anti-CD74 antibodies of the present invention as the primary composition for treatment of a CD74 expressing malignancy, where the disease or disorder is selected from the group consisting of an immune dysregulation disease, an autoimmune disease, organ graft rejection, and graft versus host disease. The CD74 expressing malignancy is selected from the group consisting of a solid tumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, another B-cell malignancy and a T-cell malignancy. The solid tumor is selected from the group consisting of a melanoma, carcinoma and sarcoma and the carcinoma is selected from the group consisting of a renal carcinoma, lung carcinoma, intestinal carcinoma, stomach carcinoma and melanoma. The B-cell malignany is selected from the group consisting of non-Hodgkins lymphoma, Hodkgin's lymphoma, indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, and multiple myeloma, B-cell disorders and other diseases. In particular, the compositions described herein are particularly useful for treatment of various autoimmune as well as indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, multiple myeloma, and Waldenstr�m's macroglobulinemia. For example, the humanized anti-CD74 antibody components and immunoconjugates can be used to treat both indolent and aggressive forms of non-Hodgkin's lymphoma.
More specifically, the invenion contemplates a method for treating a B-cell malignancy comprising administering to a subject with a B-cell related malignancy, a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one humanized, chimeric, or human anti-CD74 mAb or fragment thereof or antibody fusion protein thereof of the present invention, wherein the B-cell malignancy is lymphoma or leukemia. More specifically, the B-cell malignany is non-Hodgkin's lymphoma, indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, multiple myeloma, chronic lymphatic leukemias, or acute lymphatic leukemias. The CD74 mAb or fragment thereof is administered intravenously or intramuscularly at a dose of 20-2000 mg. The present method further comprises administereing the anti-CD74 mAb or fragment thereof before, during or after the administration of at least one therapeutic agent used to treat the B-cell malignancy. The therapeutic agent comprises a naked antibody, an immunomodulator, a hormone, a cytotoxic agent, en enzyme, an antibody conjugated to at least one immunomodulator, radioactive label, hormone, enzyme, or cytotoxic agent, or a combination thereof. The immunomodulator preferably is a cytokine and said cytotoxic agent is a drug or toxin. The antibody that is administered incombination as a naked antibody or as a supplemental immunoconjugate is reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, HM1.24, tenascin, and HLA-DR, preferably a mature HLA-DR dimer, formulated in a pharmaceutically acceptable vehicle.
The invention also contemplates treating a malignancy comprising administering to a subject with a CD74 antigen-positive malignancy other than lymphoma or leukemia, a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof as desclosed in the present invention. The anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof is administered intravenously or intramuscularly at a dose of 20-2000 mg. Further, the anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof is administered before, during or after the administration of at least one therapeutic agent used to treat the malignancy. The therapeutic agent, as described above and throughout the specification, comprises an antibody, an immunomodulator, a hormone, a cytotoxic agent, an antibody conjugated to at least one immunomodulator, radioactive label, enzyme, hormone, cytotoxic agent, antisense oligonucleotide, or a combination thereof, where the immunomodulator is a cytokine and said cytotoxic agent is a drug or toxin. When an antibody is administered in combination with the anti-CD74 mAb or fragment thereof to treat a malignancy that is not a B-cell malignancy, it should be reactive with a tumor marker other than CD74, expressed by the cells that comprise the malignancy that is treated, formulated in a pharmaceutically acceptable vehicle. Examples of antibodies that can be administered for malignant melanoma associated antigens are those antibodies reactive with MART-1, TRP-1, TRP-2 and gp100. Further, preferred antibodies to multiple myeloma-associated antigens are those reactive with MUC1 and CD38.
Naked or conjugated antibodies to the same or different epitope or antigen may also be combined with one or more of the antibodies of the present invention. For example, a humanized, chimeric or human naked anti-CD74 antibody may be combined with another naked humanized, naked chimeric or naked human anti-CD74 mAb; a humanized, chimeric or human naked anti-CD74 antibody may be combined with an anti-CD74 immunoconjugate; a naked anti-CD74 antibody may be combined with an anti-CD22 radioconjugate; or an anti-CD22 naked antibody may be combined with a humanized, chimeric or human anti-CD74 antibody conjugated to an isotope, or one or more chemotherapeutic agents, cytokines, enzymes, toxins or a combination thereof. A fusion protein of a humanized, chimeric or human CD20 antibody and a toxin or immunomodulator, or a fusion protein of at least two different B-cell antibodies (e.g., a CD74 and a CD22 mAb, a CD20 mAb or a CD19 mAb) may also be used in this invention. Reference is made to pending U.S. Ser. No. 09/965,796 filed on Oct. 1, 2001, entitled �Immunotherapy of B-Cell Malignancies Using Anti-CD-22 Antibodies,� which is a continuation of U.S. Pat. No. 6,306,393, both of which are incorporated in their entirety by reference, that discloses treatament with an anti-CD22 antibodies in combination with other naked antibodies. Many different antibody combinations, targeting at least two different antigens associated with B-cell disorders, as listed already above, may be constructed, either as naked antibodies or as partly naked and partly conjugated with a therapeutic agent or immunomodulator, or merely in combination with another therapeutic agents, such as a cytotoxic drug or with radiation.
As used herein, the term �immunomodulator� includes cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons-α, -β and -γ), the stem cell growth factor designated �S1 factor,� erythropoietin, thrombopoietin or a combination thereof. Examples of suitable immunomodulator moieties include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, and a combination thereof, and interferon-γ, TNF-α, and the like. Alternatively, subjects can receive naked anti-CD74 antibodies and a separately administered cytokine, which can be administered before, concurrently or after administration of the naked anti-CD74 antibodies. As discussed supra, the anti-CD74 antibody may also be conjugated to the immunomodulator. The immunomodulator may also be conjugated to a hybrid antibody consisting of one or more antibodies binding to different antigens.
Multimodal therapies of the present invention further include immunotherapy with naked anti-CD74 antibodies supplemented with administration of anti-CD22, anti-CD19, anti-CD21, anti-CD20, anti-CD80, anti-CD23, anti-CD46 or HLA-DR, preferably the mature HLA-DR dimer antibodies in the form of naked antibodies, fusion proteins, or as immunoconjugates. These antibodies include polyclonal, monoclonal, chimeric, human or humanized antibodies that recognize at least one epitope on these antigenic determinants. Anti-CD19 and anti-CD22 antibodies are known to those of skill in the art. See, for example, Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353 (1996) and U.S. Pat. Nos. 5,798,554 and 6,187,287, incoporated in their entirety by reference.
In another form of multimodal therapy, subjects receive naked anti-CD74 antibodies, and/or immunoconjugates, in conjunction with standard cancer chemotherapy. For example, �CVB� (1.5 g/m2 cyclophosphamide, 200-400 mg/m2 etoposide, and 150-200 mg/m2 carmustine) is a regimen used to treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitable combination chemotherapeutic regimens are well-known to those of skill in the art. See, for example, Freedman et al., �Non-Hodgkin's Lymphomas,� in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). A useful second-generation chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin), while a suitable third generation regimen is MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin). Additional useful drugs include phenyl butyrate and brostatin-1. In a preferred multimodal therapy, both chemotherapeutic drugs and cytokines are co-administered with an antibody, immunoconjugate or fusion protein according to the present invention. The cytokines, chemotherapeutic drugs and antibody or immunoconjugate can be administered in any order, or together.
Although naked anti-CD74 antibodies are the primary therapeutic compositions for treatment of B cell lymphoma and autoimmune diseases, the efficacy of such antibody therapy can be enhanced by supplementing the naked antibodies, with supplemental agents, such as immunomodulators, like interferons, including IFNα, IFNβ and IFNγ, interleukins including IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-15, IL-18, IL-21, and a combination thereof, and cytokines including G-CSF and GM-CSF. Accordingly, the CD74 antibodies can be combined not only with antibodies and cytokines, either as mixtures (given separately or in some predetermined dosing regiment) or as conjugates or fusion proteins to the anti-CD74 antibody, but also can be given as a combination with drugs or with antisense olignucleotides. For example, the anti-CD74 antibody may be combined with CHOP as a 4-drug chemotherapy regimen. Additionally, a naked anti-CD74 antibody may be combined with a naked anti-CD22 antibodies and CHOP or Fludarabine as a drug combination for NHL therapy. The supplemental therapeutic compositions can be administered before, concurrently or after administration of the anti-CD74 antibodies. The naked anti-CD74 mAb may also be combined with an antisense bcl oligonucleotide.
As discussed supra, the antibodies of the present invention can be used for treating B cell lymphoma and leukemia, and other B cell diseases or disorders as well as other malignancies in which affected or associated malignant cells are reactive with CD74. For example, anti-CD74 antibodies can be used to treat immune dysregulation diseas and related autoimmune diseases, including Class-III autoimmune diseases such as immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sj�gren's syndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis ubiterans, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.
Particularly, the humanized, chimeric or human anti-CD74 mAbs or fragments thereof or antibody fusion proteins thereof of the present invention are administered to a subject with one or more of these autoimmune diseases. The anti-CD74 antibodies of the present invention are particuarly useful in the method of treating autoimmune disorders, disclosed in pending U.S. Ser. No. 09/590,284 filed on Jun. 9, 2000 entitled �Immunotherapy of Autoimmune Disorders using Antibodiess that Target B-Cells,� which is incorporated in its entirety by reference. Preferably the anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof is administered intravenously or intramuscularly at a dose of 20-2000 mg. Further, the anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof is administered before, during or after the administration of at least one therapeutic agent used to treat the disorder. The therapeutic agent, as described above and throughout the specification, comprises an antibody, an immunomodulator, a hormone, an enzyme, a cytotoxic agent, an antibody conjugated to at least one immunomodulator, radioactive label, hormone, enzyme, or cytotoxic agent, antisense oligonucleotide or a combination thereof, where the immunomodulator is a cytokine and said cytotoxic agent is a drug or toxin. The antibody that is administered incombination as a naked antibody or as a supplemental immunoconjugate is reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, HM1.24, tenascin, and mature HLA-DR, preferably a mature HLA-DR dimer, formulated in a pharmaceutically acceptable vehicle.
A further method for treating one of the diseases selected from the group consisting of lymphoma, leukemia, myeloma, other CD-74-expressing malignancies, immune dysregulation disease, autoimmune disease and a combination thereof, comprising administering a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one anti-CD74 mAb or fragment thereof or an antibody fusion protein thereof of the pressent ivention, wherein at least one therapeutic agent is linked to the mAb or fragment thereof or the Fvs or Fab's of the antibody fusion protein thereof by chemical conjugation or by genetic fusion. The therapeutic agent may be an immunomodulator, a radioactive label, a hormone, an enzyme, or a cytotoxic agent, and the immunomodulator is a cytokine and said cytotoxic agent is a drug or toxin.
Anti-CD74 antibodies may also induce apoptosis in cells expressing the CD74 antigen. Evidence of this induction is supported in the examples of the present invention. Other antibodies have demonstrated that apoptosis could be induced using lymphoid cells that have Fc-receptors reactive with the IgG1-Fc of CD20 MAbs that crosslinked. See Shan et al., Cancer Immunol. Immunother. 48(12):673-683 (2000). Further, it was reported that aggregates of a chimeric CD20 MAb, i.e., homopolymers, induced apoptosis. See Ghetie et al., Blood 97(5): 1392-1398 (2000) and Ghetie et al., Proc. Natl. Acad. Sci USA 94(14): 7509-7514 (1997).
Antibodies specific to the CD74 surface antigen of B cells can be injected into a mammalian subject, which then bind to the CD74 cell surface antigen of both normal and malignant B cells. A mammalian subject includes humans and domestic animals, including pets, such as dogs and cats. The anti-CD74 mAbs of the present invention, i.e., humanized, chimeric, human, caninized and felinized, and even murine anti-CD74 mAbs, can be used to treat the non-human mammalian subjects when there is a species crossreactivity for the CD74 antigen. The murine mAbs, which are immunogenic in humans, are usually less immunogenic in non-human mammalian subjects. The anti-CD74 antibody bound to the CD74 surface antigen leads to the destruction and depletion of neoplastic B cells.
12. Method of /Diagnosis
Also provided for in the present invention is a method of diagnosing a disease in a subject, diagnosed with or suspected of having at least one of the diseases selected from the groups consisting of lymphoma, leukemia, myeloma, other CD-74-expressing malignancies, immune dysregulation disease, autoimmune disease and a combination thereof, comprising administering to said subject a diagnostically effective amount of a diagnostic conjugate a pharmaceutically acceptable carrier and at least one anti-CD74 mAb or fragment thereof or antibody fusion protein thereof, wherein a diagnostic agent is linked to the mAb or fragment thereof or the Fvs or Fabs of the antibody fusion protein thereof by chemical conjugation and detecting the diagnostic agent. Diagnostic agents useful in the present invention are a radioisotope, wherein the photons of the radioisotope are detected by radioscintigrapy or PET, or a metal that can be detected by MRI, or a liposome or gas filled liposome, and wherein the liposome can be detected by an ultrasound scanning device.
The internalization of murine anti-CD74 mAb, chimeric anti-CD74 mAb- and humanized anti-CD74 mAb into target cells can be followed by fluorescence labeling, essentially according to the procedure of Pirker et al., J. Clin. Invest., 76: 1261 (1985), which is incorporated by reference. Cultured Raji cells are centrifuged and the cells resuspended in fresh medium to a concentration of about 5�106 cells/ml. To each well of a 96-well microtiter plate, 100 μl of the cell suspension is added. The antibodies, 40 μg/ml, in a volume of 100 μl are added to the reaction wells at timed intervals so as to terminate all reactions simultaneously. The plate is incubated at 37� C. in a CO2 cell culture incubator. Unbound antibodies are removed by washing the cells three times with cold 1% FCS/PBS at the end of the incubation. The cells are then treated with 1 ml of Formaid-Fresh [10% formalin solution (Fisher, Fair Lawn, N.J.)] for 15 min at 4� C. After washing, antibodies present either on the cell surface or inside the cells are detected by treatment with FITC-labeled goat anti-mouse antibody (Tago, Burlingame, Calif.), or FITC-labeled goat anti-human antibody (Jackson ImmunoResearch, West Grove, Pa.), depending on whether the antibody being assayed for is murine, chimeric, or humanized, respectively. Fluorescence distributions are evaluated using a BH-2 fluorescence microscope (Olympus, Lake Success, N.Y.).
13. Expression Vectors
The DNA sequence encoding a humanized, chimeric or human anti-CD74 mAb. More specifically the DNA sequence comprises a nucleic acid encoding a mAb or fragment thereof selected from the group consisting (a) an anti-CD74 mAb or fragment described herein, (b) an immunoconjugate comprising any one of the anti-CD74 mAbs or fragment there of described herein, (c) an antibody fusion protein or fragment thereof comprising at least two of said anti-CD74 mAbs or fragments thereof described herein; (d) an antibody fusion protein or fragment described herein; (e) a vaccine as described herein; and a bispecific or multispecifc antibody described herein. Any of the DNA sequences of the present invention can be recombinantly engineered into a variety of known host vectors that provide for replication of the nucleic acid. These vectors can be designed, using known methods, to contain the elements necessary for directing transcription, translation, or both, of the nucleic acid in a cell to which it is delivered. Known methodology can be used to generate expression constructs the have a protein-coding sequence operably linked with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques and synthetic techniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel et al., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (New York). Also provided for in this invention is the delivery of a polynucleotide not associated with a vector.
The method for the expression of an anti-CD74 mAb or fragment thereof or antibody fusion protein or fragment thereof employing the present invention comprises: (a) transfecting a host cell with a DNA sequence encoding an anti-CD74 mAb or fragment thereof or an immunoconjugate, fusion protein or bispecific or multispecific antibody thereof; and (b) culturing the cell secreting the anti-CD74 mAb or fragment thereof or antibody fusion protein or fragment thereof. The host cell is derived from bacterial, yeast or mammalian cells. More preferably from a mammalian cells, which in one embodiment is a lymphocyctic cell, such as a myeloma cell.
The inventors have isolated cDNAs encoding the VL and VH regions of the murine anti-CD74 monoclonal antibody (mLL1 mAb) and recombinantly subcloned them into mammalian expression vectors containing the genes encoding kappa and IgG1 constant regions, respectively, of human antibodies. Cotransfection of mammalian cells with these two recombinant DNAs expressed a chimeric anti-CD74 mAb (cLL1) that, like the parent mLL1 mAb, bound avidly to, and was rapidly internalized by, B-lymphoma cells.
The VK and VH sequences for chimeric or humanized anti-CD74 mAb can amplified by PCR as described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)) which is incorporated by reference. VK sequences may be amplified using the primers CK3BH and VK5-3 (Leung et al., BioTechniques, 15: 286 (1993), which is incorporated by reference), while VH sequences can be amplified using the primer CH1B which anneals to the CH1 region of murine 1 gG, and VHIBACK (Orlandi et al., 1989 above). The PCR reaction mixtures containing 10 μl of the first strand cDNA product, 9 μl of 10�PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl2, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), can be subjected to 30 cycles of PCR. Each PCR cycle preferably consists of denaturation at 94� C. for 1 min, annealing at 50� C. for 1.5 min, and polymerization at 72� C. for 1.5 min. Amplified VK and VH fragments can be purified on 2% agarose (BioRad, Richmond, Calif.). See Example 3 for a method for the synthesis of an oligo A (149-mer) and an oligo B (140-mer) on an automated Cyclone Plus DNA synthesizer (Milligan-Biosearch) for use in constructing humanized V genes.
The two plasmids can be co-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14, colonies selected for hygromycin resistance, and supernatant fluids monitored for production of chimeric or humanized anti-CD74 mAbs by, for example, an ELISA assay, as described below.
Transfection, and assay for antibody secreting clones by ELISA, can be carried out as follows. About 10 μg of hLL1 pKh (light chain expression vector) and 20 μg of hLL1pG1 g (heavy chain expression vector) can be used for the transfection of 5�106 SP2/0 myeloma cells by electroporation (BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148: 1149 (1992) which is incorporated by reference. Following transfection, cells may be grown in 96-well microtiter plates in complete HSFM medium (GIBCO, Gaithersburg, Md.) at 37� C., 5% CO2. The selection process can be initiated after two days by the addition of hygromycin selection medium (Calbiochem, San Diego, Calif.) at a final concentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3 weeks post-electroporation. The cultures can then be expanded for further analysis.
Transfectoma clones that are positive for the secretion of chimeric or humanized heavy chain can be identified by ELISA assay. Briefly, supernatant samples (100 μl) from transfectoma cultures are added in triplicate to ELISA microtiter plates precoated with goat anti-human (GAH)-IgG, F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch, West Grove, Pa.). Plates are incubated for 1 h at room temperature. Unbound proteins are removed by washing three times with wash buffer (PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies (Jackson ImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl of antibody stock diluted�104, supplemented with the unconjugated antibody to a final concentration of 1.0 μg/ml). Following an incubation of 1 h, the plates are washed, typically three times. A reaction solution, [100 μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis, Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color is allowed to develop in the dark for 30 minutes. The reaction is stopped by the addition of 50 μl of 4 N HCl solution into each well before measuring absorbance at 490 nm in an automated ELISA reader (Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodies are than determined relative to an irrelevant chimeric antibody standard (obtainable from Scotgen, Ltd., Edinburg, Scotland).
EXAMPLES Example 1 Molecular Cloning and Sequence Elucidation for LL1 Heavy and Light Chain Variable Regions The V gene of mLL1 was obtained by RT-PCR using VK5′-4 and VK1FOR primers as described by Leung et al. 1993 and Orlandi et al. (PNAS 86:3833-3837 (1989), respectively, and cloned into pCR2.1 AT-cloning vector (Invitrogen). Multiple clones were sequenced to eliminate possible errors resulted from PCR reaction. Majority of clones (6) contained an identical murine V sequence, which was designated as LL1 V and the sequence is shown in FIG. 1B. Comparison with other mouse Vk sequences revealed LL1 Vk is a member of the kappa light chain subclass II.
Since RT-PCR failed to yield a full-length sequence encoding a mouse VH gene, the second cloning approach, rapid amplification of cDNA 5′-ends (5′-RACE) was employed. The adaptor-ligated cDNA prepared from LL1 hybridoma cells was amplified by PCR using a universal anchor primer (Life Technologies) and a gene specific primer, CH-1 B (Leung et al. 1994), which anneals to the CH1 region of murine heavy chain. The major PCR species of �650 bp resulted from PCR was cloned into pCR2.1 AT-cloning vector and multiple clones were sequenced by DNA sequencing. The PCR product contained a full-length VH sequence (FIG. 1A) flanked by the sequences of non-coding and secretion signal peptide at′5-end and partial coding sequence for the CH1 domain of 1 chain. No defective mutation was found within the sequence encoding the VH, which was designated as LL1 VH. Comparison of hLL1VH with other mouse VH sequences revealed that it belonged to mouse heavy chains subgroup miscellaneous (Kabat et al., 1991). By comparing the amino acid sequnences of LL1VH and V with murine Ab V genes in Kabat database and following Kabat's definition, the CDR regions of hLL1VH and Vk were identified as shown in FIGS. 1A and B, respectively. By comparing the amino acid sequnences of LL1VH and V with murine Ab V genes in Kabat database and following Kabat's definition, the CDR regions of hLL1VH and Vk were identified as shown in FIGS. 1A and B, respectively.
Example 2 Construction of the Expression Vector for Chimeric LL1 To evaluated the authenticity of the cloned Fv for LL1, a chimeric LL1 (cLL1) was constructed and expressed. The nucleotide residues 7-12 of LL1 Vk were modified to a PvuII restriction site, CAGCTG, by PCR with primers LL1VK-PvuII and VK1FOR. The resulting PCR product was digested with PvuII and BgIII (partially, due to the presence of an internal BgIII site in the Vk) and force-cloned into a pBR327-based staging vector (digested with PvuII and BcII), VKpBR2, which contained same Ig promoter, signal peptide sequence and convenient restriction sites to facilitate in-frame ligation of the VK PCR product as used by Orlandi et al., 1989 and Leung et al., 1994.
LL1VK-PvuII 5′-GAT GTT CAG CTG ACC CAA ACT CCA CTC TCC-3′ (SEQ ID NO: 26)
Similarly, the nucleotide sequences at positions 10-15 and 345-351 of LL1 VH were converted to PstI and BstEII, respectively, by PCR with primers LL1 B-1 and LL1 F-1. The VH PCR product was then digested with PstI and BstEII and ligated into PstI and BstEII digested VHpBS2, a pBluescript-based staging vector containing a signal peptide sequence and convenient restriction sites to facilitate in frame ligation of the VH PCR product {Orlandi, Gussow, et al. 1989 741/id}, modified from VHpBS (Leung, S. O., Shevitz, J., Pellegrini, M. C., Dion, A. S., Shih, L. B., Goldenberg, D. M., and Hansen, H. J. (1994)).
5′-CAG ATC CAG CTG CAG CAG TCT GGA CCT GAG-3′
5′-GA GAC GGT GAC CAG AGT CCC TTG GCC CCA A-3′
The fragment containing the Vk sequences of cLL1, together with the signal peptide sequences, were excised from LL1VKpBR2 by double restriction digestion with XbaI and BamHI. The �550 bp Vk fragments was then subcloned into the XbaI/BamHI site of a mammalian expression vector, pdHL2. The resulting vector designated as cLL1VkpdHL2. Similarly, the ca. 750 bp fragments containing the LL1VH, together with the signal peptide sequences, were excised from LL1VHpBS2 by XhoI and BamHI digestion and isolated by electrophoresis in an agarose gel. The fragment was subcloned into the XhoI and HindIII site of cLL1VkpdHL2 with the aid of linker comparable to both BamHI and HindIII ends, resulting in the final expression vectors, designated as cLL1 pdHL2.
Example 3 Transfection and Expression of cLL1 Approximately 30 μg of cLL1 pdHL2 was linerized by digestion with Sail and transfected into Sp2/0-Ag14 cells by electroporation. The transfected cells were plated into 96-well plate for 2 days and then selected for MTX resistance. Supernatants from colonies surviving selection were monitored for chimeric antibody secretion by ELISA assay. Positive cell clones were expanded and cLL1 was purified from cell culture supernatant by affinity chromatograpgy on a Protein A column.
Example 4 Binding Activity Assays A competition cell binding assay was carried out to assess the immunoreactivity of cLL1 relative to the parent mLL1. A constant amount of 125I-labeled mLL1 (100,000 cpm) was incubated with Raji cells in the presence of varying concentrations of cLL1 or mLL1 at 4� C. for 1-2 h. The radioactivity associated with cells was determined after washing. As shown in FIG. 5, cLL2 antibody exhibited comparable binding activity as that of mLL1, confirming the authenticity of the cloned V genes.
An ELISA competitive binding assay were carried out in Raji cell membrane coated plate to assess the immunoreactivity of cLL1 relative to the parent mLL1. Raji cell membrane fraction was prepared by sonication and centrifugation. The crude membrane extracts were coated in 96-well flat bottomed PVC plate by centrifugation and fixed with 0.1% glutaraldehyde. Constant amount of the biotinylated mLL1 mixed with vaying concentrations of mLL1 or cLL1 was added to the membrane coated wells and incubated at room temperature for 1-2 h. After washing, HRP-conjugated streptavidin was added and incubated for 1 h at room temperature. The amount of HRP-conjugated streptavidin bound to the membrane-bound biotinylated mLL1 was revealed by reading A490 nm after the addition of a substrate solution containing 4 mM ortho-phenylenediamine dihydrochloride and 0.04% H2O2.
Example 5 Choice of Human Frameworks and Sequence Design for the Humanization of LL1 Monoclonal Antibody By comparing the variable (V) region framework (FR) sequences of cLL1 to that of human antibodies in the Kabat data base, the FRs of cLL1 VH and Vk were found to exhibit the highest degree of sequence homology to that of the human antibodies, RF-TS3 VH and HF-21/28 Vk, respectively. The amino acid sequences of are provided in FIGS. 3A and 3B and are compared with the cLL1VH and Vk sequences. Therefore, the FRs of RF-TS3 VH and the HF-21/28 Vk and FRs were selected as the human frameworks onto which the CDRs for LL1 VH and Vk were grafted, respectively. The FR4 sequence of NEWM, however, rather than that of RF-TS3, was used to replace the RF-TS3 FR4 sequence for the humanization of LL1 heavy chain. See FIG. 3A. A few amino acid residues in the LL1 FRs that are close to the putative CDRs were maintained in hLL1 based on the guideline described previously (Qu et al., Clin. Cancer Rec. 5:3095s-3100s (1990)). These residues are L46, F87 and Q100 of V (FIG. 3B) and 136, K37, Q46, A68, F91 and S93 of VH (FIG. 3A). FIGS. 3A and 3B compare the human, chimeric and humanized VH and Vk amino acid sequences. The dots indicate the residues in the cLL1 and hLL1 that are identical to the corresponding residues in the human VH and Vk sequences. The DNA and amino acid sequences of hLL1VH and Vk are shown in FIGS. 4A and 4B, respectively.
Example 6 PCR/gene Synthesis of the Humanized V Genes A modified strategy as described by Leung et al. (Leung et al., 1994) was used to construct the designed Vk and VH genes for hLL1 using a combination of long oligonucleotide systheses and PCR as illustrated in FIG. 5. For the construction of the hLL1 VH domain, two long oligonucleotides, hLL1VHA (176-mer) and hLL1VHB (165-mer) were synthesized on an automated DNA synthesizer (Applied Biosystem). The hLL1VHA sequence represents nt 20 to 195 of the hLL1VH domain:
5′- GGTCTGAGTT GAAGAAGCCT GGGGCCTCAG TGAAGGTTTC
CTGCAAGGCT TCTGGATACA CCTTCACTAA CTATGGAGTG AACTGGATAA
AGCAGGCCCC TGGACAAGGG CTTCAGTGGA TGGGCTGGAT AAACCCCAAC
ACTGGAGAGC CAACATTTGA TGATGACTTC AAGGGA-3′
The hLL1VHB sequence represents the minus strand of the hLL1VH domain complementary to nt 173 to 337:
5′- TCCCTTGGCC CCAATAAGCA AACCAGGCTT CGTTTTTACC
CCTCGATCTT GAACAGAAAT ACACGGCAGT GTCGTCAGCC TTTAGGCTGC
TGATCTGGAG ATATGCCGTG CTGACAGAGG TGTCCAAGGA GAAGGCAAAT
CGTCCCTTGA AGTCATCATC AAATG-3′
The 3′-terminal sequences (22 nt residues) of hLL1VHA and B are complementory to each other. Under defined PCR condition, 3′-ends of hLL1VHA and B anneal to form a short double stranded DNA flanked by the rest of the long oligonucleotides. Each annealed end serves as a primer for the transcription of the single stranded DNA, resulting in a double strand DNA composed of the nt 20 to 337 of hLL1VH. This DNA was further amplified in the presence of two short oligonucleotides, hLL1VHBACK and hLL1VHFOR to form the full-length hLL1VH.
5′-GTG GTG CTG CAG CAA TCT GGG TCT GAG TTC AAG AAG CC-3′
5′-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA ATG-3′
Minimum amount of hLL1VHA and B (determined empirically) was amplified in the presence of 10 μl of 10�PCR Buffer (500 mM KCl, 100 mM Tris.HCL buffer, pH 8.3, 15 mM MgCl2), 2 mol of hLL1VHBACK and hLL1VHFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Ct). This reaction mixture was subjected to 3 cycle of PCR reaction consisting of denaturation at 94� C. for 1 minute, annealing at 45� C. for 1 minute, and polymerization at 72� C. for 1.5 minutes, and followed by 27 cycles of PCR reaction consisting of denaturation at 94� C. for 1 minute, annealing at 55� C. for 1 minute, and polymerization at 72� C. for 1 minute. Double-stranded PCR-amplified product for hLL1VH was gel-purified, restriction-digested with PstI and BstEII and cloned into the complementary PstII/BstEII sites of the heavy chain staging vector, VHpBS2.
For constructing the full length DNA of the humanized Vk sequence, hLL1VKA (159-mer) and hLL1VKB (169-mer) were synthesized as described above. hLL1VKA and B were amplified by two short oligonucleotides hLL1VKBACK and hLL1 VKFOR as described above.
CCTCCATCTC CTGCAGATCA AGTCAGAGCC TTGTACACAG AAATGGAAAC
ACCTATTTAC ATTGGTTTCA GCAGAGGCCA GGCCAATCTC CAAGGCTCCT
GATCTACACA GTTTCCAAC-3′
GAGCAGAAAT AAACCCCAAC ATCCTCAGCC TCCACCCTGC TGATTTTCAG
TGTGAAATCA GTGCCTGACC CACTGCCGCT GAATCTGTCT GGGACCCCAG
AAAATCGGTT GGAAACTGTG TAGATCAGG-3′
5′-GAT GTT CAG CTG ACT CAG TCT CCA CTC TCC CTG-3′
[]hLL1VKFOR
5′-G TTA GAT CTC CAG TCG TGT CCC AGC ACC GAA CG-3′
Gel-purified PCR products for hLL1Vk were restriction-digested with PvuII and BgIIII and cloned into the complementary Pvul/BcII sites of the light chain staging vector, VKpBR2. The final expression vector hLL1pdHL2 was constructed by sequencially subcloning the XbaI-BamHI and XhoI/BamHI fragments of hLL1Vk and VH, respectively, into pdHL2 as described above.
An ELISA competitive binding assay using Raji cell membrane extract coated plate was developed to assess the immunoreactivity of hLL1. Raji cell membrane fraction was prepared by sonication and centrifugation. The crude membrane extracts were coated in 96-well flat bottomed PVC plate by centrifugation and fixed with 0.1% glutaraldehyde. Constant amount of the biotinylated mLL1 mixed with vaying concentrations of mLL1 or cLL1 was added to the membrane coated wells and incubated at room temperature for 1-2 h. After washing, HRP-conjugated streptavidin was added and incubated for 1 h at room temperature. The amount of HRP-conjugated streptavidin bound to the membrane-bound biotinylated mLL1 was revealed by reading A490 nm after the addition of a substrate solution containing 4 mM ortho-phenylenediamine dihydrochloride and 0.04% H2O2. As shown by the competition assays in FIG. 6, mLL1 and cLL1 antibodies exhibited similar binding activities. Likewise, the competition assays in FIG. 7, hLL1 and cLL1 antibodies exhibited similar binding activities
Example 8 Internalization of hLL1 Standard antibody processing assay was used to evaluate the internalization and metabolism of hLL1 in Raji cells (Hansen et al., 1996). Cells (107) were incubated in 1 ml of tissue culture medium containing 125I-labeled hLL1 or LL1 (107 cpm) for 1 h at 37� C. To ensure the specificity of Ab binding, controls of 1/10 sample size (cells, radioactivity and medium) were set up in every experiment with and without excess unlabeled Ab (a final concentration of 100 g/ml). After the binding incubation, unbound radioactivity was removed by washing. The specificity controls were counted. In all experiments, the binding of radioactivity to cells was at least 90% blocked by the unlabeled Ab. The cells were then resuspended in 30 ml of fresh medium and dispensed in a 24-well plate with 1.5 ml/well. Samples of 1.5 ml were saved for radioactivity determination, which was the initially bound cpm. The plate was incubated in an CO2 incubator. At 3, 24, 48, and 72 h, the cells were collected as follows. Cells were resuspended by repeated pipetting and transferred to conical tubes. The wells and pipette were rinsed with 1 ml fresh culture medium, which was added to the initial cell suspension collected. The tube was centrifuged for 10 min at 600�g and 1 ml of supernatant was carefully collected (40% of the total supernatant) and counted for radioactivity. BSA was added as carrier protein to a final concentration of 1% and the protein was precipitated with 5 ml of cold 10% (w/v) trichloroacetic acid (TCA). After incubation for 30 min at 4� C. and centrifugation for 15 min at 5000 xg, the supernatant was discarded and the precipitated protein was counted for radioactivity. The radiolabeled protein that was not precipitated by TCA was considered degraded, and precipitated radioactive protein was considered intact. The cell pellet was counted for the radioactivity remaining in the cells after being washed. Radioactivity in each fraction was expressed as a percentage of that initially bound. As shown in FIG. 8A, hLL1 showed similar rapid internalizing and catabolic manner as murine LL1 after bound to the surface of Raji cells, i.e. almost all of the bound radioactivity was catabolized and released into the supernatant within 3 h. This is much faster than with other internalizing Abs, such as anti-CD22 and anti-CD19 (Hansen et al., 1996). The studies with early time points confirmed the similar processing patterns of hLL1 and mLL1. Most catabolism was accomplished within one hour (FIG. 8B).
Example 9 Cytotoxicity of hLL1 The cytotoxic effect of hLL1 was compared with that of mLL1 and cLL1 in Raji cells, a human lymphoma cell line. Goat anti-human IgG Fc fragment specific Ab (α-hFc) was used as the crosslinker for hLL1 and cLL1 and goat anti-mouse IgG Fc specific Ab (α-mFc) was used for mLL1.5�105 Raji cells were seeded at day 0 in 1 ml of culture medium containing 5 g/ml of a LL1 Ab and 50 g/ml of the appropriate crosslinker. The numbers of total and viable cells were counted daily for 3 days. As shown in FIG. 9, The total number of normal Raji cells increased 4-5 fold in 3 days and cell viability remained >80% at the end of third day. Cells treated with a crosslinker alone, a LL1 Ab alone, or a LL1 Ab with an uncomparable crosslinker (e.g. hLL1 and goat anti-mouse IgG Fc specific Ab), were indistinguishable from normal Raji cells. However, a combination of hLL1 and anti-human IgG Fc specific Ab effectively caused cell death: >40% reduction in cell viability in one day and almost total cell death in 3 days. The effectiveness of hLL1 was comparable with that of mLL1 and cLL1. Similar results were observed when Daudi cells were used (FIG. 10). No such effect was observed with another internalizing Ab, hLL2, (humanized anti-CD22 Ab). These results demonstrated that the cytotoxicity effect of hLL1 on lymphoma cell lines is specifically dependent on crosslinking of the Ab on cell surface.
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C07K16/2833, C07K2317/77, C07K2317/24, A61K51/1027, A61K47/48561European ClassificationA61K47/48T4B28, A61K51/10B28, C07K16/28Z, A61K45/06, A61K38/19, A61K38/21, A61K38/20, A61K38/18A, A61K38/21B, A61K38/21A, A61K38/19A, A61K38/21C, A61K38/32, A61K38/19B, C07K16/28A28Legal EventsDateCodeEventDescriptionJun 17, 2011FPAYFee paymentYear of fee payment: 4Apr 29, 2008CCCertificate of correctionSep 22, 2004ASAssignmentOwner name: IMMUNOMEDICS, INC., NEW JERSEYFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HANSEN, HANS J.;LEUNG, SHUI-ON;QU, ZHENGXING;AND OTHERS;REEL/FRAME:015848/0828;SIGNING DATES FROM 20040628 TO 20040715RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google