Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/72—Increased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin

Definitions

• ALL acute lymphoblastic leukemia
• CLL chronic lymphocytic leukemia
• NHL non-Hodgkin lymphoma
• mAbs monoclonal antibodies
• ADCC antibody-dependent cellular cytotoxicity
• Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen that is expressed by early pre-B cells from the time of heavy chain
• CD19 is a positive regulator of B cell- signaling that modulates the threshold for B cell activation and humoral immunity.
• CD19 is not expressed on hematopoietic stem cells or on B cells before the pro-B-cel stage.
• Figure 1 shows ADCC activity of the afucosylated anti-CD19 mAb 16C4 (16C4- afuc) and the anti-CD20 mAb, rituximab, against B cell leukemia and lymphoma cell lines. Results from in vitro ADCC assays with four cell lines are shown, which are representative fin: the activity profiles observed across a panel of 15 leukemia and lymphoma lines. The fucosylated CD19 mAb 16C4 was included for comparison. The ADCC activities of 16C4-afuc and rituximab were comparable when tested against Karpas-1106P cells (Fig. 1A); similar results were obtained with the cell lines Farage, Raji and MeC2. JVM2 (Fig.
• 1F- H shows relative expression of CD19 and CD20 (expressed as MFI; see Table I in Example 2) plotted against the % maximal cell kill observed (CD19, Fig, IF; CD20, Fig. 1G) and against the EC50 values (CD19, Fig. 1H; CD20, Fig. II) determined for 16C4- afuc and rituximab.
• Cell line/mAb combinations for which an EC50 could not be determined were excluded (see also Table I in Example 2). Across this diverse panel of cell lines no significant correlation of EC50 or maximal cell killing with antigen expression could be determined.
• FIG. 3 shows that patient-derived acute lymphoblastic leukemia (ALL) cells are sensitive to 16C4-afuc mediated ADCC.
• Fig.3 A illustrates expression of CD19 and CD20 on patient ALL cells. The number of antigenic sites for CD19 and CD20 was determined for three individual ALL samples. For comparison, the number of antigenic sites on normal human peripheral blood B cells from four individual donors is shown.
• 16C4-afuc has potent in vitro ADCC activity against primary ALL cells. The results from FACS-based assays with samples from four patients are shown. KC1333 NK cells were used as effector cells at an E:T ratio of 2.5:1;
• rituximab was included for comparison. All measurements were performed in triplicate with mean values (+/- standard deviation) presented.
• rituximab or isotype control (3 mg/kg) started on day 7 post cell injection and continued for 5 doses.
• survival time or the time to paralysis was used as endpoint.
• Multiple parameters can be indicative of treatment efficacy, e.g anti-tumor activity. These include, but are not limited to, a reduction in the size of the tumor mass; a reduction in metastatic invasiveness of the tumor; a reduction in the rate of tumor growth; a decrease in severity or incidence of tumor-related sequelae such as cachexia and ascites production; a decrease and/or prevention of tumor-related complications such as pathologic bone fractures, autoimmune hemolytic anemia, prolymphocytic
• the combined effect of two or more agents results in “synergistic inhibition" of an activity or process, for example, tumor growth
• the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent
• the term "synergistic therapeutic effect” refers to a therapeutic effect observed with a combination of two or more therapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.
• synergistic effect means that the effect observed when employing a combination of a CD19 antibody and a CD20 antibody administered on a comparable dosing schedule or regime is (1) greater than the effect achieved when that CD19 antibody or CD20 antibody is employed alone (or individually) and (2) greater than the sum added (additive) effect for that CD19 antibody and CD20 antibody.
• comparable dosing schedule refers to a dosing schedule or regime that is used to evaluate or compare the results of at least two different treatments and as such is designed to be the same as between the treatments being compared, i.e. patients are being dosed in the same way (e.g., day, time between dosing, concentration of antibody agent) but with a different antibody or combination therapy.
• the variables of the dosing schedule will be determined by one of skill in the art depending on the B cell malignancy being treated and choice of treatment
• Such synergy or synergistic effect can be determined by way of a variety of means known to those in the art.
• the synergistic effect of a CD19 antibody and a CD20 antibody can be observed using in vitro or in vivo assay formats examining reduction of tumor cell number or tumor size, or by inhibition of tumor growth or a depletion of tumor cells.
• a synergistically effective amount of each individual component may be determined by testing a range of concentrations of each component.
• administering at the same time refers to administering the antibodies together in same formulation or in separate formulations wherein the administration may be a few minutes to a few hours apart, but no more than one day.
• administering at different times refers to administering the antibodies of the combination therapy a few hours to days, weeks and even months apart.
• the two antibodies are administered simultaneously, they can be administered as separate pharmaceutical compositions, each comprising either the anti- CD20 antibody (or antigen-binding fragment thereof) or the anti-CD19 antibody (or antigen-binding fragment thereof), or can be administered as a single pharmaceutical composition comprising both of these antibodies.
• the methods of the disclosure comprise using combination therapy which confers a positive therapeutic response to a subject in need thereof a treatment for B cell diseases.
• a positive therapeutic response with respect to the combination treatment using anti- CD19 and anti-CD20 antibodies is intended to mean an improvement in the disease in association with the anti-tumor activity of these antibodies or fragments thereof, and/or an improvement in the symptoms associated with the disease. That is, an antiproliferative effect, the prevention of further tumor outgrowths, a reduction in tumor size, a reduction in the number of cancer cells, and/or a decrease in one or more symptoms mediated by neoplastic B cells can be observed.
• an improvement in the disease may be characterized as a complete response.
• Tumor response can be assessed for changes in tumor morphology (e.g., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MBS) scan, x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis,
• MFS magnetic resonance imaging
• CT computed tomographic
• FACS fluorescence-activated cell sorter
• the combination therapy disclosed herein is administered at a therapeutically effective dose.
• the term ''therapeutically effective dose,” “therapeutically effective amount,” or “effective amount” is intended to be an amount of the anti-CD19 antibody (or antigen-binding fragment thereof) that, when administered in combination with an amount of the anti-CD20 antibody (or antigen-binding fragment thereof), brings about a positive therapeutic response with respect to treatment of a subject for a cancer comprising neoplastic B cells.
• a therapeutically effective dose of either the anti-CD20 antibody (or antigen-binding fragment thereof) or anti-CD19 antibody (or antigen-binding fragment thereof) is in the range from about 1 mg/kg to about 200 mg/kg. It is recognized that the method of treatment may comprise a single administration of a therapeutically effective dose of the antibody combination useful in the practice of the methods or multiple administrations of a therapeutically effective dose of the antibody combination.
• the combination therapy with anti-CD19 antibody and an anti-CD20 antibody provides prolonged anti-tumor activity relative to treatments involving either an anti-CD19 antibody alone or an anti-CD20 antibody alone.
• a combination therapy may provide anti-tumor activity that lasts for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 2 years longer than the anti-tumor activity obtainable with either an anti-CD19 antibody alone or an anti-CD20 antibody alone.
• the relative duration of antitumor activity may be determined based on statistical analysis of a test population.
• an anti-CD19 antibody exhibits a certain level of anti-tumor activity for a mean of 6 weeks in a test population and an anti-CD20 antibody exhibits a certain level of anti-tumor activity for a mean of 8 weeks in a test population
• the combination exhibits anti-tumor activity that is at least 4 weeks longer than either antibody therapy administered alone if the combination therapy exhibits at least the same level of antitumor activity as that seen for the single antibody therapies for a mean of at least 12 weeks in a test population.
• tumors present in the peripheral lymph nods can be obtained by body scans, using instruments described above, that provide diameter or estimated tumor volume measurements ⁇ Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition). Vol 22, No 14S (July 15 Supplement), 2004: 6606)). Therefore, the presently disclosed combination therapy exhibits anti-tumor activity when there is a lack of an increase in either a diameter measurement of a tumor or in an estimated tumor volume measurement. In another embodiment, anti-tumor activity may be reflected in an actual decrease in tumor size, or maintenance of a tumor at a fixed size over a period of time.
• anti-tumor activity may be determined by inhibition of tumor growth of more than a 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25% or 30% relative to the size of tumor before treatment, e.g. diameter measurements or estimated tumor volume measurements.
• anti-tumor activity may be measured by determining the level of B cell depletion obtained with a given therapy. Circulating B cells, including malignant B cells, are most easily measured by flow cytomery, or other cell counting devices described above and well known in the art, resulting in a count number of circulating B cells. It is further contemplated that any method providing for a number of circulating B cells can be used to determine the depletion of B cells after treatment with the combination therapy.
• the combination therapy described herein may be achieved by various means of administration.
• the anti-CD19 antibody and the anti-CD20 antibody may be separately formulated and administered to the patient Alternatively, the anti-CD19 and anti-CD20 antibody may be formulated together in a single formulation.
• the antibodies may be administered on the same or different dosing schedules.
• Suitable anti-CD19 antibodies include, for example, known anti-CD19 antibodies, commercially available anti-CD19 antibodies, or anti- CD19 antibodies developed using methods well known in the art.
• antibody and “antibodies”, also known as immunoglobulins, encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab”)2 fragments, antibody fragments that exhibit the desired biological activity (e.g.
• antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain at least one antigen-binding site.
• an anti-CD19 antibody used in the compositions and methods of the disclosure may be a human, humanized or chimeric antibody having an IgG isotype, particularly an IgGl , IgG2, IgG3, or IgG4 human isotype or any IgGl ,
• IgG2, IgG3, or IgG4 allele found in the human population.
• Antibodies of the human IgG class have functional characteristics such as a long half-life in serum and the ability to mediate various effector functions (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)).
• the human IgG class antibody is further classified into the following 4 subclasses: IgGl, IgG2, IgG3 and IgG4.
• an anti-CD19 antibody of the disclosure is any of the anti-CD19 antibodies described in U.S. Publication Nos. 2008/0138336 and 2009/0142349 and US Patent Nos. 7,462,352 and 7,109,304.
• an anti-CD19 antibody is the 16C4 antibody, or an antigen binding fragment thereof, as described in U.S. Publication No.2008/0138336 and below.
• Kd dissociation constant
• anti-CD19 antibodies for use in compositions and methods of the disclosure may be able to reduce or deplete B cells in a human treated therewith.
• Depletion of B cells can be in circulating B cells, or in particular tissues such as, but not limited to, bone marrow, spleen, gut-associated lymphoid tissues, and/or lymph nodes.
• anti-CD19 antibody of the disclosure may deplete circulating B cells, blood B cells, splenic B cells, marginal zone B cells, follicular B cells, peritoneal B cells, and/or bone marrow B cells.
• an anti-CD19 antibody of the disclosure may achieve depletion of progenitor B cells, early pro-B cells, late pro-B cells, large-pre-B cells, small pre-B cells, immature B cells, mature B cells, antigen stimulated B cells, and/or plasma cells.
• depletion may be achieved via various mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), and/or by blocking of CD19 interaction with its intended ligand, and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g., via apoptosis).
• ADCC antibody-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• B cell depletion by an anti-CD19 antibody of the disclosure may persist for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months or at least 12 months.
• B cell malignancies are characterized by the pathological expansion of specific B cell subsets, for example, precursor B cell acute lymphoblastic leukemia is characterized by an abnormal expansion of B cells corresponding to pro-B cell/ Pre-B cell
• an anti-CD19 antibody may therefore deplete malignant B cells in a human subject
• an anti-CD19 antibody of the disclosure may achieve at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% depletion of malignant B cells in a human subject
• anti-CD19 antibodies are modified with respect to effector function, so as to enhance the effectiveness of the antibody in treating B cell
• Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N- acetylglucosaminyltransferase ⁇ (GnTIl 1), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed.
• one or more enzymes for example DI N- acetylglucosaminyltransferase ⁇ (GnTIl 1)
• An antibody can also be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
• Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. BioL Chem.
• an anti-CD19 antibody of the disclosure comprises a variant
• useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
• PBMC peripheral blood mononuclear cells
• NK Natural Killer
• ADCC activity of the molecules of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. (Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998)).
• the assay may also be performed using a commercially available kit, e.g. CytoTox 96TM (Promega).
• Exemplary Anti-CD19 Antibodies In certain embodiments, the methods and compositions described herein utilize the anti-CD19 antibody 16C4 (see e.g., U.S. Publication No.
• 16C4 is a CD19 mAb that has been shown to have potent ADCC effector function. 16C4 is the afucosylated form of the CD19 mAb anti-CD19-2, which was developed by humanization and affinity optimization of the HB12b mAb (Kansas GS and Tedder TF. J Immunol, 1991; 147:4094-4102; Yazawa et al., Proc Natl AcadSci, 2005; 102(42): 15178-15183; Herbst et al., J Pharmacol Exp Ther, 2010, 335(l):213-222).
• an anti-CD19 antibody of the disclosure comprises a heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:2, a CDR2 comprising the amino acid sequence of SEQ ID NO:3, and a CDR3 comprising the amino acid sequence of SEQ ID NO:4.
• an anti-CD19 antibody of the disclosure comprises a heavy chain comprising a CDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:2, a CDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity whith SEQ ID NO:3, and a CDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:4.
• an anti-CD19 antibody of the disclosure comprises a heavy chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 1.
• an anti- CD ⁇ antibody comprises a heavy chain comprising a variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 1.
• an anti-CD19 antibody of the disclosure comprises a light chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8.
• an anti-CD19 antibody of the disclosure comprises a light chain comprising a variable region comprising the amino acid sequence of SEQ ID NO:5.
• an anti- CD19 antibody comprises a light chain comprising a variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:5.
• an anti-CD19 antibody of the disclosure comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:8.
• the VH and/or VK CDRs derivatives may include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less man 4 amino acid substitutions, less man 3 amino acid substitutions, less than 2 amino acid substitutions, or 1 amino acid substitution relative to the original VH and/or VK CDRs of the 16C4 anti-CD19 antibody.
• the VH and/or VK CDRs derivatives may have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues (e.g., amino acid residues which are not critical for the antibody to specifically bind to human CD19).
• Mutations can also be introduced randomly along all or part of the VH and/or VK CDR coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined. The percent identity of two amino acid sequences can be determined by any method known to one skilled in the art, including, but not limited to, BLAST protein searches.
• CD20 is present on both normal B cells as well as malignant B cells, for example the cells in B-cell non-Hodgkin's lymphoma (NHL), where CD20 is expressed on greater than 90% of NHL (Anderson et al. Blood 63(6):1424-1433 (1984)). CD20 is not found on hematopoietic stem cells, pro- B cells, normal plasma cells or other normal tissues (Tedder et al. J. Immunol.
• NHL B-cell non-Hodgkin's lymphoma
• CD20 regulates an early step(s) in the activation process for cell cycle initiation and differentiation (Tedder et al., supra) and possibly functions as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D:195 (1990)).
• Suitable anti-CD20 antibodies include, for example, known anti-CD20 antibodies, commercially available anti-CD20 antibodies, or anti- CD20 antibodies developed using methods well known in the art
• a CD20 antibody of the present disclosure may be a monoclonal human, humanized or chimeric anti-CD20 antibody.
• Anti-CD20 antibodies used in compositions and methods of the disclosure can be naked antibodies, immunoconjugates or fusion proteins.
• an anti-CD20 antibody of the disclosure may mediate human antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cell- mediated cytotoxicity (CDC), and/or apoptosis in an amount sufficient to deplete circulating B cells.
• an anti-CD20 antibody of the disclosure is an anti-CD20 antibody that has been engineered to have enhanced ADCC activity relative to the parent antibody. Methods for creating antibody variants having enhanced ADCC activity are described above.
• an anti-CD20 antibody of the disclosure is an afucosylated antibody having enhanced ADCC activity.
• an anti-CD20 antibody used in the compositions and methods of the disclosure may be a human, humanized or chimeric antibody having an IgG isotype, particularly an IgGl, IgG2, IgG3, or IgG4 human isotype or any IgGl, IgG2, IgG3, or IgG4 allele found in the human population.
• compositions of the disclosure utilize rituximab, or an antigen binding fragment thereof, in combination with an anti-CD19 antibody, or fragment thereof.
• RITUXANTM is indicated for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B-cell non-Hodgkdn's lymphoma. In vitro mechanism of action studies have
• rituximab or “RITUXANTM” herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated “C2B8" in U.S. Pat. No. 5,736,137, expressly incorporated herein by reference.
• the complete nucleic acid and amino acid sequences for the light chain variable region and the heavy chain variable region or rituximab are disclosed in U.S. Patent No. 5,736,137.
• the nucleic acid and amino acid sequences for the light chain variable region of rituximab are disclosed in Fig.4 and SEQ ID NO:6 of U.S. Patent No. 5,736,137.
• rituximab The nucleic acid and amino acid sequence for the heavy chain variable region of rituximab are disclosed in Fig. 5 and SEQ ID NO:9 of U.S. Patent No. 5,736,137.
• the nucleic acid and amino acid sequences of SEQ ID NOs: 6 and 9 and Figs.4 and 5 of U.S. Patent No.5,736,137 are expressly incorporated herein by reference.
• Rituximab may also be made by a CHO cell transfectoma comprising the vector DNA present in the E. coli host cell deposited with the American Type Culture Collection (ATCC) under accession number.69119.
• Rituximab may also be produced from hybridoma 2B8, which is deposited with the ATCC under accession number HB 11388.
• B cell malignancy includes any malignancy that is derived from a cell of the B cell lineage.
• Exemplary B cell malignancies include, but are not limited to: B cell subtype non-Hodgkin's lymphoma (NHL) including low
• grade/folhcular NHL small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL; mantle-cell lymphoma, and bulky disease NHL; Buridtt's lymphoma; multiple myeloma; pre-B acute lymphoblastic leukemia and other malignancies that derive from early B cell precursors; common acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL) including immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL; hairy cell leukemia; Null-acute lymphoblastic leukemia; Waldenstrom's Macroglobulinemia;
• DLBCL diffuse large B cell lymphoma
• GCB germinal center B cell-like
• ABSC activated B cell-like
• type 3 DLBCL type 3 DLBCL
• pro-lymphocytic leukemia light chain disease
• plasmacytoma osteosclerotic myeloma
• plasma cell leukemia monoclonal gammopathy of undetermined significance
• SMM smoldering multiple myeloma
• IM indolent multiple myeloma
• Hodgkin's lymphoma including classical and nodular lymphocyte pre-dominant type
• marginal-zone lymphoma including gastric mucosal-associated lymphoid tissue (MALT) lymphoma. Treatment of relapses of these cancers is also contemplated.
• LPHD is a type of
• CLL Hodgkin's disease that tends to relapse f equently despite radiation or chemotherapy treatment and is characterized by CD20-positive malignant cells.
• CLL is one of four major types of leukemia A cancer of mature B-cells called lymphocytes, CLL is manifested by progressive accumulation of cells in blood, bone marrow and lymphatic tissues.
• Indolent lymphoma is a slow-growing, incurable disease in which the average patient survives between six and 10 years following numerous periods of remission and relapse.
• the desired level of B cell depletion will depend on the disease. For the treatment of a B cell malignancy, it may be desirable to maximize the depletion of the B cells which are the target of the anti-CD19 and anti-CD20 antibodies of the disclosure. Thus, for the treatment of a B cell neoplasm, it is desirable that the B cell depletion be sufficient to at least prevent progression of the disease which can be assessed by the physician of skill in the art, e.g., by monitoring tumor growth (size), proliferation of the cancerous cell type, metastasis, other signs and symptoms of the particular cancer.
• the B cell depletion is sufficient to prevent progression of disease for at least 2 months, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 or more months. In even more preferred embodiments, the B cell depletion is sufficient to increase the time in remission by at least 6 months, more preferably 9 months, more preferably one year, more preferably 2 years, more preferably 3 years, even more preferably 5 or more years. In a most preferred embodiment, the B cell depletion is sufficient to cure the disease. In preferred embodiments, the B cell depletion in a cancer patient is at least about 75% and more preferably, 80%, 85%, 90%, 95%, 99% and even 100% of the baseline level before treatment
• a patient is alleviated or successfully treated of a B cell neoplasm by the present methods of the disclosure if there is a measurable improvement in the symptoms or other applicable criteria after administration of the compositions of the disclosure compared to before treatment
• the effect of treatment may be apparent within 3-10 weeks after administration of the compositions of the disclosure.
• the applicable criteria for each disease will be well known to the physician of skill in the appropriate art For example, the physician can monitor the treated patient for clinical, or serologic evidence of disease such as serologic markers of disease, complete blood count including B cell count, and serum immunoglobulin levels.
• the patient may show observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (e.g., slow to some extent and preferably stop) of cancer cell infiltration into organs; inhibition (e.g., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer, reduced morbidity and mortality, and improvement in quality of life issues.
• the improvement is at least 20% over the baseline for a particular symptom or criterion taken before treatment by the methods of the disclosure, more preferably, 25- 30%, even more preferably 30-35%, most preferably 40% and above.
• Parameters can include median time to disease progression, time in remission and stable disease.
• measurable criteria may include, e.g., time to disease progression, an increase in duration of overall and/or progression-free survival.
• a bone marrow biopsy can be conducted to determine the degree of remission. Complete remission can be defined as the leukemia cells making up less than 5 percent of all cells round in a patient's bone marrow 30 days following treatment
• the invention provides pharmaceutical compositions comprising an anti-CD19 antibody, an anti-CD20 antibody, or a combination thereof and a pharmaceutically acceptable excipient
• the pharmaceutical compositions of the disclosure are used as a medicament
• an anti-CD19 antibody, an anti-CD20 antibody, or a combination thereof may be formulated with a pharmaceutically acceptable carrier, excipient or stabilizer, as pharmaceutical (therapeutic) compositions, and may be administered by a variety of methods known in the art As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
• pharmaceutically acceptable carrier means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
• Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
• Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
• Other contemplated carriers, excipients, and/or additives, which may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium and the like.
• the formulations described herein comprise an anti-CD19 antibody, an anti-CD20 antibody, or a combination thereof in a concentration resulting in a w/v appropriate for a desired dose.
• an anti-CD19 antibody or an anti-CD20 antibody is present in a formulation at a concentration of about 1 mg/ml to about 200 mg/ml, about 1 mg/ml to about 100 mg/ml, about 1 mg/ml to about SO mg/ml, or 1 mg/ml and about 25 mg/ml.
• the concentration of an anti-CD19 or anti-CD20 antibody in a formulation may vary from about 0.1 to about 100 weight %.
• the concentration of an anti-CD19 or anti-CD20 antibody is in the range of 0.003 to 1.0 molar.
• an anti-CD19 antibody and an anti-CD20 antibody are formulated together and each of the antibodies is present in a formulation at a concentration of about 1 mg/ml to about 200 mg/ml, about 1 mg/ml to about 100 mg/ml, about 1 mg/ml to about 50 mg/ml, or 1 mg/ml and about 25 mg/ml.
• the concentration of each of the antibodies in the formulation may vary from about 0.1 to about 100 weight %. In certain embodiments, the concentration of each of the antibodies is in the range of 0.003 to 1.0 molar.
• the endotoxin and pyrogen levels in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg.
• the formulations of the disclosure When used for in vivo administration, the formulations of the disclosure should be sterile.
• the formulations of the disclosure may be sterilized by various sterilization methods, including sterile filtration, radiation, etc.
• the formulation is filter-sterilized with a presterilized 0.22-micron filter.
• Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy", 21*ed., Lippincott Williams & Wilkins, (200S).
• compositions of the present disclosure can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
• routes of administration such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
• parenteral administration and “administered parenteralry” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
• Fonnulations of the present disclosure which are suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the antibody(ies) may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellents which may be required (US Patent No. 7,378, 110; 7,258,873; 7,135,180; US PubhcationNo.2004-0042972; and 2004-0042971).
• compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (e.g., "a therapeutically effective amount").
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• Suitable dosages may range from about 0.0001 to about 100 mg/kg of body weight or greater, for example about 0.1, 1, 10, or 50 mg/kg of body weight, with about 1 to about 10 mg/kg of body weight being preferred.
• the method comprises administration of multiple doses of anti- CD20 antibody (or antigen-binding fragment thereof) in combination with multiple doses of anti-CD19 antibody (or antigen-binding fragment thereof).
• the method may comprise administration of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective doses of a pharmaceutical composition comprising either anti-CD20 antibody (or antigen-binding fragment thereof) or anti-CD19 antibody (or antigen-binding fragment thereof), or both.
• the frequency and duration of administration of multiple doses of the pharmaceutical compositions can be readily determined by one of skill in the art without undue experimentation.
• the dosing regimen includes administration of a therapeutically effective dose of the anti-CD20 antibody (or antigen-binding fragment thereof) in combination with a therapeutically effective dose of the anti-CD19 antibody (or antigen-binding fragment thereof), where the combination is administered on days 1, 8, IS, and 22 of a treatment period.
• Further embodiments include a dosing regimen where a therapeutically effective dose of the anti-CD20 antibody (or antigen-binding fragment thereof) is administered in combination with a therapeutically effective dose of the anti- CD19 antibody (or antigen-binding fragments thereof), where the combination is administered on days 1, 3, 5, and 7 of a week in a treatment period; a dosing regimen that includes administration of a therapeutically effective dose of the anti-CD20 antibody (or antigen-binding fragment thereof) in combination with a therapeutically effective dose of the anti-CD19 antibody (or antigen-binding fragment thereof), where the combination of antibodies is administered on days 1 and 3 of a week in a treatment period; and a preferred dosing regimen that includes administration of a therapeutically effective dose of the anti-CD20 antibody (or antigen-binding fragment thereof) in combination with the anti-CD19 antibody (or antigen-binding fragments thereof) on day 1 of any given week in a treatment period.
• the treatment period may comprise 1 week, 2 weeks, 3 weeks, a month, 3 months, 6 months, or a year. Treatment periods may be subsequent or separated from each other by a day, a week, 2 weeks, a month, 3 months, 6 months, or a year.
• Treatment using a combination of anti-CD19 antibody (or antigen-binding fragment thereof) and anti-CD20 antibody (or antigen-binding fragment thereof) may comprise administration of one or both antibodies simultaneously or concurrently, as long as the treatment includes the combination of anti-CD20 antibody (or antigen-binding fragment thereof) and anti-CD19 antibody (or antigen-binding fragment thereof) at some point during treatment.
• the effect of the combination therapy can also be optimized by varying the liming of administration of either the anti-CD20 antibody and/or the anti-CD19 antibody treatment
• Treatment with an anti-CD20 antibody or antigen-binding fragment thereof in combination with an anti-CD19 antibody or antigen-binding fragment thereof can be simultaneous (concurrent), consecutive (sequential), or a combination thereof. Therefore, a subject undergoing combination antibody therapy can receive both the anti- CD20 antibody (or antigen-binding fragment thereof) and anti-CD19 (or antigen-binding fragment thereof) at the same time (e.g., simultaneously) or at different times (e.g., sequentially, in either order, on the same day, or on different days).
• the anti-CD20 antibody such as Rituximab (or antigen-binding fragment thereof) is administered simultaneously with the anti-CD19 antibody, such as the 16C4 (or antigen-binding fragment thereof).
• the anti-CD20 antibody, such as Rituximab (or antigen-binding fragment thereof) is administered first and then the anti-CD19 antibody, such as 16C4 (or antigen-binding fragment thereof) is adrninistered next.
• the anti-CD19 antibody, such as 16C4 (or antigen- binding fragment thereof) is adrninistered first, and the anti-CD20 antibody, such as Rituximab (or antigen-binding fragment thereof) is administered next.
• the combination of anti-CD20 antibodies and anti-CD19 antibodies is given concurrently for one dosing, but other closings include sequential administration, in either order, on the same day, or on different days.
• the anti-CD20 antibody such as Rituximab and the anti-CD19 antibody such as 16C4 are administered simultaneously, they can be administered as separate pharmaceutical compositions, each comprising either the anti-CD20 antibody (or antigen-binding fragment thereof) or the anti-CD19 antibody (or antigen-binding fragment thereof), or can be administered as a single pharmaceutical composition comprising both of these anti-cancer agents.
• Example 1 Anti-CD19 mAb 16C4-afuc has potent in vitro ADCC activity against multiple B leukemia and lymphoma cell lines
• 16C4-afuc Compared to the fucosylated 16C4 mAb, 16C4-afuc has ⁇ 9-fold increased affinity to the activating human Fc ⁇ RIIIA and mouse Fc ⁇ RTV and enhanced ADCC effector function. In contrast to rituximab, 16C4 does not mediate CDC. (Herbst et al., J Pharmacol Exp Ther, 2010, 335(l):213-222.)
• ADCC activity of 16C4-afuc was compared with that of the fucosylated precursor, mAb 16C4, in a large panel of B leukemia and lymphoma cell lines.
• the CD20 mAb rituximab was included in all assays as a positive control. With all cell lines tested, 16C4-afuc was significantly more potent in mediating ADCC than the parental mAb anti-CD19 mAb (Figs. 1A-1E).
• the cell lines were also analyzed for their relative expression of CD19 and CD20 to determine whether the surface levels of the two antigens determines their in vitro sensitivity to CD19 and CD20 mAbs, respectively (Table I).
• Fig. IB the ADCC activities observed with 16C4-afuc and rituximab maximal percentage of cytotoxicity, Fig. 1 E for CD19 mAb and Fig. IF for CD20 mAb; EC50 values, Fig. 1G for CD19 mAb and Fig. 1H for CD20 mAb) are plotted against the relative surface expression of CD19 and CD20, as determined by flow cytometry with the mAbs 16C4 and rituximab as the primary antibodies for detection.
• Example 2 16C4-afuc is effective against patient derived CLL and ALL cells in vitro
• CD19 mAb against primary leukemic cells Given the activity of 16C4-afuc against B cell lines, the effects of CD19 mAb against primary leukemic cells were also examined.
• Six PBMC samples were obtained from patients diagnosed with CLL and the surface antigen densities for CD19 and CD20 were determined. As shown in Fig.2A, B cells in these samples expressed CD19 and CD20 to varying degrees. In some of these samples the number of CD20 antigenic sites was greater than the number of CD19 sites.
• An in vitro FACS-based cytotoxicity assay was used to evaluate the ability of 16C4-afuc to kill B cells in the CLL samples, with rituximab as a positive control.
• Figs.2B-2D shows results from ADCC assays with 16C4-afuc and rituximab for three representative CLL samples (CLL#106, Fig.2B; CLL #104, Fig. 2C; CLL #107, Fig. 2D).
• the ECso values for 16C4-afuc ranged from 0.007 nM to 0.063 nM
• the ECso values for rituximab ranged from 0.639 nM to 0.682 nM.
• the sensitivity of the CLL cells to ADCC mediated by 16C4-afuc and rituximab was compared to their surface expression of CD19 and CD20, respectively (Figs.
• results from this analysis show a clear trend towards more efficient cell killing with increasing antigen density for both CD19 and CD20.
• the results with these primary CLL samples also show that 16C4-afuc is more effective than rituximab in mediating depletion in vitro at relatively low levels of surface antigen expression.
• the activity of 16C4-afuc was also tested in FACS-based ADCC assays with PBMC samples from four patients with ALL. For three of these assays, there were sufficient cell numbers to determine antigen densities for CD19 and CD20, in comparison to B cells from four healthy donors (Fig. 3A). For normal peripheral blood B cells, the average density of CD19 and CD20 was determined to be -20,000 and -200,000 antigenic sites per cell, respectively. Compared to normal B cells, CD19 expression was somewhat less in two and increased by about two-fold in the third ALL sample (Fig. 3A). The number of CD20 antigenic sites, however, varied more broadly. In the ADCC B-cell depletion assays for samples from donors with ALL (Figs. 3B-3E), the ECso values with 16C4-afuc ranged from 0.002 nM to 0.131 nM. These values were 116 to less than 1/100 of ECso values obtained with rituximab.
• Example 3 16C4-afuc inhibits tumor growth in SCID-lymphoma models by an Fc- dependent mechanism
• 16C4-afuc was tested.
• the antitumor efficacy of 16C4 was evaluated in multiple human CD19+ lymphoma xenografts grown in SCTD mice.
• mAbs against CD19 have anti-proliferative activity.
• the mAb 16C4 was shown to inhibit proliferation of transformed B cell lines as well as primary B cell from healthy donors.
• the efficacy of 16C4-afuc was compared to mAb 16C4-TM, a version of the CD19 mAb engineered for the elimination of Fc- mediated effector function.
• Example 4 16C4-afuc inhibits tumor growth in multiple SOD mouse models of human B cell lymphoma
• the doses of 16C4-afuc required to suppress the growth of tumors in mouse models were determined.
• a range of mAb doses and administration schedules were tested in the SCID/Raji s.c. xenograft model.
• the mAb dose range included 0.3, 1, 3, and 10 mg/kg 16C4-afuc.
• the dosing schedule variations included 1, 3, and 5 doses, with the first dose given on day 5 after cell implantation (Fig. 5).
• the in vivo efficacy of 16C4-afuc was dose and schedule dependent In the model presented here, five doses of 16C4-afuc (Fig. SB) resulted in stronger antitumor activity than 3 doses (Fig. SA).
• Fig. 6 shows the results from tumor models with Namalwa (Fig. 6 A), Daudi (Fig. 6B), and Toledo cells (Fig. 6G.
• Namalwa tumors responded poorly to rituximab treatment
• Daudi xenografts were inhibited in their growth somewhat better with the CD20 mAb than with 16C4-afuc.
• the two mAbs showed comparable efficacy.
• the effect of the mAb combination was less pronounced in the Oci-LY19 s.c. xenograft model (Fig. 7B), which may have been the result of the poor activity of rituximab in this model.
• the CD19 mAb showed good efficacy, and the combination of CD19 mAb with rituximab resulted in greater tumor suppression.
• the effect of the mAb combination was less pronounced in the Ramos xenograft model (Fig. 7D), which may be been the result of the relatively poor activity of 16C4-afuc in this model.
• 16C4-afuc (1 mg/kg) + control (e.g., no rituximab); 16C4-afuc (l0mg/kg) + control; 16C4-afuc (lmg/kg) + rituximab (10 mg/kg); 16C4-afuc (l0mg/kg) + rituximab (10 mg/kg).
• Fig. 8 A shows the pharmacokinetic results of the experiment. Whether administered alone or with rituximab, the higher dose of 16C4-afuc (l0mg/kg) was retained for longer in the blood than the lower dose of 16C4-afuc (lmg/kg).
• Example 7 B cell depletion in huCD19/CD20 transgenic mice
• the human B leukemia and lymphoma cell lines Raji, Daudi, Ramos, Namalwa, Toledo, Farage, and RL were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
• the KC1333 NK cell line (expressing human CD16) was obtained from BioWa Inc. (Princeton, NJ).
• the mouse IgGl mAb HB12b (Kansas and Tedder, J Immunol, 1991; 147:4094-4102), which recognizes human CD19, was humanized and affinity optimized, resulting in mAb 16C4.
• the humanized IgGl mAb 16C4 was expressed in a fiicosyltransferase-deficient producer CHO cell line (BioWa Potelligent® Technology, BioWa Inc.; Princeton, NJ) to generate 16C4-afuc.
• the antigen densities of CD19 and CD20 on B cells from frozen PBMC from donors with CLL or ALL and PBMC samples from healthy adult donors were determined by flow cytometry using QIFIKIT® (Dako, Glostrp, Denmark) manufacturer's instructions with anti-CD19 clone HD37 and anti-CD20 clone 2H7 as the primary antibodies.
• SCID immunodeficient-lymphoma xenograft models.
• mice Four to six week old female CB17-SCID mice were bred at Taconic Farms (Germantown, NY, USA) and maintained in the Laboratory Animal Resources facility at MedImmune. All mouse experiments were conducted in accordance with IACUC approved protocols. The studies were carried out using both the localized and disseminated SCID-lymphoma xenograft models.
• mice cohorts of 10 were inoculated with SxlO6 tumor cells on day 0. Mice were treated with mAbs or vehicle on day 5 or 7, as indicated, by intraperitoneal (i.p.) injection.

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