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Anti cd37 antibodies

a technology of anti-cd37 antibodies and antibodies, applied in the field of immunotherapies, to achieve the effect of exquisite antigen specificity

Inactive Publication Date: 2011-07-07
BOEHRINGER INGELHEIM INT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0003]CD37, a member of the tetraspanin superfamily, is a heavily glycosylated cell surface molecule with four transmembrane domains and two extracellular loops. CD37 is almost exclusively expressed on mature B cells, with highest expression levels on peripheral blood B cells, reduced levels on plasma cells and non-detectable levels on CD10+ precursor B cells in the bone marrow. Low level expression of CD37 has also been reported on resting and activated T cells, granulocytes, and monocytes. In B cell neoplasm, CD37 expression is mainly observed in aggressive non-Hodgkin's lymphoma (NHL) and chronic lymphoid leukemia (CLL). High level of CD37 expression is also found on mantle cell lymphoma (MCL). This expression pattern makes CD37 an attractive target for antibody-mediated cancer therapy.
[0084]Representatives of CD37 antibodies of the invention show potent pro-apoptotic activity without cross-linking; in this respect, antibodies with this property are superior to the anti-CD37 SMIP Tru16.4, which does not show apoptosis without cross-linking (Zhao et al., 2007). Induction of apoptosis without cross-linking, which could be shown for antibodies of the invention both with and without Fc engineering, is advantageous in the absence of a cross-linking agent in vivo (e.g. effector cells harboring Fcγ receptors) or at low density of the target antigen CD37 (e.g. tumor cells with low level expression of CD37). An antibody which induces apoptosis without cross-linking may still cause cell death, whereas an antibody dependent on cross-linking does not.
[0099]Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. The expression vector and expression control sequences are selected to be compatible with the host cell. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector. Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above. The constant chain is usually kappa or lambda for the antibody light chain, for the antibody heavy chain, it can be, without limitation, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.
[0113]An anti-CD37 antibody molecule may also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g. to increase serum half-life or to increase tissue binding.

Problems solved by technology

However, only a subset of patients respond to therapy and the majority of those eventually relapse following rituximab treatment.
Third, the antibody may alter the ability of B cells to respond to antigen or other stimuli.

Method used

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Examples

Experimental program
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Effect test

example 2

[0190]Chimeric mAb A0 Specifically Recognizes the CD37 Antigen

[0191]The specificity of MAb A0 for cellular CD37 is tested in a FACS competition assay on Ramos Burkitt lymphoma cells (ATCC #CRL-1596). Cells are grown in tissue culture flasks (175 cm2) using RPMI-1640+GlutaMAX supplemented with 10% heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM non-essential amino acids as culture medium. Cells are cultivated with an initial density of 3×105 cells / ml at 37° C. and 5% CO2 in a humidified atmosphere for three days. The cultures are maintained at a cell concentration between 3×105 and 1.8×106 / ml by sub-cultivation in a ratio of 1:6 with fresh culture medium 2-3 times a week. For FACS competition analysis, the CD37-specific mAb HH1 (Santa Cruz) directly labeled with phycoerythrin (PE) is used at a concentration of 1 μg / ml. The antibody is preincubated with the unlabelled competitor antibody A0 for 10 min at 4° C. at the indicated molar ratio. Thereafter, 1...

example 3

[0192]Binding of Humanized Versions of mAb A0 to Cellular CD37 Antigen

[0193]Humanized versions of A0 are tested for their binding to cellular CD37 antigen by FACS analysis. Antibodies are added to Ramos cells at the indicated concentrations and allowed for binding for 30 min at 4° C. Thereafter, bound antibody is detected with PE-labelled goat-anti-human IgG antibody (Sigma), cells are washed twice with PBS, and thereafter cells are resuspended in FACS buffer and analyzed by FACS on a BD FACS Canto. Examples are shown in FIGS. 2 and 3 (antibodies A, B, C, D, I or A, H, I, J, K, L and M, respectively; see Table 1). Several of the humanized versions of A0 show similar binding to Ramos cells as the parental antibody A0, indicating that humanization does not reduce binding to cellular CD37 antigen.

example 4

[0194]FACS Scatchard Analysis of Humanized Versions of Chimeric mAb A0

[0195]The affinity of humanized versions of antibody A0 (designated B, C, D, H, I and K; see Table 1) to cellular CD37 antigen is determined by FACS scatchard analysis as described elsewhere (Brockhoff et al., 1994). Briefly, antibody dilutions are prepared in a 96 well plate starting with 100-400 nM in the first well (80 μl), followed by 11 dilution steps (1:2, 40+40 μl). 50 μl of mAb dilutions are added to FACS tubes, 150 μl cells (0.8×106 / ml=1.2×105 cells / tube) are added to each FACS tube. Cells are gently mixed and incubated for 1 h on ice. Thereafter 50 μl FITC conjugated secondary antibody (conc.15 μg / ml; mouse mAb anti-hu IgG all subclasses, Zymed 05-4211) is added, mixed, and incubated for 30 min on ice. 4 ml PBS ph7.2 containing 0.02% acid are added thereafter, cells are pelleted and resuspended in 300 μl PBS pH 7.2 and subjected to FACS analysis using a BD FACS Canto. All experimental steps are performed...

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Abstract

Chimeric and humanized anti-CD37 antibodies and pharmaceutical compositions containing them are useful for the treatment of B cell malignancies and autoimmune and inflammatory diseases that involve B cells in their pathology.

Description

INTRODUCTION [0001]The present invention relates to immunotherapies that are based on B cell depletion. In particular, the present invention relates to anti-CD37 antibody molecules for use in such therapies, e.g. in the treatment of B cell malignancies and autoimmune conditions.[0002]Immunotherapy using monoclonal antibodies (mAbs) has been emerging as a safe and selective method for the treatment of cancer and other diseases. In particular, the role of monoclonal antibodies in therapies that are based on B cell depletion, e.g. in the treatment of B cell malignancies, has expanded since the introduction of rituximab (Rituxan®), an antibody that is directed against the CD20 antigen on the B cell surface. Numerous studies have confirmed the efficacy of rituximab as a single agent and in combination therapy in low-grade NHL (Hiddemann et al., 2005a; Hiddemann et al., 2005b; Hainsworth 2004; McLaughlin et al., 1998), mantle cell lymphoma (Forstpointner et al., 2004; Kahl et al., 2006; F...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/32C07H21/04C12N15/63C12N5/10C12N1/00C12P21/02C12N5/0781A61P35/00A61P35/02
CPCA01K67/0275A01K2207/15A01K2217/072A01K2227/105A61K2039/505C07K16/2896C07K16/3061C07K2317/24C07K2317/72C07K2317/92A61K39/39558A61K45/06C07K2316/96C07K2317/732C07K2317/73A61P29/00A61P35/00A61P35/02A61P37/00A61P37/02A61P43/00C07K16/28C07K2317/76
Inventor HEIDER, KARL-HEINZBORGES, ERICOSTERMANN, ELINBORG
Owner BOEHRINGER INGELHEIM INT GMBH
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