HK1191350A - Anti cd37 antibodies - Google Patents

Description

anti-CD 37 antibodies

The application is a divisional application of an invention application with the application date of 2008/8, the Chinese application number of 200880102642.5 and the invention name of "anti-CD 37 antibody".

Introduction to

The present invention relates to B cell depletion (B cell depletion) based immunotherapy. In particular, the invention relates to anti-CD 37 antibody molecules for use in such therapies, e.g., for the treatment of B cell malignancies and autoimmune diseases.

Immunotherapy using monoclonal antibodies (mabs) has emerged as a safe and selective method for treating cancer and other diseases. In particular, rituximab (rituximab) was introduced by itself(antibodies directed against the CD20 antigen on the surface of B cells) the role of monoclonal antibodies in B cell ablation-based therapies (e.g., treatment of B cell malignancies) has been expanded. A number of studies have demonstrated that rituximab in the form of single agents and combination therapies is indicated in mild NHL (Hiddemann et al, 2005 a; Hiddemann et al, 2005 b; Hainsworth 2004; McLaughlin et al, 1998), mantle cell lymphoma (Forstpointner et al, 2004; Kahl et al, 2006; Foran et al, 2000; Howard et al, 2002; Romaguera et al, 2005), Diffuse Large Cell Lymphoma (DLCL) (Coiffier et al, 1998; Feugier et al, 2005) and Burkitt (Burkitt) leukemia/lymphoma(Thomas et al, 2006). However, only a fraction of patients respond to therapy and most of them eventually relapse after rituximab treatment. Thus, new B cell therapeutic targets have been sought that are potentially more effective than CD20 for B cell malignancy therapy (Zhao et al, 2007). The CD37 antigen is a cell surface antigen that has not heretofore been considered to be a target of B cell malignancies to the same extent as the B cell antigen CD 20.

CD37 is a member of the tetraspanin superfamily, a highly glycosylated cell surface molecule with four transmembrane domains and two extracellular loops. CD37 was expressed almost exclusively on mature B cells, with the highest level on peripheral blood B cells, reduced on plasma cells, and undetectable on CD10+ precursor B cells in bone marrow. Low expression of CD37 on resting and activated T cells, granulocytes, and monocytes has also been reported. In B-cell neoplasia, CD37 expression is observed primarily in aggressive non-Hodgkin's lymphoma (NHL) and Chronic Lymphocytic Leukemia (CLL). High levels of CD37 expression were also found on Mantle Cell Lymphoma (MCL). This pattern of expression makes CD37 an attractive target for antibody-mediated cancer therapy.

CD37 was first described in 1986 and was characterized by the murine monoclonal antibody MB-1(Link et al, 1986).

The physiological role of CD37 is unknown. Although mice lacking CD37 do not show changes in lymphoid development and cellular composition, they have reduced IgG1 levels and attenuated T cell-mediated immune responses (Knobeloch et al, 2000). For CD37^-/-T cell studies indicate a role for CD37 in T cell proliferation (van Spriel et al, 2004).

CD37 has been reported to be expressed on malignant B cells in various diseases. CD37 is expressed in most mature B cell malignancies (e.g., Burkitt's lymphoma, follicular lymphoma, and lymphocytic lymphoma) (Link et al, 1986). High levels of CD37 expression have been observed in hairy cell leukemia and patient samples with Chronic Lymphocytic Leukemia (CLL) and different subtypes of non-Hodgkin's lymphoma (NHL), including Mantle Cell Lymphoma (MCL) (Schwartz-Albiez et al, 1988; Barrena et al, 2005). One report of immunophenotypic classification using antibody microarrays states that CD37 is a good discriminator between malignant CLL cells (high CD37 expression) and normal Peripheral Blood (PB) lymphocytes (low CD37 expression) (Belov et al, 2001).

Binding of a mAb specific for CD37 to cancer cells can trigger various mechanisms of action: first, after the antibody binds to the extracellular domain of the CD37 antigen, it can activate the complement cascade and lyse the target cells. Second, the anti-CD 37 antibody can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) to the target cells following recognition of the Fc portion of the bound antibody by appropriate receptors on the cytotoxic cells of the immune system.

Third, the antibody may alter the ability of the B cell to respond to an antigen or other stimulus. Eventually, anti-CD 37 antibodies can trigger programmed cell death (apoptosis).

The anti-CD 37mAb MB-1 was evaluated in B-NHL (B-cell non-Hodgkin's lymphoma) patients using two radioimmunotherapy trials, Press et al, 1989; Kaminski et al, 1992). In one trial, 6 patients with relapsed NHL were administered a therapeutic dose¹³¹I-MB-1, and all 6 patients achieved complete Clinical Remission (CR), with a median duration of 7 months. Notably, 2 of 6 patients showed clinical regression after administration of only tracer doses of MB-1, suggesting that the antibody itself had a direct anti-tumor effect. In a second trial, radiolabeled MB-1 was used to treat refractory NHL patients and elicit a targeted response of limited duration in 3 of 9 evaluable patients (Kaminski et al, 1992). In both experiments, rapid and transient peripheral B cell removal following injection of tracer-labeled MB-1 antibody doses was reported. These observations support the conclusion that MB-1 exerts cytotoxic activity independently. In summary, these clinical trials highlight the feasibility of targeting CD37 for B cell malignancies and suggest a potential clinical approach to anti-CD 37 therapyAnd (4) correlation.

Experimental evidence for a CD 37-specific antibody-like single-chain molecule ("Small modular immunopharmaceutical", SMIP) suggests that treatment with this molecule induces apoptosis in vitro and delays burkitt lymphoma growth in an in vivo xenograft model. Recently, the anti-apoptotic activity of recombinant anti-CD 37SMIP tru16.4 from trubium was described (Zhao et al, 2004). Tru16.4 induces caspase-independent apoptosis on primary CLL cells from tumor patients. The induction of apoptosis in these cells was greater than that of rituximab and comparable to Alemtuzumab (CD52 antagonist). The degree of induction of apoptosis is proportional to the cell surface expression of CD37 and can be further enhanced by cross-linking with anti-human IgG antibodies. For in vitro cell lines, the correlation of CD37 expression with ADCC was demonstrated. Treatment with anti-CD 37scFv demonstrated therapeutic efficacy in the burkitt lymphoma mouse model (Raji) (Zhao et al, 2007). These data demonstrate for the first time that targeting CD37 is a desirable approach for targeted anti-tumor therapy by inducing apoptosis and ADCC.

In summary, it has been shown that the CD37 antigen is commonly expressed on tumor cells and mature normal B lymphocytes in several human B cell malignancies and that anti-CD 37 based therapies may be a desirable approach for the treatment of B cell malignancies. Removal of normal B cells positive for CD37 is not considered critical because clinical data from a large number of patients show that even prolonged removal of B cells with anti-CD20 mAb for 6 months does not significantly reduce IgG serum levels or increase the risk of infection (Van der Kolk et al, 2002).

Although the above-described anti-CD 37 antibodies or antibody-like molecules (MB-1 and SMIP tru16.4) have shown anti-tumor efficacy in B-cell malignancies and the potential to target CD37, there remains a need for alternative anti-CD 37 inhibitors that improve B-cell ablation-based therapies.

Summary of The Invention

The present invention relates to the following items.

1. An antibody molecule that binds to human CD37 and is derived from an antibody that:

a) murine monoclonal antibodies were defined as follows:

i) a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO 2; and

ii) a variable light chain comprising the amino acid sequence shown in SEQ ID NO. 4, or

b) A non-human antibody recognizing the same epitope of human CD37 as the antibody defined in a), or recognizing an epitope similar or overlapping to the epitope;

wherein the antibody molecule is a chimeric or humanized antibody.

2. The antibody molecule of item 1, which is a chimeric antibody defined by:

i) a chimeric antibody defined by a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO 2;

ii) a chimeric antibody defined by a variable light chain comprising the amino acid sequence set forth in SEQ ID NO 4;

iii) chimeric antibodies defined by constant heavy and light chains of human origin.

3. The antibody of item 2, wherein:

i) the constant heavy chain is an IgG1 chain; and is

ii) the constant light chain is a kappa chain.

4. The antibody of item 3, wherein the constant heavy chain i) comprises the amino acid sequence shown in SEQ ID NO. 24 and the constant light chain ii) comprises the amino acid sequence shown in SEQ ID NO. 26.

5. The antibody of item 1, which is a humanized antibody defined by:

a) a humanized antibody defined by the CDRs in the variable heavy chain set forth in SEQ ID NO. 2;

b) a humanized antibody defined by the CDRs in the variable light chain set forth in SEQ ID NO. 4;

c) a humanized antibody defined by a framework derived from a human antibody and supporting the CDRs;

d) a humanized antibody defined by a constant heavy chain and a light chain from a human antibody.

6. The antibody of item 5, which comprises a variable heavy chain having the sequence shown in SEQ ID NO 6.

7. The antibody of item 6, which comprises a variable light chain having a sequence set forth in SEQ ID NO 12, 14, 16, 18, 20 or 22.

8. The antibody of item 5, which comprises a variable heavy chain having the sequence shown in SEQ ID NO. 8.

9. The antibody of item 8, which comprises a variable light chain having a sequence set forth in SEQ ID NOs 12, 14, 16, 18, 20, or 22.

10. The antibody of item 5, which comprises a variable heavy chain having the sequence shown in SEQ ID NO. 10.

11. The antibody of item 10, comprising a variable light chain having a sequence set forth in SEQ ID NOs 12, 14, 16, 18, 20, or 22.

12. The antibody of any one of items 1 to 11, wherein the antibody has one or more mutations in the Fc region that alter one or more effector functions.

13. The antibody of item 12, wherein the altered effector function is an increase in antibody-dependent cell-mediated cytotoxic activity.

14. The antibody of item 12 or 13, wherein the one or more mutations of the Fc region is a single substitution at position 332, numbered according to the Kabat EU numbering index.

15. The antibody of item 12 or 13, wherein the one or more mutations of the Fc region is a combination of substitutions at positions 239 and 332, numbered according to the Kabat EU numbering index.

16. The antibody of item 12 or 13, wherein the one or more mutations of the Fc region is a combination of substitutions at positions 236 and 332, numbered according to the Kabat EU numbering index.

17. The antibody of item 12 or 13, wherein the one or more mutations of the Fc region is a combination of substitutions at positions 236, 239 and 332, numbered according to the Kabat EU numbering index.

18. The antibody of any one of claims 14-17, wherein the substitutions are I332E, S239D, and G236A.

19. An antibody that binds to human CD37 and has a heavy chain comprising the amino acid sequence SEQ ID NO 28.

20. The antibody of item 19, having a light chain comprising the amino acid sequence of SEQ ID NO 30.

21. An antibody that binds to human CD37 and has a heavy chain comprising the amino acid sequence of SEQ ID NO 36.

22. The antibody of item 21, having a light chain comprising the amino acid sequence of SEQ ID NO 38.

23. An antibody that binds to human CD37 and has a heavy chain comprising the amino acid sequence of SEQ ID NO 32.

24. The antibody of item 23, having a light chain comprising the amino acid sequence of SEQ ID NO 34.

25. An antibody that binds to human CD37 and has a heavy chain comprising the amino acid sequence SEQ ID NO 40.

26. The antibody of item 25, having a light chain comprising the amino acid sequence of SEQ ID NO 42.

A DNA molecule comprising a region encoding the variable heavy chain of the antibody of any one of items 1 to 26.

28. The DNA molecule of item 27, wherein the variable heavy chain coding region is fused to a region encoding a constant heavy chain of human origin.

29. The DNA molecule of item 28, wherein the human constant heavy chain is IgG 1.

30. The DNA molecule of item 29, wherein the IgG1 is encoded by the sequence set forth in SEQ ID NO. 23.

31. The DNA molecule of any one of claims 28 to 30, wherein the human constant heavy chain has one or more substitutions in the Fc region as defined in any one of claims 14 to 18.

A DNA molecule comprising a region encoding a variable light chain of an antibody of any one of claims 1 to 26.

33. The DNA molecule of item 32, wherein the variable light chain coding region is fused to a region encoding a constant light chain of human origin.

34. The DNA molecule of item 33, wherein the constant light chain is a chain.

35. The DNA molecule of item 34, wherein the kappa light chain is encoded by the sequence set forth in SEQ ID NO: 25.

36. An expression vector comprising a DNA molecule according to any one of claims 27 to 31 and/or a DNA molecule according to any one of claims 32 to 35.

37. A host cell carrying one or more of the vectors of item 36.

38. The host cell of item 37, which carries an expression vector comprising the DNA molecule of any one of items 27 to 31 and a second expression vector comprising the DNA molecule of any one of items 32 to 35.

39. The host cell of item 37, which is a mammalian cell.

40. A method of producing an antibody according to any one of claims 1 to 26, comprising transfecting a mammalian host cell with one or more vectors according to item 36, culturing the host cell, and recovering and purifying the antibody molecule.

41. A pharmaceutical composition comprising as active ingredient one or more anti-CD 37 antibody molecules according to any one of claims 1 to 26, and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of item 41, further comprising one or more additional therapeutic agents.

43. The pharmaceutical composition of item 42, wherein the one or more additional therapeutic agents are selected from agents that target B cell antigens other than CD 37.

44. The pharmaceutical composition of item 43, wherein the B cell antigen is CD 20.

45. The pharmaceutical composition of clause 42, wherein the one or more additional therapeutic agents are selected from agents that induce apoptosis.

46. The pharmaceutical composition of item 45, wherein the agent is a modulator of a TRAIL receptor.

47. The pharmaceutical composition of any one of items 41 to 46 for use in depleting B cells that express CD37 on their surface.

48. The pharmaceutical composition of item 47, for use in treating a B cell malignancy.

49. The pharmaceutical composition of item 48, wherein the B cell malignancy is selected from the group consisting of B cell non-Hodgkins lymphoma, B cell chronic lymphocytic leukemia and multiple myeloma.

50. The pharmaceutical composition of item 47, which is used for treating an autoimmune disease or an inflammatory disease whose pathology involves B cells.

51. A method of removing a CD 37-expressing B cell from a population of cells, comprising administering to the population of cells the antibody molecule of any one of items 1 to 26 or a pharmaceutical composition comprising the antibody molecule.

52. The method of item 51, which is performed in vitro.

53. A method of treating a patient having a B cell malignancy selected from the group consisting of: a B-cell non-hodgkin's lymphoma, a B-cell chronic lymphocytic leukemia, and multiple myeloma, said method comprising administering to the patient an effective amount of the pharmaceutical composition according to any one of items 41 to 46.

It is an object of the present invention to provide novel CD37 antagonists for the treatment of B cell malignancies and other disorders responsive to the removal of CD37 positive B cells.

Furthermore, it is an object of the present invention to provide anti-CD 37 antibodies with improved effector function. In particular, the present inventors have attempted to provide anti-CD 37mAB with antibody-dependent cell-mediated cytotoxicity (ADCC).

To address the problems underlying the present invention, the murine monoclonal anti-CD 37 antibody was used as a starting antibody for the generation of chimeric and humanized anti-CD 37 antibodies that can be used in human therapy.

In a first aspect, the invention provides antibody molecules that bind to human CD37 and are derived from antibodies that are:

a) a murine monoclonal antibody defined by the structure:

i) a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO 2; and

ii) a variable light chain comprising the amino acid sequence shown in SEQ ID NO 4, or

b) A non-human antibody recognizing the same epitope of human CD37 as the antibody defined in a), or recognizing an epitope close (close) or overlapping (overlap) to the epitope;

wherein the antibody molecule is a chimeric or humanized antibody.

As will be understood from the following, an antibody "derived from" another antibody (i.e., the starting antibody) refers to an antibody that has been prepared by modifying the starting antibody as described below.

In a preferred embodiment, said antibody molecule is a chimeric or humanized antibody molecule derived from said starting antibody of a). Antibodies with related sequences are referred to as G28.1 and are described in WO 2005/017148.

b) The starting antibody may be selected from, for example, CD37 specific antibodies characterized as CD37 antigen in Third HLDA works, as G28.1; these antibodies are designated HD28, HH1, BI14, F97-3G6(Ling and MacLennan, 1987). Other described antibodies specific for CD37 include RFB-7, Y29/55, MB-1, M-B371, M-B372 and IPO-24. All of these antibodies (including G28.1) recognize the same or partially identical or similar epitope of CD37 according to Moldenhauer,2000 and Schwartz-Albiez et al, 1988. Schwartz-Albiez et al, 1988, indicated that this epitope is located in the carbohydrate portion of CD 37. A number of the above antibodies are commercially available, for example HH1(Santa Cruz), RFB-7(Biodesign), Y29/55(Biogenesis), M-B371(BD Biosciences), M-B372(Santa Cruz) and IPO-24 (AbCam).

Other antibodies specific for CD37 are S-B3(Biosys), NMN46(Chemicon) and ICO-66 (BioProbe). Whether an antibody recognizes the same epitope as G28.1 can be determined by competitive binding assays or by cross-suppression radioimmunoassay as described by Moldenhauer et al, 1987 and Moldenhauer, 2000.

For example, competitive binding can be determined in an ELISA using plates coated with CD37 protein or CD37 peptide or CD37 positive cells (cell ELISA) and measuring the binding of the biotin-labeled antibody in the presence of a competitive candidate antibody. The binding of biotin-labeled G28.1 (or another antibody known to recognize the same epitope) is reduced in the presence of a competing antibody or antibody-derived fragment under conditions in which the antibodies recognize the common epitope. To identify the G28.1 epitope peptide, a fragment derived from the CD37 sequence or a short polypeptide or recombinant protein can be synthesized or produced and the binding of G28.1 to the peptide/polypeptide measured in an ELISA assay. Competitive binding can also be determined by FACS analysis, as described in the examples.

b) The antibody as defined in (1) can be used as a starting antibody in a similar manner to G28.1 to produce a chimeric or humanized antibody molecule.

b) The starting antibody may also be generated de novo by immunizing with a peptide or protein fragment containing the relevant epitope, or a DNA molecule encoding said peptide/fragment, respectively, to obtain an antibody reactive with the epitope of G28.1.

The starting antibody b) can also be prepared by immunization with whole cells carrying the relevant epitope; and screening the hybridoma cells thus obtained for competitive binding of the secreted antibody.

The term "anti-CD 37 antibody molecule" encompasses anti-CD 37 antibodies, anti-CD 37 antibody fragments, and conjugates with said antibody molecules. Within the meaning of the present invention, antibodies include chimeric monoclonal antibodies and humanized monoclonal antibodies. The terms "antibody" and "antibody molecule" are used interchangeably and shall include intact immunoglobulins (which are produced by lymphocytes and are present in, for example, serum), monoclonal antibodies secreted by hybridoma cell lines, polypeptides recombinantly expressed by host cells that have the binding specificity of an immunoglobulin or monoclonal antibody, and molecules derived from modifications or further processing of the antibody that retain its binding specificity.

In one embodiment of the invention, the anti-CD 37 antibody molecule is a chimeric antibody defined by:

i) a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO 2;

ii) a variable light chain comprising the amino acid sequence set forth in SEQ ID NO 4;

iii) constant heavy and light chains of human origin.

The construction and production of chimeric mouse/human antibodies is known in the art (Boulianne et al, 1984). The variable region of a non-human antibody is typically linked to at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. Human constant region DNA sequences can be isolated from a variety of human cells, preferably immortalized B cells, according to well known methods (see Kabat et al, 1991; and WO 87/02671). The antibody molecule may contain all or only a portion of the constant region, so long as it exhibits specific binding to the CD37 antigen and the Fc receptor. The type and length of the constant region is selected depending on whether effector functions such as complement fixation or antibody-dependent cell-mediated toxicity are desired and the desired pharmacological properties of the antibody molecule.

In certain embodiments, the antibody molecule of the invention is a chimeric CD 37-specific antibody having the heavy chain variable region of a non-human antibody as defined in a) or b) fused to a human heavy chain constant region IgG1 and the light chain variable region of a non-human antibody as defined in a) or b) fused to a human light chain constant region κ.

In another embodiment, the antibody molecule is a chimeric CD 37-specific antibody having the heavy chain variable region of SEQ ID NO:2 fused to human heavy chain constant region IgG1, which is an IgG1 molecule having the sequence shown in SEQ ID NO:24 (DNA coding sequence: SEQ ID NO:23) or a mutant IgG1 molecule derived therefrom, and having the light chain variable region of SEQ ID NO:4 fused to human light chain constant region kappa (DNA coding sequence: SEQ ID NO:25) shown in SEQ ID NO: 26.

Other human constant regions for the chimerization of the non-human starting antibodies defined under a) or b) are available to the person skilled in the art, for example IgG2, IgG3, IgG4, IgA, IgE or IgM (replacing IgG1) or lambda (replacing kappa). The constant region may also be chimeric, for example, heavy chain IgG1/IgG2 or IgG1/IgG3 chimeras.

In certain embodiments of the invention, the anti-CD 37 antibody molecule is a humanized antibody defined by:

CDRs within the variable heavy chain shown in SEQ ID NO. 2, and

CDRs within the variable light chain shown in SEQ ID NO 4,

a human antibody-derived framework supporting the CDRs,

constant heavy and light chains from human antibodies.

Humanized forms of non-human antibodies (e.g., murine, rat, or rabbit antibodies) are immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab ', F (ab')₂Or other antigen binding molecules having antibody subsequences).

Humanized antibodies include human immunoglobulins (from an acceptor antibody) in which residues from a Complementarity Determining Region (CDR) of the acceptor antibody are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues.

In the humanized antibodies of the invention, sequences encoding the CDRs of the non-human starting antibody defined in a) or b) have been grafted into respective genes of human immunoglobulin heavy and light chains.

"complementarity determining regions" (CDRs) of monoclonal antibodies are to be understood as those amino acid sequences involved in specific antigen binding according to Kabat et al, 1991 and Chothia and Lesk (1987). In the variable region sequences shown in SEQ ID NO. 2 and SEQ ID NO. 4, the CDR sequences can be determined conventionally by searching for sequence features in the Kabat sequence database.

Techniques for obtaining humanized antibodies are generally known to those skilled in the art, see, e.g., US5,225,539, US6,548,640, and US6,982,321.

Suitable framework residues of the CDR-grafted antibody can be reverted to murine residues to improve binding affinity. As described above, according to methods known in the art, experts know how to obtain CDRs from a given non-human antibody, select and obtain appropriate human immunoglobulin genes, graft CDRs into these genes, modify selected framework residues, express the CDR-grafted antibody in an appropriate host cell (e.g., Chinese Hamster Ovary (CHO) cell), and test the resulting recombinant antibody for binding affinity and specificity.

To obtain a humanized antibody, the antigen binding site formed by the heavy chain CDRs and the light chain CDRs is excised from the DNA of a cell secreting a rodent (murine) monoclonal antibody and grafted into the DNA encoding the human antibody framework.

Alternatively for CDR grafting, non-human, especially murine, anti-CD 37 antibodies can be humanized by the so-called "resurfacing" technique, as described in US5,639,641, whereby the rodent framework remains unchanged except for surface exposed residues.

In another aspect, the invention relates to a humanized antibody comprising a variable heavy chain having the sequence shown in SEQ ID NO. 6 and a variable light chain having a sequence selected from the group consisting of the sequences shown in SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20 and SEQ ID NO. 22.

In another aspect, the invention relates to a humanized antibody comprising a variable heavy chain having the sequence shown in SEQ ID NO. 8 and a variable light chain having a sequence selected from the group consisting of the sequences shown in SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20 and SEQ ID NO. 22.

In another aspect, the invention relates to a humanized antibody comprising a variable heavy chain having the sequence shown in SEQ ID NO. 10 and a variable light chain having a sequence selected from the group consisting of the sequences shown in SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20 and SEQ ID NO. 22.

The humanized antibodies defined above are shown in table 1.

In certain embodiments, the humanized antibody has a human heavy chain constant region IgG1 and a human light chain constant region κ. As described above for chimeric antibodies, the constant regions may be selected from other classes and subclasses.

In certain embodiments, in the humanized antibodies of the invention, human constant heavy chain IgG1 is an IgG1 molecule having the sequence shown in SEQ ID NO. 24 or a mutant IgG1 molecule derived therefrom, and human light chain constant region κ has the sequence shown in SEQ ID NO. 26.

The anti-CD 37 antibody molecule of the present invention may also be a variant of an antibody defined by the amino acid sequence shown in the sequence listing. Using commonly available techniques, one skilled in the art will be able to prepare, test and utilize functional variants of the antibodies defined above. Examples are variant antibodies with a change in at least one position in the CDR and/or framework, variant antibodies with a single amino acid substitution in the framework region and deviating from the germline sequence, antibodies with conservative amino substitutions, antibodies encoded by DNA molecules hybridizing under stringent conditions with DNA molecules encoding the variable chains of the antibodies in the sequence listing.

Where the properties of individual amino acids are specified, reasonable substitutions can be made to obtain antibody variants that preserve the overall molecular structure of the starting antibody. Amino acid substitutions, i.e., "conservative substitutions," may be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the individual amino acids. Those skilled in the art are familiar with commonly practiced amino acid substitutions (as described, for example, in WO 2007/042309) and methods of obtaining antibodies modified thereby. Given the genetic code and recombinant and synthetic DNA techniques, DNA molecules encoding variant antibodies with one or more conservative amino acid exchanges can generally be designed and individual antibodies readily obtained.

The antibody variants encompassed by the present invention have at least 60%, more preferably at least 70% or 80%, more preferably at least 90% and most preferably at least 95% sequence identity in the CDR regions as compared to the antibody defined by the variable chains in the sequence listing. Preferably, the antibody also has at least 80%, more preferably 90% and most preferably 95% sequence similarity in the CDR regions. Preferably, antibody variants have at least 60%, more preferably at least 70% or 80%, more preferably at least 90% and most preferably at least 95% sequence identity in the variable region. Preferably, the antibodies also have at least 80%, more preferably 90% and most preferably 95% sequence similarity in the variable region.

"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between these sequences. "sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions.

Variants may also be obtained by using an antibody having the sequence specified in the sequence listing as a starting point, optimizing and diversifying one or more amino acid residues, preferably in one or more CDRs, and screening the resulting collection of antibody variants for variants having improved properties. Diversification of one or more amino acid residues in the variable light chain CDR3, variable heavy chain CDR3, variable light chain CDR1, and/or variable heavy chain CDR2 has proven useful. Diversification can be carried out by methods known in the art (e.g., the so-called TRIM technique mentioned in WO 2007/042309).

In another embodiment, the anti-CD 37 antibody molecule of the invention is an "affinity matured" antibody.

An "affinity matured" anti-CD 37 antibody is an anti-CD 37 antibody derived from an antibody having the sequences in the sequence listing, which has one or more alterations in one or more CDRs which result in an improvement in affinity for the antigen as compared to the respective original immature antibody. One method for generating such antibody mutants includes phage display (Hawkins et al, 1992; and Lowman et al, 1991). Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are presented monovalent from filamentous phage particles as fusions with the gene III product of M13 encapsulated in each particle. The phage-displayed mutants are then screened for biological activity (e.g., binding affinity) as disclosed herein.

Affinity matured antibodies can also be produced by methods as described below: for example, Marks et al, 1992 (affinity maturation by shuffling of Variable Heavy (VH) and Variable Light (VL) domains); or Barbas et al, 1994; shier et al, 1995; yelton et al, 1995; jackson et al, 1995; and Hawkins et al, 1992 (random mutagenesis of CDR and/or framework residues). Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen.

In another embodiment, the anti-CD 37 antibody molecule of the invention is a "de-immunized" antibody.

A "deimmunized" anti-CD 37 antibody is an antibody derived from a humanized or chimeric antibody having the sequence shown in the sequence listing, which has one or more changes in the amino acid sequence that result in a reduction in the immunogenicity of the antibody as compared to the respective original non-deimmunized antibody. One method for generating such antibody mutants involves the identification and removal of T-cell epitopes from antibody molecules (Baker and Jones, 2007). In a first step, the immunogenicity of the antibody molecule can be determined by several methods as described in the literature (Jones et al, 2004; Jones et al, 2005; Reche et al, 2004; Hertz et al, 2006), for example by in vitro determination of T-cell epitopes or by in silico prediction (in silico prediction). Once critical residues for T-cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity (Jones et al, 2005; Tangri et al, 2005). Methods for introducing mutations into proteins are known in the art, for example by overlapping PCR techniques.

Because the Fc region of an antibody interacts with a variety of Fc receptors, giving rise to a variety of important functional capabilities (referred to as "effector functions"), in certain embodiments, the antibody is a full-length antibody or an antibody containing an Fc region, the latter of which exhibits specific binding to both the relevant portion of the antigen and to the Fc receptor. The type and length of the constant region is selected depending on whether the effector function (e.g., complement fixation or antibody-dependent cell-mediated cytotoxic activity) is of the desired character and the desired pharmacological properties of the antibody protein.

In one embodiment of the invention, the anti-CD 37 antibody is a chimeric or humanized antibody having an Fc region or a related portion thereof that has been engineered to modulate effector function, particularly to enhance binding of the antibody to one or more Fc receptors, thereby enhancing effector function ADDCs. The engineering of the Fc region mediates the effector function of the antibody in the presence of effector cells more effectively than the parent antibody that has not been Fc engineered. In one embodiment, the antibody variant mediates ADCC that is greater than the ADCC mediated by the parent antibody. (hereinafter, if not otherwise stated, the term "parent" in the case of an antibody molecule or in the case of an IgG or Fc region refers to the non-engineered antibody molecule, Fc region or IgG, respectively, from which the mutated (engineered) molecule was derived).

Modifications of various Fc regions have been proposed in the art (both scientific and patent literature), for example in EP0307434, WO9304173, WO9734631, WO9744362, WO9805787, WO9943713, WO9951642, WO9958572, WO02060919, WO03074679, WO2004016750, WO2004029207, WO2004063351, WO2004074455, WO2004035752, WO2004099249, WO2005077981, WO2005092925, WO2006019447, WO2006031994, WO2006047350, WO2006053301, WO2006088494 and WO 2007041635.

In a preferred embodiment, the antibody of the invention is an Fc variant with amino acid substitutions at positions 332 and/or 239 and/or 236. In a preferred embodiment, the antibody of the invention has a mutation in the Fc region selected from the group consisting of:

i) a single substitution at position 332, preferably I332E;

ii) a combination of substitutions at positions 239 and 332, preferably S239D/I332E;

iii) a combination of substitutions at positions 236 and 332, preferably G236A/I332E;

iv) combinations of substitutions at positions 236, 239 and 332, preferably G236A/S239D/I332E.

Substitutions as defined above can be found, for example, in Lazar et al, 2006, WO2004029207 and WO 2007041635.

The Fc variants of the antibodies of the invention are defined in terms of the amino acid modifications that they comprise. Thus, for example, I332E is an Fc variant having an I332E substitution relative to a parent Fc polypeptide. Likewise, S239D/I332E defines an Fc variant having substitutions S239D and I332E relative to the parent Fc polypeptide, and S239D/I332E/G236A defines an Fc variant having substitutions S239D, I332E and G236A relative to the parent Fc polypeptide.

The numbering is according to the EU numbering scheme (Kabat et al, 1991), which refers to the numbering of EU antibodies (Edelman et al, 1969). One skilled in the art will appreciate that these conventions consist of non-sequence numbering in specific regions of immunoglobulin sequences, enabling normalized reference to conserved positions of immunoglobulin families.

In the above-defined antibody, the substituted positions 236, 239 and 332 correspond to positions 119, 122 and 215, respectively, of the heavy chain of IgG1 depicted in SEQ ID NO. 24. (in the full length sequence of the heavy chains of antibodies A2, A4, B2 and B4 shown in SEQ ID NOs: 28, 32, 36 and 40, the substituted amino acids are at positions 235, 238 and 331).

In certain embodiments, the Fc variants of the invention are based on human IgG sequences, and thus human IgG sequences are used as "base" sequences compared to other sequences. For the antibodies of the invention, the engineered Fc region is preferably an IgG, particularly IgG1, but it may also be an IgG2 or a variant sequence from other immunoglobulin classes (such as IgA, IgE, IgGD, IgM) or chimeric versions of two or more immunoglobulin classes (e.g., IgG2/IgG1) and analogs thereof. Although the Fc variants of the invention are engineered in the context of one parent IgG, the variants may also be engineered or "transferred" to another second parent IgG context in the context of another second parent IgG context. This is done by determining "equivalent" or "corresponding" residues and substitutions between the first and second IgG, typically based on sequence or structural homology between the first and second IgG sequences. To determine homology, the amino acid sequence of a first IgG as outlined herein is directly compared to the sequence of a second IgG. After aligning the sequences, one or more homology alignment programs known in the art are used, allowing for the necessary insertions and deletions to maintain the alignment (i.e., to avoid elimination of conserved residues through any deletions and insertions), thereby identifying residues equivalent to the particular amino acids in the initial sequence of the first Fc variant. Regardless of how equivalent or corresponding residues are determined, and regardless of the identity of the parent IgG from which the IgG is made, it is intended to demonstrate that the Fc variants used in the present invention can be engineered to any second parent IgG that has significant sequence or structural homology to the Fc variant. Thus, for example, if a variant antibody whose parent antibody is human IgG1 is produced by using the above-described method or other methods for determining equivalent residues, the variant antibody can be engineered in, for example, a human lgG2 parent antibody, a human IgA parent antibody (see WO 2007041635).

The antibody of the invention targets antigen CD37, targeting antigen CD37 may be advantageous over targeting CD20 in diseases where the degree of CD37 expression is higher than the degree of CD20 expression, for example, as in chronic lymphocytic leukemia where the sample has shown a high degree of CD37mRNA expression compared to a low degree of CD20mRNA expression.

The antibodies of the invention have been shown to be superior to rituximab (registered anti-CD20 antibody) in ADCC activity on Ramos (Ramos) cells, normal B-cell depletion in whole blood and removal of ramosburkitt lymphoma cells. As can be seen in the experiments of the present invention, the antibodies of the present invention (both non-Fc engineered and Fc engineered) have B cell depleting activity that is superior to that of rituximab. Antibodies with a mutated Fc region showed about 10-fold increased B cell removal activity as compared to rituximab (fig. 11B).

Representative of the CD37 antibodies of the invention exhibit potent pro-apoptotic activity without cross-linking; in this respect, antibodies with this property are superior to anti-CD 37SMIP tru16.4(Zhao et al, 2007) which do not show apoptosis in the absence of cross-linking. Inducing apoptosis without crosslinking, which may be shown by Fc-engineered or non-Fc-engineered antibodies of the invention, is advantageous in the absence of crosslinking agents (e.g., effector cells with Fc γ receptors) in vivo or in low density target antigen CD37 (e.g., tumor cells with low expression levels of CD 37). Antibodies that induce apoptosis without crosslinking may still cause cell death, whereas antibodies that rely on crosslinking do not.

In another aspect, the anti-CD 37 antibody molecule of the invention is an antibody fragment derived from a humanized or chimeric CD37 specific antibody according to the invention. To obtain antibody fragments, e.g., Fab fragments, digestion can be achieved by conventional techniques (e.g., using papain). Examples of papain digestion are described in WO94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments (so-called Fab fragments), each having a single antigen-binding site and a residual Fc fragment. Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking with antigen₂And (3) fragment.

Fab fragments obtained by antibody digestion also contain the constant light domain and the first constant heavy domain (CH)₁). Fab' fragments differ from Fab fragments in that they differ in the presence of the heavy chain CH₁The carboxy terminus of the domain contains other residues, including one or more cysteines from the antibody hinge region. Fab '-SH is used herein to denote Fab' in which the cysteine residues of the constant domains carry free thiol groups. F (ab')₂Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Antibody fragments can also be generated by molecular biological methods that produce the respective coding DNA fragments.

Antibody molecules are typically tetramers consisting of two light/heavy chain pairs, but may also be dimeric, i.e., consist of light/heavy chain pairs (e.g., Fab or Fv fragments), or they may be monomeric single chain antibodies (scFv; Johnson and Bird,1991), minibodies, or diabodies.

The anti-CD 37 antibody molecule may also be in the form of a conjugate, i.e., an antibody molecule chemically coupled to a cytotoxic agent, particularly a cytotoxic agent that induces the cytotoxic activity (e.g., apoptosis or mitotic arrest) of tumor cells. Due to normal pharmacological clearance mechanisms, antibodies used in drug conjugates ("immunoconjugates") contact and bind to target cells only in limited amounts. Thus, the cytotoxic agent used in the conjugate must have a high degree of cytotoxic activity such that sufficient cell killing occurs to elicit a therapeutic effect. Examples of such cytotoxic agents include taxanes (taxanes) (see, e.g., WO01/38318 and WO03/097625), DNA alkylating agents (e.g., CC-1065 analogs), anthracyclines (anthracyclines), tubulicin (tubulysin) analogs, duocarmycin (duocarmycin) analogs, daunorubicin (doxorubicin), auristatin E (auristatin E), ricin A toxin, and cytotoxic agents comprising reactive polyethylene glycol moieties (see, e.g., Sasse et al, 2000; Suzawa et al, 2000; Ichimura et al, 1991; Francisco et al, 2003; U.S. Pat. No. 5,475,092, U.S. Pat. No. 6,340,701, U.S. Pat. No. 6,738,738,78, and U.S. Pat. No. 3/5639, U.S. Pat. No. 6/0199519 and WO 49 01/698), as described in US 2004/0241174.

In a preferred embodiment, the cytotoxic agent is a maytansinoid (i.e., a derivative of maytansine (CAS35846538)), which is known in the art and includes maytansine, maytansinol, C-3 esters of maytansinol and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163).

anti-CD 37 antibody immunoconjugates can be designed and synthesized as described in WO2007077173 for anti-FAP immunoconjugates.

In another embodiment, the anti-CD 37 molecules of the invention can be radiolabeled to form radioimmunoconjugates, a method proposed for the anti-CD 37 antibody MB-1 (Buchsbaum et al, 1992, supra). Radionuclides with advantageous radioactive properties are known in the art, examples being phosphorus-32, strontium-89, yttrium-90, iodine-131, samarium-153, erbium-169, ytterbium-175, rhenium-188, which have been successfully and stably coupled to MAbs. The anti-CD 37 antibody molecules of the invention may be labeled with various radionuclides using direct labeling or indirect labeling methods known in the art, as described in US6,241,961. A review of the techniques for generating and applying the novel radiolabeled antibody conjugates suitable for use in the present invention is given by golden and Sharkey, 2007.

The antibody molecules of the invention (whether Fc engineered or not) may also be bispecific, i.e., the antibody molecule binds to two different targets, one of which is CD37 and the other is selected, for example, from T cell expressed surface antigens such as CD3, CD16 and CD 56.

The invention also relates to DNA molecules encoding the chimeric or humanized anti-CD 37 antibody molecules of the invention. The sequences encoding the variable heavy chain of the antibody molecule of the invention are shown in SEQ ID NO 1, SEQ ID NO 5, SEQ ID NO 7 and SEQ ID NO 9. The sequences encoding the variable light chains of the antibody molecules of the invention are shown in SEQ ID NO 3, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21.

Nucleic acid molecules encoding the light and heavy chains can be synthesized chemically and enzymatically (PCR amplification) by standard methods. First, suitable oligonucleotides can be synthesized by methods known in the art (e.g., Gait,1984), which can be used to generate synthetic genes. Methods for generating synthetic genes from oligonucleotides are known in the art (e.g., Stemmer et al, 1995; Ye et al, 1992; Hayden et al, Mandecki, 1988; Frank et al, 1987).

The DNA molecule of the invention includes (but is not limited to) the DNA molecule shown in the sequence table. Thus, the present invention also relates to a nucleic acid molecule as defined in WO2007/042309 which hybridizes under high stringency binding and washing conditions with a DNA molecule as described in the sequence listing, wherein said nucleic acid molecule encodes an antibody or functional fragment thereof having properties equivalent or superior to those of the antibody encoded by the sequence listed in the sequence listing. Preferably the molecules (from an mRNA perspective) are those having at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity to one of the DNA molecules described herein.

Another class of DNA variants within the scope of the present invention may be defined in terms of the polypeptide that it encodes. The sequences of these DNA molecules deviate from those described in the sequence listing, but due to the degeneracy of the genetic code, encode antibodies with identical amino acid sequences. For example, in view of expression of antibodies in eukaryotic cells, the DNA sequences shown in the sequence listing are designed to conform to codon usage habits in eukaryotic cells. If it is desired to express the antibody in e.coli (e.coli), these sequences can be varied to match e.coli codon usage. For example, variants of the DNA molecules of the invention can be constructed in several different ways, as described in WO 2007/042309.

To produce the recombinant anti-CD 37 antibody molecules of the invention, DNA molecules encoding the full-length light and heavy chains or fragments thereof are inserted into expression vectors such that the sequences are operably linked to transcriptional and translational control sequences.

To make antibodies of the invention, one skilled in the art can select from a variety of expression systems well known in the art, such as those reviewed by Kipriyanow and Le Gall, 2004.

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 may be inserted into separate vectors. In certain embodiments, the two DNA sequences are inserted into the same expression vector. Suitable vectors are those encoding functionally intact human CH or CL immunoglobulin sequences with appropriate restriction sites engineered so that any VH or VL sequence can be readily inserted and expressed as described above. For antibody light chains, the constant chains are typically kappa or lambda, and for antibody heavy chains, the constant chains may be (without limitation) of any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulin, including allelic variants.

The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The DNA encoding the antibody chain may be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the mature antibody chain DNA. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein. Alternatively, the DNA sequence encoding the antibody chain may already contain a signal peptide sequence.

In addition to the DNA sequence encoding the antibody chain, the recombinant expression vector also carries regulatory sequences, including promoters, enhancers, termination and polyadenylation, and other expression control components that control expression of the antibody chain in a host cell. Examples of promoter sequences, exemplified with respect to expression in mammalian cells, are those derived from (CMV), such as CMV monkey virus 40(SV40), such as the SV40 promoter/enhancer, adenovirus (e.g., adenovirus major late promoter (AdMLP)), promoters and/or enhancers of polyoma and strong mammalian promoters, such as native immunoglobulin and actin promoters. Examples of polyadenylation are BGH polyA, SV40 late or early polyA; alternatively, 3' UTR of immunoglobulin gene or the like can be used.

Recombinant expression vectors may also carry sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Nucleic acid molecules encoding the heavy and/or light chain or antigen-binding portion thereof of an anti-CD 37 antibody, and vectors comprising such DNA molecules, can be introduced into a host cell (e.g., a bacterial cell or a higher eukaryotic cell, such as a mammalian cell) according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer via a viral vector.

The DNA molecules encoding the heavy and light chains are preferably present on two vectors which are co-transfected into a host cell, preferably a mammalian cell.

Mammalian cell lines that can be used as hosts for expression are known in the art and include chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells (HeLa cells), Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human cancer cells (e.g., Hep G2), a549 cells, 3T3 cells, or derivatives/progeny of any of these cell lines. Other mammalian cells can be used, including but not limited to human, mouse, rat, monkey, and rodent cell lines; or other eukaryotic cells, including but not limited to yeast, insect, and plant cells; or prokaryotic cells, such as bacteria. The anti-CD 37 antibody molecules of the invention are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody molecule in the host cells.

The antibody molecule is preferably recovered from the culture medium as a secreted polypeptide or, if expressed, for example, in the absence of a secretion signal, it may be recovered from the host cell lysate. It is necessary to purify the antibody molecule in such a way that a substantially homogeneous antibody preparation is obtained, using standard protein purification methods for recombinant proteins and host cell proteins. For example, state of the art purification methods suitable for obtaining the anti-CD 37 antibody molecules of the invention include the removal of cells and/or particulate cell debris from the culture medium or lysate as a first step. The antibodies are then purified from contaminating soluble proteins, polypeptides and nucleic acids, for example, by fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or cation exchange resins. As a final step in the process of obtaining a preparation of the anti-CD 37 antibody molecule, the purified antibody molecule may be dried, e.g. freeze-dried, as described below for therapeutic applications.

In another aspect, the invention relates to a pharmaceutical composition comprising as an active ingredient an anti-CD 37 antibody molecule of the invention.

For use in therapy, the anti-CD 37 antibody is included in a pharmaceutical composition suitable for facilitating administration to an animal or human. Typical formulations of anti-CD 37 antibody molecules can be prepared as lyophilized or otherwise dried formulations or aqueous solutions or aqueous or non-aqueous suspensions by mixing the anti-CD 37 antibody molecule with a physiologically acceptable carrier, excipient, or stabilizer. The carrier, excipient, modifier or stabilizer is non-toxic at the dosages and concentrations employed. It includes: buffer systems such as phosphates, citrates, acetates and other inorganic or organic acids and their salts; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexa hydroxy quaternary ammonium chloride; benzalkonium chloride; phenol; butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; mono-, di-, oligo-or polysaccharides and other carbohydrates including glucose, mannose, sucrose, trehalose, dextrins or dextrans (dextrans); chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium;metal complexes (e.g., Zn-protein complexes); and/or ionic or nonionic surfactants, such as TWEEN^TM(Polysorbate), PLURONICS^TMOr fatty acid esters, fatty acid ethers or sugar esters. The antibody formulation may also contain an organic solvent, such as ethanol or isopropanol. Excipients may also have release modifying or absorption modifying functions.

The anti-CD 37 antibody molecule may also be dried (freeze-dried, spray-freeze-dried, dried by near or supercritical gas, vacuum-dried, air-dried), precipitated or crystallized or embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, using hydroxymethylcellulose or gelatin and poly (methylmethacrylate), respectively, embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), embedded in macroemulsions, or precipitated or immobilized on carriers or surfaces, for example, by pcmc techniques (protein-coated microcrystals). The technique is disclosed in Remington, The Science and Practice of Pharmacy, 21 st edition, Hendrickson R.

Naturally, the formulation to be used for in vivo administration must be sterile; sterilization may be accomplished by conventional techniques, such as by filtration through sterile filtration membranes.

Applicable is increasing the anti-CD 37 antibody concentration to reach a so-called High Concentration Liquid Formulation (HCLF); various ways of generating the HCLF have been described.

The anti-CD 37 antibody molecule may also be contained in a sustained release formulation. The formulations comprise a solid, semi-solid or liquid matrix of hydrophobic or hydrophilic polymers and may be in the form of shaped articles, such as films, sticks or microcapsules, and may be applied via an application device. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or sucrose acetate butyrate or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-gamma-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic-glycolic acidCopolymers (such as lupronepot)^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibody remains in the body for a long time, it may denature or aggregate due to exposure to moisture at 37 ℃, resulting in a loss of biological activity and a possible change in immunogenicity. Depending on the mechanism involved, reasonable strategies can be devised to achieve stability. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions (e.g., as described in WO 89/011297), controlling moisture content, using appropriate additives, and forming specific polymer matrix compositions.

Formulations of anti-CD 37 antibody molecules that can also be used in the present invention are described in US7,060,268 and US6,991,790.

The CD37 antibody molecule may also be incorporated into other forms of use, such as into dispersions, suspensions or liposomes, tablets, capsules, powders, sprays, transdermal or intradermal patches or creams with or without permeation enhancing means, wafers, nasal, buccal or pulmonary formulations, or may be produced by implanted cells or by the individual's own cells following gene therapy.

The anti-CD 37 antibody molecule may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological characteristics of the antibody, such as increasing serum half-life or increasing tissue binding.

The preferred mode of application is parenteral by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of application such as by inhalation, transdermal, intranasal, buccal, oral may also be suitable.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, the administration of antibody for prophylactic or therapeutic purposes, previous therapy, patient history and response to the antibody, and the judgment of the attending physician. Suitably, the antibody is administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 0.01 μ g/kg to 40mg/kg (e.g., 0.1mg/kg-20mg/kg) of antibody is an initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosing regimens may also be useful. The progress of this therapy is readily monitored by conventional techniques and experimentation, such as by determining the extent of B cell removal (e.g., using a flow cytometer).

A "therapeutically effective amount" of an antibody to be administered is the minimum amount required to prevent, ameliorate, or treat a disease or disorder.

The anti-CD 37 antibody molecules of the invention and pharmaceutical compositions containing them are useful for the removal of B cells that express CD37 on their surface and cause cancer or autoimmune/inflammatory diseases.

In a first aspect, the pharmaceutical composition of the invention may be used for the treatment of cancer, in particular any CD37 positive malignancy.

B cell malignancies include, but are not limited to, B cell lymphomas (e.g., various forms of Hodgkin's disease), B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g., Waldenstrom macroglobulinemiamacroglobulinaemia) (also known as lymphoplasmacytoma or immunocytoma) or central nervous system lymphoma), leukemia (e.g., Acute Lymphoblastic Leukemia (ALL), chronic lymphocytic leukemia (CLL; also known as B fineChronic lymphocytic leukemia, BCLL), hairy cell leukemia, and chronic myoblastic leukemia), and myeloma (e.g., multiple myeloma). Other B cell malignancies include small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt's lymphoma/leukemia, gray zone lymphoma, B cell proliferation of undetermined malignancy potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

In another aspect, pharmaceutical compositions comprising anti-CD 37 antibodies are useful for treating autoimmune and inflammatory diseases that involve B cells in their pathology.

Such diseases include (but are not limited to): arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, reactions associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, Adult Respiratory Distress Syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T cell infiltration and chronic inflammatory reactions, atherosclerosis, autoimmune myocarditis, lack of leukocyte adhesion, Systemic Lupus Erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus (discotus), Lupus myelitis, lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis including Wegener's granulomatosis and Churg-Strauss disease, granulosa, vasculitis including allergic vasculitis, ANCA and rheumatoid vasculitis, aplastic anemia, bunyasu anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red blood cell development (PRCA), acute and delayed hypersensitivity associated with cytokines and T-lymphocytes, and immune responses including acute and delayed hypersensitivity Factor VIII deficiency, hemophilia A, autoimmune neutropenia, total cytopenia, leukopenia, diseases involving leukocyte extravasation, Central Nervous System (CNS) inflammatory disorders, multiple organ injury Syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody Syndrome, allergic neuritis, Behcet's disease, Cassierman's Syndrome, Goodpasture's Syndrome, Lambert-Easton's Syndrome, Reauynd's Syndrome, Sjorgen's Syndrome, Stejorgen's Syndrome, Stewart's Syndrome, Stephan-Johnson Syndrome, anti-organ transplant GVHD, Bullous pemphigoid, pemphigus, autoimmune polyendocrine lesion, seronegative spondyloarthropathy, Reiter's disease, systemic myotonic syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Henoch-Schonlein purpura (Henoch-Schonlein purpura), autoimmune thrombocytopenia, autoimmune diseases of testis and ovary (including autoimmune orchitis and oophoritis), primary hypothyroidism, autoimmune endocrine diseases (including autoimmune Thyroiditis), chronic Thyroiditis (Hashimoto's Thyroiditis), subacute Thyroiditis, idiopathic hypothyroidism, Addison's disease (Addison's disease), Grave's disease, autoimmune multiglandular Syndrome (or multiglandular endocrine Syndrome), type I diabetes (also known as insulin-dependent diabetes mellitus (IDDM)) and Sheehan's Syndrome, autoimmune hepatitis, lymphoid interstitial pneumonia (HIV), NSIP versus bronchiolitis obliterans (non-transplant), Guillain-Barre Syndrome (Guillain-Barre's Syndrome), macrovasculitis (including polymyalgia rheumatica and giant cell (Takayasu's)) arteritis), intermediate vasculitis (including Kawasaki's disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (Berger's disease) (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary sclerosis, sprue (gluten diarrhea), and other diseases, Glomerulosclerosis, hepatitis-related glomerulosclerosis, Amyotrophic Lateral Sclerosis (ALS), coronary artery disease, familial mediterraneanferver, microvascular vasculitis, cochleoveal vestibular syndrome (Cogan's syndrome), viso-odpasture syndrome (Whiskott-Aldrich syndrome) and thromboangiitis obliterans (see WO 2007014278).

Depending on the condition to be treated, the anti-CD 37 antibody molecules of the invention may be used alone or in combination with one or more other therapeutic agents, especially selected from DNA damaging or tubulin binding agents or therapeutically active compounds that inhibit angiogenesis, signal transduction pathways or mitotic checkpoints in cancer cells.

Other therapeutic agents may be administered concurrently with the anti-CD 37 antibody molecule, or before or after administration of the anti-CD 37 antibody molecule, as part of the same pharmaceutical formulation.

In certain embodiments, the additional therapeutic agent may be, but is not limited to, one or more inhibitors selected from the group consisting of: an inhibitor of the EGFR family, VEGFR family, IGF-1R, insulin receptor, auroraA, auroraB, PLK and PI3 kinases, FGFR, PDGFR, Raf, KSP or PDK 1.

Other examples of other therapeutic agents are CDKs, Akts, Src, Bcr-Abl, cKit, cMet/HGF, c-Myc, Flt3, inhibitors of HSP90, hedgehog antagonists, JAK/STAT, Mek, mTor, NF κ B, proteasomes, inhibitors of Rho, inhibitors of Wnt signaling or Notch signaling, or inhibitors of the ubiquitination pathway.

Examples of Aurora inhibitors are, but are not limited to, PHA-739358, AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735, SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237 and ENMD-2076.

An example of a PLK inhibitor is GSK-461364.

Examples of RAF inhibitors are BAY-73-4506 (also a VEGFR inhibitor), PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFR inhibitor), XL-281, and Nevavar (also an inhibitor of VEGFR).

Examples of KSP inhibitors are ipine filab (ispinesib), ARRY-520, AZD-4877, CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355, and EMD-534085.

Examples of src and/or bcr-abl inhibitors are dasatinib (dasatinib), AZD-0530, bosutinib (bosutinib), XL-228 (also IGF-1R inhibitors), nilotinib (also nilotinib) (also PDGFR and cKit inhibitors), imatinib (also cKit inhibitors), NS-187, KX2-391, AP-245634 (also inhibitors of EGFR, FGFR, Tie2, Flt 3), KM-80 and LS-104 (also inhibitors of Flt3, Jak 2).

An example of a PDK1 inhibitor is AR-12.

An example of a Rho inhibitor is BA-210.

Examples of PI3 kinase inhibitors are PX-866, PX-867, BEZ-235 (also an mTor inhibitor), XL-147 and XL-765 (also an mTor inhibitor), BGT-226, CDC-0941.

Examples of cMet or HGF inhibitors are XL-184 (also an inhibitor of VEGFR, cKit, Flt 3), PF-2341066, MK-2461, XL-880 (also an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie 2), SU-11274, PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607, JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, INCB-028060 and E-7050.

An example of a c-Myc inhibitor is CX-3543.

Examples of Flt3 inhibitors are AC-220 (also an inhibitor of cKit and PDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl and Jak 2), MC-2002, SB-1317, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK 2), XL-999 (also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR and cKit).

Examples of HSP90 inhibitors are tanespimycin (tanespimamycin), adriamycin (alvespimycin), IPI-504, STA-9090, MEDI-561, AUY-922, CNF-2024 and SNX-5422.

Examples of JAK/STAT inhibitors are CYT-997 (also interacting with tubulin), TG-101348 (also Flt3 inhibitors), and XL-019.

Examples of Mek inhibitors are ARRY-142886, AS-703026, PD-325901, AZD-8330, ARRY-704, RDEA-119, and XL-518.

Examples of mTor inhibitors are tacrolimus (temsirolimus), deforolimus (which also acts as a VEGF inhibitor), everolimus (furthermore, VEGF inhibitors), XL-765 (also a PI3 kinase inhibitor) and BEZ-235 (also a PI3 kinase inhibitor).

Examples of Akt inhibitors are perifosine (perifosine), GSK-690693, RX-0201 and triciribine (triciribine).

Examples of cKit inhibitors are masitinib, OSI-930 (also used as a VEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR), tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also an inhibitor of VEGFR and PDGFR), sunitinib (also an inhibitor of Flt3, PDGFR, VEGFR) and XL-820 (also used as an inhibitor of VEGFR and PDGFR), imatinib (also an inhibitor of bcr-abl), nilotinib (also an inhibitor of bcr-abl and PDGFR).

Examples of hedgehog antagonists are IPI-609, CUR-61414, GDC-0449, IPI-926 and XL-139.

Examples of CDK inhibitors are Sellissie (seliciclib), AT-7519, P-276, ZK-CDK (which also inhibits VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, PHA-848125, and SCH-727965.

Examples of proteasome inhibitors are bortezomib (bortezomib), carfilzomib (carfilzomib) and NPI-0052 (also inhibitors of NF-. kappa.B).

Examples of proteasome inhibitors/inhibitors of the NF-. kappa.B pathway are bortezomib, carfilzomib, NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957, AVE-8680 and SPC-839.

An example of an inhibitor of the ubiquitination pathway is HBX-41108.

Examples of anti-angiogenic agents are inhibitors of FGFR, PDGFR and VEGF (R) and thalidomide (thalidomide) selected from, but not limited to, bevacizumab (bevacizumab), motinib (motesanib), CDP-791, SU-14813, tiratinib (telatinib), KRN-951, ZK-CDK (also a CDK inhibitor), ABT-869, BMS-690514, RAF-265, IMC-KDR, IMC-18F1, IMiD, thalidomide, CC-4047, lenalidomide (lenalidomide), ENMD-0995, IMC-D11, Ki-23057, brivarnib (brivanib), cediranib (cediranib), 1B3, CP-868596, IMC-3G3, R-D (also Flt 2), sunitinib (kivatib) (also kic 3), and inhibitors of thalidomide (also found in) (also inhibitors of fibrat) and inhibitors of Flt (tilinib) (also inhibitors of bevacizumab) and inhibitors of PKI-493-1530, also found in, Vantalanib (vatalanib), tandutinib (also inhibitors of Flt3 and cKit), pazonib (pazopanib), PF-337210, aflibercept (aflibercept), E-7080, CHIR-258, sorafenib tosylate (also inhibitors of Raf), vandetanib (vandetanib), CP-547632, OSI-930, AEE-788 (also inhibitors of EGFR and Her 2), BAY-57-9352 (also a Raf inhibitor), BAY-73-4506 (also a Raf inhibitor), XL-880 (also a cMet inhibitor), XL-647 (also an inhibitor of EGFR and EphB 4), XL-820 (also an inhibitor of cKit), nilotinib (also an inhibitor of cKit and brc-abl), CYT-116, PTC-299, BMS-584622, CEP-11981, dovidinib, CY-2401401, and ENMD-2976.

The other therapeutic agent may also be selected from an EGFR inhibitor, which may be a small molecule EGFR inhibitor or an anti-EGFR antibody. Examples of anti-EGFR antibodies are, but are not limited to, cetuximab (cetuximab), panitumumab (panitumumab), nimotuzumab (nimotuzumab), zalutumumab (zalutumumab); examples of small molecule EGFR inhibitors are gefitinib (gefitinib), erlotinib (erlotinib) and fantadinib (also VEGFR inhibitors). Another example of an EGFR modulator is EGF fusion toxin.

Other EGFR and/or Her2 inhibitors that may be used in combination with the anti-CD 37 antibody molecules of the invention are lapatinib (lapatinib), trastuzumab (trastuzumab), pertuzumab (pertuzumab), XL-647, neratinib (neratinib), BMS-599626, ARRY-334543, AV-412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also VEGFR inhibitors), AZD-8931, ARRY-380ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769.

Other drugs may also be selected from agents that target the IGF-1R and insulin receptor pathways. The agents include antibodies that bind IGF-1R (e.g., CP-751871, AMG-479, IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab IGFR) and novel chemical entities that target the kinase domain of IGF1-R (e.g., OSI-906 or BMS-554417, XL-228, BMS-754807).

Other agents that may be advantageously combined in therapy with the anti-CD 37 antibody molecules of the invention are molecules that target CD20, including CD 20-specific antibodies (e.g., rituximab, LY-2469298, ocrelizumab (ocrelizumab), MEDI-552, IMMU-106, GA-101(= R7159), XmAb-0367, ofatumumab), radiolabeled CD20 antibodies (e.g., tositumumab (tositumumab) and temepumumab (ibrinomtan)) or other proteins directed against CD20, such as SMIP Tru015, PRO-131921, FBT-a05, valtuzumab (veltuzumab), R-7159.

The CD37 antibody may be combined with inhibitors of other surface antigens expressed on leukocytes, in particular antibodies or antibody-like molecules, such as anti-CD2 (spirizumab)), anti-CD 4 (zanolimumab), anti-CD 19(MT-103, MDX-1342, SAR-3419, XmAb-5574), anti-CD 22 (epratuzumab), anti-CD 23 (lumiximab), anti-CD 30 (imatumab), anti-CD 32B (MGA-321), anti-CD 38(HuMax-CD38), anti-CD 40(SGN40), anti-CD 52 (alemtuzumab), anti-CD 80 (galiximab)). The antibodies of the invention can also be combined with another CD37 antagonist (e.g., TRU-016).

Other agents to be combined with the CD37 antibody are immunotoxins (e.g. BL-22 (anti-CD 22 immunotoxin)), eltuzumab ozogamicin (anti-CD 23 antibody-calicheamicin (calicheamicin) conjugate), rft5.dga (anti-CD 25 ricin a chain), SGN-35 (anti-CD 30 auristatin E conjugate) and gemtuzumab ozogamicin (gemtuzumab ozogamicin) (anti-CD 33 calicheamicin conjugate), MDX-1411 (anti-CD 70 conjugate) or radiolabeled antibodies (e.g. BL-22 (anti-CD 22 immunotoxin)), rituzumab ozogamicin (anti-CD 23 antibody-calicheamicin conjugate), rft5.dga (anti-CD 25 ricin a chain), SGN-35 (anti-CD 30 auristatin E conjugate), gem⁹⁰Y-epratuzumab (anti-CD 22 radioimmunoconjugate)).

Furthermore, anti-CD 37 antibodies can also be combined with immunomodulators, such as antibodies that induce apoptosis or modify signaling pathways, e.g., the TRAIL receptor modulator mapatumab (mapatumumab) (TRAIL-1 receptor agonist), lexatuzumab (lexatuzumab) (TRAIL-2 receptor agonist), tegafuzumab (tigatuzumab), Apomab (Apomab), AMG-951, and AMG-655; anti-HLA-DR antibodies (e.g., 1D09C3), anti-CD 74, inhibitors of osteoclast differentiation factor ligands (e.g., denosumab), BAFF antagonists (e.g., AMG-623a), or Toll-like receptor agonists (e.g., TLR-4 or TLR-9).

Other drugs that may be used in combination with the anti-CD 37 antibody molecule of the invention are selected from, but are not limited to, hormones, hormone analogs and anti-hormones (e.g., tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), fulvestrant (fulvestrant), megestrol acetate (megestrocetate), flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), cyproterone acetate (cyproterone acetate), finasteride (finasteride), buserelin acetate (buserelin acetate), fludrocortisone (fluucocortistin), fludroxymesterone (fludroxymesterone), medroxyprogesterone (medroxyprogesterone), hydroxyprogesterone hexanoate (oxyprogesterone), diethylstilbestrol (dihydrotestosterone), testosterone (ketorolotriacetate), ketoprofen (ketoprofen) antagonists, ketoprofen (ketoprofen), ketoprofen (ketoprofen, ketoprofen (equivalent), ketoprofen (ketoprofen), ketoprofen (ketoprofen, ketoprofen, Dexamethasone (dexamethasone), aminoglutethimide (ainogluthimide)); aromatase inhibitors (e.g., ammedadadine (anastrozole), letrozole (letrozole), liazole (liarozole), exemestane (exemestane), atamestane (atamestane), formestane (formestane)); LHRH agonists and antagonists (e.g., goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin, histrelin, triptorelin); antimetabolites (e.g., antifolates such as methotrexate, trimetrexate, pemetrexed, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, capecitabine, decitabine, nelarabine, 5-azacytidine, gemcitabine, purine and adenosine analogs such as mercaptopurine, thioguanine, azathioprine, cladribine, and stastastaudin, penethazine, fludarabine, chlorambucil, and epirubicin, e.g., epirubicin, and erythromycin), and analogs such as epirubicin, and epirubicin, Bleomycin (bleomycin), actinomycin D (dactinomycin), plicamycin (plicamycin), serromycin (spicamycin), apramycin D (actimycin D), mitoxantrone (mitoxantrone), mitoxantrone idarubicin (mitoxantrone), pyranthrone (pixantrone), streptozotocin (streptozocin), aphidicolin (aphidicolin)); platinum derivatives (e.g., cisplatin (cissplatin), oxaliplatin (oxaliplatin), carboplatin (carboplatin), lobaplatin (lobaplatin), satraplatin (satraplatin)); alkylating agents (e.g., estramustine (estramustine), semustine (semustine), mechlorethamine (mechlorethamine), melphalan (melphalan), chlorambucil (chlorambucil), malilan (busulphan), dacarbazine (dacarbazine), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), hydroxyurea (hydroxyurea), temozolomide (temozolomide), nitrosourea (nitrourea) (such as carmustine (carmustine) and lomustine (lomustine)), thiotepa (thiotepa)); antimitotic agents (e.g., vinblastine (vincalekaloid), such as vinblastine (vinblastine), vinblastine amide (vindesine), vinorelbine (vinorelbine), vinflunine (vinflunine) and vincristine (vinrisine), and taxanes, such as paclitaxel (paclitaxel), docetaxel (docetaxel) and its formulations ralfataxel (larotaxel), simetaxel (simotaxel) and epothilones (epothilones), such as ixabepilone (ixabepilone), patupilone (patupilone), ZK-EPO); topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etoposide phosphate, teniposide, amsacrine, topotecan, irinotecan, banoanthrone, camptothecin (camptothecin)); and miscellaneous chemotherapeutic agents such as retinoic acid derivatives, amifostine (amifostine), anagrelide (anagrelide), interferon alpha, interferon beta, interferon gamma, interleukin-2 (inteleukin-2), procarbazine (procarbazine), N-methylhydrazine (N-methylhydrazine), mitotane (mitotane) and porphine (porfimer), bexarotene (benxotene), celecoxib (celecoxib), ethylenimine (ethylenimine)/methylmelamine, triethylenemelamine (triethylenemelamine), triethylenethiophosphoramide (triethylenephosphoramide), hexamethylmelamine (hexamethylenetetramine), and enzymes L-asparaginase, L-arginase and metronidazole (metazoxazine), misonidazole (metronidazole), metronidazole (methylmetronidazole), metronidazole (thiamethoxazole), nicotinamide-355, nicotinamide (uracil-5), nicotinamide (uracil-ethylene-carbazide), nicotinamide-N-methyl-carbazide (R-carbazide), nicotinamide-carbazide (ethylenediamine-5, nicotinamide-carbazide (R-carbazide), nicotinamide-carbazide (thiamethoxide), nicotinamide-carbazide (thiamethoxazole), nicotinamide-9, nicotinamide-carbazide (thiamethoxazole), nicotinamide-carbazide (thiamethoxazole), nicotinamide-9), nicotinamide-carbazone (thiamethoxide, nicotinamide-carbazide (thiamethoxazole), and (thiamethoxazole), nicotinamide-carbazide (thiamethoxazole), and (thiamethox, 5-iododeoxyuridine (5-iododeoxyuridine), bromodeoxycytidine (bromodeoxycytidine), erythrohydroxynonyladenine (erythrohydroxynonyloxy-adenine), anthracenedione (anthracenedione), GRN-163L (competitive telomerase template antagonist), SDX-101(PPAR agonist), Talarobot (Talarostat) (DPP inhibitor), forodesine (Forodesine) (PNP inhibitor), Alatecept (atacept) (soluble receptor targeting TNF family members BLyS and APRIL), TNF- α neutralizer (Enbrel), famila (Humira), Remicard (Remicadde)), SPC-844 (CHK1/2 inhibitor), VNP-40101M (DNA alkylating agent), SPC-2996 (antisense bci 2 inhibitor), Babytustoura (HDAC 2) (HDAC inhibitor), Wolsartorin (Remustine) inhibitor (Remusosaponin) (Wolson inhibitor), Wolson (Wolson inhibitor) (Wolsins inhibitor), Wolsane (Wolsane) (Wolson inhibitor), Wolson-Na inhibitor (Wolson-60) and Wolson-163L (Wolson-Na inhibitor) (Wolson-2 inhibitor), Wolson-Pvolson-Pk inhibitor (Wolson-Px inhibitor), Wolson-PyS, Wolson-Pl (Wolson, AT-101(Bcl-2/Bcl-xL inhibitor), pridilysin (pletidepsin) (multi-acting depsipeptide), SL-11047 (polyamine metabolism regulator).

In certain embodiments, the anti-CD 37 antibody molecule is used with "CHOP" (a combination of cyclophosphamide, erythromycin hydroxydaunorubicin, vincristine, and prednisone).

The anti-CD 37 antibody molecules of the invention can also be used in combination with other therapies, including surgery, radiation therapy, endocrine therapy, biological response modifiers, hyperthermia and cryotherapy, and agents that attenuate any adverse effects (e.g., antiemetics), G-CSF, GM-CSF, photosensitizers (such as hematoporphyrin derivatives, anti-tumor agents, anti-tumor,Benzoporphyrin derivatives, Npe6, stannsoporphyrin, phenylborolide a (phenobioside-a), bacteriochlorophyll a (bacteriochlorophyl-a), naphthalocyanine (naphthalocyanine), phthalocyanine (naphthalocyanine), zinc phthalocyanine.

Monoclonal antibodies exhibit sensitive antigen specificity and typically react only with human target antigens and not with homologous proteins from animal species. In support of the development of therapeutic antibodies, appropriate animal models for assessing toxicity and pharmacodynamic behavior in vivo may be required. One possibility for in vivo models is a transgenic mouse with an endogenous target antigen replaced with a human homolog ("knockout/knock-in" mouse). In particular, to develop a therapeutic anti-CD 37 antibody, the murine CD37 gene may be replaced by the human CD37 gene. This can be achieved by constructing a targeting vector containing the coding genomic sequence of the human CD37 gene flanked by untranslated sequences. The targeting vector can be used for homologous recombination using mouse ES cells. Homozygous transgenic animals for human CD37 expression can be used to assess the pharmacodynamic effects of antibodies with respect to human CD37, for example, by monitoring the number of peripheral B cells following antibody application. Alternatively, they can be used to study the potential toxic effects of human CD 37-specific antibodies after intravenous (i.v.) use.

In the case of animal cross-reactivity in the absence of monoclonal antibodies, another possibility is the generation of so-called surrogate antibodies. Alternative antibodies are antibodies reactive with homologous proteins of the relevant animal species (e.g., mouse or cynomolgus monkey) that can be used to study drug efficacy and toxic effects. In the case of CD37, monoclonal antibodies specific for cynomolgus CD37 or mouse CD37, respectively, were developed. Ideally, such alternative antibodies should have similar binding and functional properties as the developed antibody. This can be studied by using a test system that utilizes cynomolgus monkey or mouse cells expressing CD37 as target cells, for example for binding FACS Scatchard analysis, ADCC and apoptosis assays can be used. Finally, surrogate antibodies can be selected based on the B cell depleting activity in the blood of cynomolgus monkeys or mice in vitro.

Drawings

FIG. 1: chimeric antibody a0 specifically recognized CD37 antigen as determined by FACS competition assay.

FIG. 2: binding of humanized form of a0 to cellular CD37 antigen as determined by FACS.

FIG. 3: binding of humanized form of a0 to cellular CD37 antigen as determined by FACS.

FIG. 4: affinity of the humanized form of a0 for the cellular CD37 antigen was determined by FACS scatchard analysis.

FIG. 5: ADCC activity of the humanized form of a0 on ramus cells.

FIG. 6: the pro-apoptotic activity of humanized forms of a0 on ramus cells.

FIG. 7: ADCC activity of the Fc-engineered version of mAb a0 on ramus cells.

FIG. 8: ADCC activity of the Fc-engineered version of mAb B0 on ramus cells.

FIG. 9: pro-apoptotic activity of mabs a0 and B0.

FIG. 10: pro-apoptotic activity of Fc-engineered version of mAb a 0.

FIG. 11A: removal of normal human B cells by Fc engineered antibodies a2 and B2, whole blood assay, compared to rituximab.

FIG. 11B: compared with rituximab, the antibody has better B cell removal activity after Fc engineering.

FIG. 11C: antibodies a2 and B2 did not remove T cells and monocytes in the whole blood assay.

FIG. 12: compared with rituximab, ADCC activity is better after Fc engineering.

FIG. 13: removal of ramsbury lymphoma cells by Fc engineered antibodies a2 and B2, whole blood test, compared to rituximab.

FIG. 14: in vivo tumor growth inhibition of ramus xenograft tumors in nude mice by Fc engineered antibodies a2 and B2.

FIG. 15: expression of CD37 on multiple myeloma cells.

FIG. 16: ADCC activity of antibodies a2 and B2 on multiple myeloma cells.

FIG. 17: pro-apoptotic activity of antibodies a2 and B2 on CLL cells from patients.

Example 1

Preparation of chimeric and humanized anti-CD 37 antibodies

a) Preparation of chimeric antibody A0

Based on the variable heavy and light chain amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, the corresponding DNA sequences were synthesized using the optimal codon usage law of mammalian cells (GeneArt, Regensburg, Germany), with the addition of a HindIII cloning site at the 5 'end and a BamH1 cloning site at the 3' end. The synthetic DNA molecules were digested with HindIII and BamHI, and the resulting DNA fragments (SEQ ID NO:1 and SEQ ID NO:3 plus restriction sites) were cloned into pcDNA3.1 based on expression vectors encoding the human IgG1 constant region and the human kappa light chain constant region (SEQ ID NO:24 and SEQ ID NO:26), respectively. EndoFree plasmid preparations (Qiagen) were prepared and the heavy and light chain plasmids were co-transfected into HEK293 free (freestyle) cells (Invitrogen) at a concentration of 1mg/L of each plasmid according to the supplier's protocol. After 72 hours, supernatants were collected and IgG concentrations were determined by ELISA. The resulting chimeric anti-CD 37 antibody (designated a0) was purified on a modified protein a column (GE Healthcare), eluted into citrate buffer, and then dialyzed in PBS.

b) Preparation of a humanized version of chimeric antibody A0

The chimeric mAb a0 obtained in a) was humanized using a CDR grafting method as described, for example, in US5,225,539, US6,548,640, US6,982,321.

To model the structure of mAb A0VL domain, structural templates were selected from the Protein Database (PDB) of Brookhaven National Laboratory. Selection from the murine monoclonal antibody pathway "1KB5" had 88% sequence identity/81% similarity and 2.5VL domain of resolution. For mAb A0VH domain, the identical mouse monoclonal antibody structure "1KB5" with 90% sequence identity and 91% similarity was selected as the primary modeling template. Human vk 1(hVK1) and human VH1(hVH1) types were found to fit the human consensus framework most optimally. As an alternative design, grafts were selected that most stabilized the human consensus domain hVK3 and hVH 3. For transplantation, mAb a0_ VL and mAb a0_ VH models were combined with human consensus domain models hVK1, hVK3, hVH1A and hVH3, and combined to generate Fv models. Loop grafting was performed by embedding the murine mAb A0CDR regions into a human antibody framework, and DNA molecules of the humanized chain constructs were synthesized.

The respective humanized variable regions were synthesized, cloned into immunoglobulin expression vectors, transiently expressed in HEK293 free expression system (Invitrogen) as described a) along with the heavy and light chain sequences shown in table 1, and purified on a protein a column.

Variable heavy and light chain sequences of chimeric and humanized anti-CD 37 antibodies used in the examples of Table 1

c) Preparation of Fc engineered chimeric and humanized anti-CD 37 antibodies

Fc mutants were made as described by Lazar et al, 2006. The resulting Fc-engineered heavy chain sequence was introduced into the expression vector pAD-CMV1 (described in EP 393438) and co-transfected into CHO-DG44 cells along with a plasmid containing the light chain coding sequence. Antibodies were collected from cell culture media 5 to 7 days post transfection and purified via protein a chromatography, eluting into citrate buffer, followed by dialysis in PBS. The protein content of the samples was determined by protein A HPLC and the endotoxin content was determined by Kinetic-QCL Kinetic Chromogenic Assay (Lonza). The monomer content of the samples was determined by HP-SEC, all samples used for the functional test showed a monomer content of > 95%.

TABLE 2

The sequences of the heavy chain variable region and the light chain variable region of the chimeric and humanized anti-CD 37 antibody (columns III and IV) and Fc mutation (column II) (antibodies a0, B0, C0, etc. are identical to antibodies a and B, C, etc. in table 1).

The full length sequences of the heavy and light chains are listed in columns V and VI. (the sequences marked with @, column V, refer to the sequences of IgG1 of SEQ ID NOs 24 and 23 (wild type sequences) modified to have substitutions corresponding to column II, and corresponding mutations in the encoding DNA).

Example 2

Chimeric mAb A0 specifically recognizes CD37 antigen

The specificity of MAb a0 for cellular CD37 was tested in FACS competition experiments on ramus burkitt lymphoma cells (ATCC # CRL-1596). Grouping cellsTissue culture bottle (175 cm)²) Medium, RPMI-1640+ GlutaMAX in flasks as medium and supplemented with 10% heat-inactivated fetal bovine serum, 12.5mM HEPES, 1mM sodium pyruvate, 1% MEM non-essential amino acids. In a humid atmosphere, cells were grown at 3X 10⁵Initial density of individual cells/ml at 37 ℃ with 5% CO₂Cultured for 3 days. Cultures were maintained at 3X 10 by inoculating 2-3 times per week with fresh medium at a 1:6 ratio⁵-1.8×10⁶Cell concentration per ml. FACS competition analysis was performed with the CD 37-specific mAb HH1(Santa Cruz) directly labeled with Phycoerythrin (PE) at a concentration of 1. mu.g/ml. The antibody was preincubated with unlabeled competitor antibody A0 at the indicated molar ratio for 10min at 4 ℃. Thereafter, 1 × 10⁵Individual ramus cells were incubated with the antibody mixture on ice for 30 min. Thereafter, cells were washed twice in Phosphate Buffered Saline (PBS), resuspended in FACS buffer and measured on a BD FACS Canto. The results of such tests are shown in figure 1. Addition of control human IgG1 antibody (Sigma IgG1 κ) in 20-fold molar excess did not significantly reduce the Mean Fluorescence Intensity (MFI) of ramus cells. Addition of unlabelled HH1 antibody or a0 antibody at a 20-fold molar excess almost completely abolished binding of the directly labeled HH1 antibody. This indicates that the a0 and HH1 antibodies recognize the same or similar epitope on ramus cells and compete for binding to the cellular CD37 antigen.

Example 3

Binding of humanized form of mAb A0 to cellular CD37 antigen

The humanized form of a0 was tested for binding to the cellular CD37 antigen by FACS analysis. The antibody was added to the ramus cells at the indicated concentration and allowed to bind for 30min at 4 ℃. Thereafter, bound antibodies were detected with PE-labeled goat anti-human IgG antibody (Sigma), cells were washed 2 times with PBS, after which the cells were 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 forms of a0 showed binding to ramus cells similar to the parent antibody a0, indicating that humanization did not reduce binding to the cellular CD37 antigen.

Example 4

FACS scatchard analysis of humanized versions of chimeric mAb A0

The affinity of the humanized form of antibody A0 (designated B, C, D, H, I and K; see Table 1) for the cellular CD37 antigen was determined by FACS scatchard analysis as described elsewhere (Brockhoff et al, 1994). Briefly, antibody dilutions were prepared in 96-well plates, starting with 100-400nM in the first well (80. mu.l), followed by 11 dilution steps (1:2,40+ 40. mu.l). Add 50. mu.l mAb dilution to FACS tubes and 150. mu.l cells (0.8X 10)⁶/ml=1.2×10⁵Individual cells/tube) were added to each FACS tube. Cells were gently mixed and incubated on ice for 1 h. Thereafter, 50. mu.l of FITC-conjugated secondary antibody (concentration 15. mu.g/ml; mouse mAb anti-hu IgG subclass, Zymed05-4211) was added, mixed, and incubated on ice for 30 min. After this time, 4ml of PBS (ph7.2) containing 0.02% acid was added, the cells were sedimented, resuspended in 300. mu.l of PBS (pH7.2), and FACS analysis was performed using BD FACS Canto. All experimental steps were performed on wet ice and all antibody dilutions were performed in PBS/0.5% BSA +0.02% acid. FACS calibration was performed using Quantum FITC MESF (Premix) high content beads (Bangs Laboratories). All samples were measured using the same FACS parameters. The ratio of bound IgG to free IgG was calculated from the MFI values for different antibody concentrations and displayed as scatchard dots. Fig. 4 shows the MFI/antibody concentration relationship for several of the a0 humanized variants. The results show that some humanized forms bind to ramus cells similarly to the starting antibody with dissociation constant (K)_d) Is 2.15-4.90 nmol/l.

Example 5

ADCC Activity of humanized versions of chimeric mAb A0

The ability of the humanized form of a0 (designated B, C, D, H, J, K; see table 1) to mediate antibody-dependent cell-mediated cytotoxic Activity (ADCC) was evaluated using ramus cells as target cells and IL 2-stimulated human PBMC as effector cells. Lamus cells (Burkitt's lymphoma; ATCC # CRL-1596) were purchased from ATCC. Cells were plated in tissue culture flasks (175 cm)²) Medium, RPMI-1640+ GlutaMAX in flasks as medium and supplemented with 10% heat-inactivated fetal bovine serum, 12.5mM HEPES, 1mM sodium pyruvate, 1% MEM non-essential amino acids. In a humid atmosphere, cells were grown at 3X 10⁵Initial density of individual cells/ml at 37 ℃ with 5% CO₂Cultured for 3 days. Cultures were maintained at 3X 10 by inoculating 2-3 times per week with fresh medium at a 1:6 ratio⁵-1.8×10⁶Cell concentration per ml. The cell density was 1.5X 10⁶/ml-1.8×10⁶Aliquots of logarithmic growth phase cell cultures in ml were centrifuged (200 Xg, i.e., 1000rpm) for 10 min. The cells were washed once with washing medium (RPMI1640, without L-glutamine) and sedimented (200 Xg, i.e.1000 rpm; 10 min). The cell pellet was resuspended in test medium [ RPMI, 1% BSA, L-glutamine free]And counting the cells. Adjusting the cell concentration to 2X 10⁵/ml。

Approximately 50-80ml of whole blood was withdrawn from healthy donors for PBMC isolation. In a 50ml tube, 10ml of whole blood was diluted 1:3.6 with 26ml of HBSS (Hanks' balanced salt solution, without calcium and magnesium). 18ml of diluted whole blood was added to the top of 12ml Lymphoprep (Nycomed Pharma) in a 50ml tube and centrifuged at 370 Xg (1400rpm) for 35 min. Monocytes were aspirated from the interface, washed first with HBSS (750 Xg, i.e. 1900 rpm; 10min), followed by a second wash with HBSS (300 Xg, i.e. 1200 rpm; 10min), and finally with HBSS (160 Xg, i.e. 900 rpm; 10 min). The settled cells were gently resuspended in medium/test medium (RPMI1640 with 10% heat-inactivated human AB serum, without L-glutamine) using a pipette and the cell number was determined in a cell counter. Adjusting PBMC concentration to 1 × 10⁷And/ml. At 37 ℃ and CO₂Tissue culture flasks in a thermostat (75 cm)²) In the process, theFreshly isolated PBMC (5X 10)⁵/ml) was maintained overnight in medium (RPMI1640 with 10% human AB serum, without L-glutamine). The following day, cells were stimulated with hIL-2 at a final concentration of 1U/ml for an additional 3 days. IL-2 stimulated PBMC were isolated from cell debris on a Lymphoprep gradient. Purified and IL-2 stimulated PBMC at 1X 10⁷The suspension was suspended in medium/test medium.

Co-culture of effector cells and target cells in the presence of specific or non-specific antibodies was performed in duplicate or triplicate in 96-well round-bottomed microtiter plates, with a final volume of 200. mu.l of assay medium per well, consisting of 10% human AB serum and 1% BSA in RPMI solution at a 1:1 ratio. First, effector cells (freshly isolated PBMC cells in 100. mu.l per well in 10% human AB serum in RPMI) were plated, followed by target cells and antibody solution diluted in 50. mu.l RPMI containing 1% BSA. As a control, effector cells were cultured in test medium alone (effector cell control) and target cells were cultured in test medium alone (spontaneous lysis) or in test medium supplemented with 1% Triton X-100 (maximal lysis). CO-culture in humid CO₂Incubate at 37 ℃ for 3 hours in an incubator. At the end of the incubation, the cells were removed from the medium by centrifugation (200 Xg, i.e.1000 rpm; 10min) at room temperature. Cell-free supernatant (100. mu.l per well) was transferred to corresponding wells of a 96-well flat-bottom plate. To determine the LDH activity in these supernatants, 100. mu.l of the reaction mixture (250. mu.l of catalyst freshly mixed with 11.25ml of dye solution) was added to each well and incubated at room temperature in the dark for 30 min. Subsequently, the absorbance was measured as follows.

ADCC activity was measured using a cytotoxic activity detection kit (LDH; Roche). The detection of cytotoxic activity is based on the measurement of LDH enzyme activity released by plasma membrane damaged cells. LDH released into culture supernatant reduced the tetrazolium salt from the kit to formazan (formazan). The absorbance maximum of formazan dye at 490nm was measured in an ELISA plate reader relative to a reference wavelength of 650 nm. To calculate the percent cell-mediated cytotoxic activity, five controls were performed in each set of experiments.

Background control I (1): the LDH activity in the medium was tested and subtracted from the values (3) and (5).

Background control II (2): LDH activity in the test medium containing 1% Triton-X100 was subtracted from the maximum LDH release value (4).

Spontaneous LDH release value (3): LDH activity released only by target cells.

Maximum LDH release value (4): maximal releasable LDH activity in target cells.

Effector cell control (5): LDH activity released only when effectively responding to the cells.

To determine the percent cell-mediated cytotoxic activity, the average absorbance of triplicate or duplicate experiments was calculated according to the manufacturer's instructions and the background was subtracted. In figure 5, the results of ADCC assays using 25: 1E: T ratio and ramus target cells are shown. Antibody was added at a concentration of 30 ng/ml. The starting mabs and humanized forms thereof showed similar ADCC activity against ramus cells. It can be seen that humanization of anti-CD 37mAb a does not significantly alter its ability to induce ADCC.

Example 6

Pro-apoptotic Activity of humanized forms of chimeric mAb A0

The pro-apoptotic activity of mAb A0(= A) and humanized forms thereof (B, C, D and I; see Table 1) was assessed by incubating Lamus cells with mAb and measuring Annexin (Annexin) V/PI positive cells. The Lamos cells (Burkitt's lymphoma; ATCC # CRL-1596) were derived from ATCC.

Cells were plated in tissue culture flasks (175 cm)²) Medium, RPMI-1640+ GlutaMAX in flasks as medium and supplemented with 10% heat-inactivated fetal bovine serum, 12.5mM HEPES, 1mM sodium pyruvate, 1% MEM non-essential amino acids. In a humid atmosphere, cells were grown at 3X 10⁵One cell/milliInitial density in liters at 37 ℃ 5% CO₂Cultured for 3 days. Cultures were maintained at 3X 10 by inoculating 2-3 times per week with fresh medium at a 1:6 ratio⁵-1.8×10⁶Cell concentration per ml. The cell density was 1.5X 10⁶/ml-1.8×10⁶Aliquots of logarithmic growth phase cell cultures in ml were centrifuged (200 Xg, i.e., 1000rpm) for 10 min. The cells were washed once with washing medium (RPMI1640, without L-glutamine) and sedimented (200 Xg, i.e.1000 rpm; 10 min). The cell pellet was resuspended in medium and cell counting was performed. Adjusting the cell concentration to 1X 10⁶And/ml. 100 μ l of cell suspension per well was plated in a 96-well round bottom plate. The antibody was diluted in cell culture medium containing 10% FBS, and 100. mu.l of the antibody solution was added per well. Cells in CO₂Incubations were carried out at 37 ℃ for 20 to 24 hours in an incubator, after which staining was carried out with Vybrant apoptosis test kit # 2. Alexa Fluor 488-labeled annexin V and propidium iodide solution were added to the cells and incubated for 15min in the dark. Thereafter, cells were resuspended in 400. mu.l annexin V binding buffer and FACS analysis was performed using BD FACS Canto. The percentage of annexin V positive/PI negative cells and annexin V/PI positive cells was determined by two-dimensional dot blot using FL1/FL2 channel. Isotype matched unbound antibody (Sigma human IgG1) was used as a negative control.

FIG. 6 shows the pro-apoptotic effect of various humanized forms of mAb A on Lamus cells. Cells were incubated with 10 μ g/ml of antibody for 24 hours, showing the total percentage of annexin V positive cells (PI positive and PI negative). The parent mAb a showed strong pro-apoptotic activity. Surprisingly, the humanized form showed a significantly reduced number of annexin V positive cells compared to the parent mAb a, indicating that the pro-apoptotic activity of the humanized antibody was altered. It can be seen that humanization of MAb a reduced its pro-apoptotic activity in this set of experiments.

Example 7

ADCC Activity of Fc engineered forms of chimeric mAb A0

ADCC activity of Fc-engineered forms of mAb a0 (designated a1, a2, A3, a4, see table 2) were evaluated using ramus cells as target cells. ADCC assays were performed as described above (example 5). The results of the experiment are shown in fig. 7. The Fc-engineered version of a0 was a significant improvement over the parent mAb a0 in both potency and efficacy (efficcy). Some Fc variants have maximal lytic amplification of up to 100%, EC, compared to the parent mAb₅₀The amplification is up to 10 times. It can be seen that the introduction of specific Fc mutations greatly increased the ADCC activity of the chimeric mAb a 0.

Example 8

ADCC Activity of Fc-engineered forms of mAb B0

The ADCC activity of the Fc-engineered form of mAb B0 (designated B1, B2, B3, B4; see Table 2) was evaluated using Lamus cells as target cells. ADCC assays were performed as described above (example 5). The Fc-engineered version of B0 was a significant improvement in both potential and efficacy compared to the parent mAb B0. Some Fc variants showed maximal lytic amplification of up to 80%, EC, compared to the parental mAb₅₀The amplification is up to 20 times. It can be seen that the introduction of specific Fc mutations greatly increased the ADCC activity of the chimeric mAb B0. The results of the experiment are shown in FIG. 8.

Example 9

Proapoptotic activity of mAbs A0 and B0

The pro-apoptotic activity of mabs a0 and B0 on ramus cells before and after cross-linking with anti-IgG mabs is shown in fig. 9. For antibody cross-linking, an anti-human IgG antibody (gamma-chain specificity; Sigma) was added to the antibody at a ratio of 1:1, incubated at 37 ℃ for 15min, followed by addition of target cells. In FIG. 9, both crosslinked and uncrosslinked CD 37-specific mAbs were added at a concentration of 1. mu.g/ml. Chimeric mAb a0 was a potent inducer of apoptosis even when not cross-linked, this effect being significantly enhanced after mAb cross-linking. Surprisingly, the non-crosslinked humanized mAb B0 completely lacked pro-apoptotic activity, yet showed potent pro-apoptotic activity after crosslinking with anti-IgG Ab. In summary, this experiment shows that the pro-apoptotic activity of humanized mAb a0 can be restored after antibody cross-linking.

Example 10

Fc engineered forms of mAb A0 for pro-apoptotic activity

The pro-apoptotic activity of the Fc-engineered form of chimeric mAb a0 on ramus cells was assessed by annexin V/PI staining as described in example 6. The parent antibody a0 and the Fc engineered variants a2 and a4 were titrated at a concentration range of 0.1 to 10.000 ng/ml. As can be seen in fig. 10, all 3 antibodies showed similar pro-apoptotic activity. In conclusion, this experiment shows that Fc engineering of mAb a0 does not alter its pro-apoptotic activity.

Example 11

a) B cell removal Activity of Fc engineered antibodies A2 and B2 in Whole blood assay

The potential and efficacy of removing normal B cells from human blood was assessed using a whole blood assay. In this assay format, the test antibody is added to an EDTA-treated sample of a healthy human blood sample, and after 3 to 4 hours of incubation at 37 ℃, the number of B cells is quantitatively measured by a 4-color FACS assay. The extent to which the test agent removes B cells can be calculated by comparison to a buffer or IgG control. This type of assay is believed to be highly relevant for predicting the in vivo efficacy of the test antibody due to the presence of human IgG levels and effector cells similar to those found in humans.

The number of B cells and/or the number of added (spiked) Lamos cells in blood samples of healthy individuals was determined by quantitative FACS assay. Quantitative analysis was performed using BD trount tubes containing known numbers of fluorescent beads as internal standards for quantification of the target cell population. 4-color analysis was performed with 4 different CD markers (CD3/CD14/CD19/CD45) in combination with FSC/SSC analysis to identify B cells.

Mu.l of fresh blood per well was incubated in duplicate in 48-well plates with 30. mu.l of antibody dilution (in PBS) or PBS (buffer control). The samples were incubated at 37 ℃ for 4h, immediately thereafter on ice. Mu.l of CD-labeled master mix was added to the Trucount tubes and 50. mu.l of blood-antibody mix was added. The sample was vortexed and incubated at room temperature for 15 minutes. Thereafter, 450. mu.l lysis buffer was added, vortexed and incubated at room temperature for a further 15 minutes. Samples were placed on ice and immediately processed with a BD FACS Canto^TMFACS analysis was performed with flow cytometry. Data evaluation was performed with BD FACSDiva software (version 5.0.2).

Fc-engineered chimeric and humanized mAbs A2 and B2 show excellent potential for normal B cell depleting capacity with EC₅₀The value was 0.15-0.35 nM. Normal B cell depletion levels ranged from 57% to 65%. Rituximab (an antibody approved for treatment of B-NHL) was tested in parallel in this assay format and its B-cell depleting activity was significantly lower (fig. 11A).

b) Fc engineering B cell removal activity of A0 and B0 was better than rituximab

The effect of mAb on B cell depletion in blood of healthy humans was evaluated as described under a). The non-Fc engineered mabs a0 and B0 showed B cell removal activity of 13% -26%, similar to rituximab. Fc engineering significantly increased B cell removal activity for both mabs, with an average percent removal of 75%. This clearly demonstrates the superiority of a2 and B2 over rituximab (fig. 11B).

c) Antibodies A2 and B2 did not remove T cells and monocytes in whole blood assays

In parallel to the evaluation of the effect on B lymphocytes, the effect of a2 and B2 on T lymphocytes (CD3+) and monocytes (CD14+) was also evaluated. No significant change in T cell number or monocyte number was observed, but a significant reduction in B cell number was observed (fig. 11C). This indicates that a2 and B2 specifically remove B cells from human blood.

Example 12

Fc engineering results in better ADCC activity than rituximab

ADCC activity of Fc-engineered form a2 of mAb a0 was assessed using ramus cells as target cells. ADCC assays were performed as described above (example 5). The maximum lysis of the ramus target cells by antibody a0 without Fc engineering was inferior to rituximab (an antibody specific for CD20 approved for the treatment of patients suffering from B-cell lymphoma). It was surprisingly found that Fc engineering of a0 allowed a2 to be a significant improvement over rituximab in both potency and efficacy. This shows that the ADCC activity of Fc-engineered anti-CD 37mAb a2 was significantly better than that of rituximab when the antigen densities of CD20 and CD37 on ramus cells were similar (figure 12).

Example 13

Lymphoma cell removal activity of Fc engineered antibodies A2 and B2 in whole blood assay

The potential and efficacy of ramus cell (burkitt lymphoma cell line from human blood) for inactivating activity was assessed using the whole blood assay described in example 11. In a modified version of this assay, ramus tumor cells are added (spike in) to the whole blood matrix in about a ten-fold excess compared to endogenous B cells, the removal of which is monitored by FACS analysis. Fc engineered chimeric and humanized mAbs A2 and B2 show good potential for Ramopsis cell removal activity, their EC₅₀The values were 0.35-0.54 nM. The ramus cell removal level was 36% -55%. Rituximab (antibody approved for treatment of B-NHL) was tested in parallel in this assay format and its ramus cell removal activity was significantly lower (fig. 13).

Example 14

In vivo efficacy of Fc engineered antibodies A2 and B2 in disease-related models

The in vivo anti-tumor efficacy of mAbs A2 and B2 was assessed using a Ramophilt lymphoma nude mouse model. CD37 positive ramus cells were injected subcutaneously into the animal's flank and when tumors formed, intravenous treatment of the animals was initiated. Twice weekly treatment regimens (q3/4d) were selected and two different doses (8mg/kg and 25mg/kg) were tested in parallel. Both mabs showed significant anti-tumor efficacy with T/C values of 0.2% -26%. No significant differences were observed between the two dose levels and between the two antibodies. However, there was a tendency for better efficacy in animals treated with high doses of a2, with a T/C of 0.2%, and a complete regression of 5/10 tumors. All treatments were well tolerated without significant weight loss. In summary, mabs a2 and B2 showed significant anti-tumor efficacy in a ramosburkitt lymphoma model, with maximal activity obtained at the 8mg/kg dose level. This activity was comparable to that of rituximab tested in parallel. It should be noted that the in vivo activity observed in Fc engineered antibodies a2 and B2 may be underestimated because these mabs are optimized for interaction with human effector cells rather than murine effector cells. This optimized interaction results in a substantial improvement of ADCC activity in vitro when human effector cells are used (example 8), but is not reflected in the mouse model used. However, the data obtained in this experiment (shown in figure 14) provides in vivo evidence that the concept of targeting CD37, and thus can be used to assess the therapeutic dose in humans.

Example 15

Association of Pharmacokinetics (PK) and Pharmacodynamics (PD) of A2 and B2 in mice for evaluation of therapeutic doses in humans

Using a ramus tumor xenograft model, correlations between serum concentrations of a2 and B2 and their pharmacodynamic effects were established in mice. These studies demonstrated that doses of 8mg/kg A2 and B2 (formulated in citrate buffer: 25mM sodium citrate, 115mM NaCl, 0.04% Tween80, pH6.0) caused significant delays in tumor growth in this aggressive subcutaneous (s.c.) tumor model when using standard q3 or 4d antibody dosing regimens in mice, indicating sustained activity throughout the dosing interval. In addition, pharmacokinetic data for the same dose were also established.

Using this PK/PD correlation in mice, the estimated dose in humans can be calculated using published data for humanized antibody Clearance (CL) in humans (Lobo et al, 2004).

Complete calculation of a 2:

mean AUC (0- ∞) = 6099. mu.g.h/mL after single dose of 8mg/kg

The specified AUC (0- ∞) = AUC (ss, τ) in mice, and AUC (ss, τ)/τ = C (ave, ss) in mice

C (ave, ss) (τ =84 hours) =73 μ g/mL in mice, assuming it is equivalent to C (ave, ss) (τ =168h) in humans.

Because AUC (ss, τ) = D/CL in humans, and using the humanized antibody Clearance (CL) range in humans reported by Lobo et al 2004: CL =7mL/h/70kg to 15mL/h/70 kg.

For 7mL/h/70 kg: 168hr × 7=1176mL × 73 μ g =86 mg.

For 15mL/h/70 kg: 168hr × 15=2520mL × 73 μ g =184 mg.

Thus, for a 70kg person, the weekly estimated dose of A2 is 86-184 mg. Using the same assumptions as above, the weekly estimated human dose of B2 was calculated to be 189 to 404mg for a 70kg human.

Example 16

Antibodies A2 and B2 exhibit ADCC activity against multiple myeloma cells

Expression of CD37 on a panel of multiple myeloma cell lines by FACS analysis using CD37 specific antibodiesTo evaluate. Cells were incubated with either a direct fluorescently labeled anti-CD 37 antibody or with an unlabeled CD37 specific antibody, followed by incubation with a fluorescently labeled secondary antibody against the primary antibody. Fluorescence activity of the labeled cells was measured with a FACS Canto flow cytometer (BD Biosciences) and fluorescence intensity was recorded as MFI with FACS Diva software. Of the 11 multiple myelomas tested, 6 showed cell surface expression of CD37 (fig. 15). Next, the cell line (RPMI8226) was tested with CD 37-specific antibodies a2 and B2 according to the ADCC assay described in example 5. Both antibodies demonstrated potent ADCC activity on RPMI8226 cells, EC₅₀The value was 25ng/ml, with a maximum cell lysis of about 20% (FIG. 16). This example demonstrates that CD 37-positive multiple myeloma cells are susceptible to ADCC-mediated cell lysis using CD 37-specific mabs a2 and B2.

Figure 15 shows FACS analysis of CD37 expression for 6 multiple myeloma cell lines. Open curves indicate reactivity with antibodies specific for CD37, and filled curves indicate negative control antibodies.

Example 17

Proapoptotic activity of antibodies A2 and B2 on CLL cells from patients

The pro-apoptotic activity of a2 and B2 was evaluated on Chronic Lymphocytic Leukemia (CLL) cells derived from patients. Peripheral Blood Mononuclear Cells (PBMC) were prepared from patients diagnosed with CLL who had previously had informed consent under the declaration of Helsinki. By usingThe plus method (StemCell Technologies, maylan, France) purified primary CLL cells from freshly collected blood and stored at 4 ℃ in RPMI1640 medium with 10% heat-inactivated human AB serum (Sigma, France) for use. The culture medium of primary CLL cells was RPMI1640 supplemented with 2 ml-glutamine and 10% heat-inactivated human AB serum. For the experiments, primary CLL cells were used in a hemocytometerThe counts were counted by a counter and their viability was assessed by the 0.25% trypan blue exclusion method. The viability of CLL samples was above 90%. The percentage of annexin V positive cells was determined after incubation of the cells with 30. mu.g/ml antibody for 24 hours at 37 ℃ as described in example 6. As shown in fig. 17, Fc-engineered antibodies a2 and B2 showed potent pro-apoptotic activity on primary CLL cells with annexin V positive cells at about 90% (a2) and 40% (B2), respectively. Both mabs are significantly superior to rituximab, a B-cell specific antibody that has been approved for treatment of B-NHL. Mab a2 was also significantly better active than alemtuzumab, an antibody approved for treatment of B-CLL.

Example 18

A transgenic mouse model was generated in which the endogenous CD37 gene was replaced by its human homolog.

A targeting vector was constructed containing the coding sequence of human CD37(BAC (bacterial artificial chromosome) ID: RP11-433N13, RP11-50I11) flanked by non-translated sequences. The targeting vector also contains loxP sites flanking exons 3-4, and a neo selection marker flanked by frt sites. Homologous recombination is then performed using the targeting vector and mouse ES cells according to standard techniques to replace exons 1-8 of the mouse genomic sequence with the corresponding human sequences. To this end, C57BL/6N ES cell lines were grown on mitotically inactive feeder layers containing Mouse Embryonic Fibroblasts (MEFs) in DMEM high glucose medium supplemented with 20% FBS (PAN) and 1200u/mL leukemia inhibitory factor (Millipore ESG 1107). Under 240V and 500F, 1X 10⁷Cells and 30g of linearized DNA vector were electroporated (Biorad Gene Pulser). The G418 selection (200G/mL) was started on day 2. Counter-selection with ganciclovir (2M) started on day 5 after electroporation (d 5). ES clones were isolated on day 8 (d8), amplified and frozen in liquid nitrogen, and analyzed by Southern blotting according to standard methods, for example, by using radiolabeled DNA probes specific for the target gene. Then, by standards known in the artMethods, such as the production of transgenic animals by blastocyst injection (blastocyst injection) followed by the production of chimeric animals. The hybrid and homozygous animals of human CD37 are obtained by conventional breeding of chimeric and heterozygous animals respectively. Successful gene knock-out of the murine CD37 gene and knock-in of the human CD37 gene were monitored at the protein level using standard methods, such as FACS analysis of peripheral blood lymphocytes or immunohistochemical analysis of tissue sections.

Example 19

Preparation of surrogate antibody (survivate antibody)

Mice and rabbits were genetically immunized with the complete coding sequence of the macaque (macaque) CD37 antigen (accession number ENSMMUT00000020744) to produce macaque CD37 specific monoclonal antibodies. Specific antibodies were selected using recombinant HEK293 or CHO cells expressing the cynomolgus CD37 antigen using, for example, standard ELISA or FACS techniques. The variable heavy and light chain coding sequences of these antibodies were recovered by PCR cloning and used (as described in example 1) to generate chimeric antibodies having VH and VL regions derived from murine or rabbit starting antibodies and an Fc portion identical to that of an antibody of the invention (e.g., a2 or B2). Binding and functional properties can be studied using assays such as assay systems using cynomolgus CD37 expressing cells as target cells for e.g. binding, FACS, Scatchard analysis, ADCC and apoptosis assays. Finally, surrogate antibodies were selected based on the in vitro B cell depletion activity of Cynomolgus monkey (Cynomolgus monkey) blood.

Example 20

Preparation of clones producing the antibodies

To prepare clones for use in generating antibodies of the invention (e.g., antibodies A2, A4, B2, or B4), DNA molecules encoding the entire heavy chain (e.g., having the sequence shown in SEQ ID NOs: 27, 31, 35, or 39, respectively) are inserted into eukaryotic expression vector pBI-26, which also encodes a hamster-derived dihydrofolate reductase selectable marker.

DNA molecules encoding the complete light chain shown in SEQ ID Nos 29, 33, 37 and 41, respectively, were inserted into eukaryotic expression vector pBI-49, which also encodes a neomycin phosphotransferase selection marker. The DNA sequences of the entire heavy and light chains were fully sequenced.

For the hamster cell line CHO-DG44, which grew in suspension in chemically synthesized medium (chemically defined media), the eukaryotic expression vectors encoding the antibody heavy and light chains described above were co-transfected. Transfected cells were selected in medium without hypoxanthine and thymidine but with the G418 antibiotic. The cells were then stepwise selected and expanded with increasing concentrations of Methotrexate (MTX). In the 800nM MTX amplification step, one Cell line was selected based on growth performance and antibody production in a spinner run and cryopreserved in Safety Cell Bank (SCB).

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Claims (9)

1. An antibody molecule that binds to human CD37 and is derived from an antibody that:

a) murine monoclonal antibodies were defined as follows:

i) a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO 2; and

ii) a variable light chain comprising the amino acid sequence shown in SEQ ID NO 4,

wherein the antibody is a humanized antibody defined as:

a) the CDRs contained in the variable heavy chain shown in SEQ ID NO. 2,

b) CDRs contained in the variable light chain shown in SEQ ID NO. 4,

c) a framework derived from a human antibody and supporting the CDRs,

d) from the constant heavy and light chains of human antibodies.

2. The antibody of claim 1, comprising a variable heavy chain having the sequence shown in SEQ ID NO 6 and preferably a variable light chain having the sequence shown in SEQ ID NO 12.

3. The antibody of claim 1 or 2, wherein the antibody has one or more mutations in the Fc region that alter one or more effector functions, preferably the altered effector function is an increase in antibody-dependent cell-mediated cytotoxic activity.

4. The antibody of claim 3, wherein the one or more mutations of the Fc region are a combination of substitutions at positions 239 and 332, as numbered by the Kabat EU numbering index, or wherein the one or more mutations of the Fc region are a combination of substitutions at positions 236, 239 and 332, as numbered by the Kabat EU numbering index, and wherein the substitutions are preferably I332E, S239D and G236A.

5. An antibody which binds to human CD37 and has a heavy chain comprising the amino acid sequence SEQ ID NO 36 and preferably a light chain comprising the amino acid sequence SEQ ID NO 38.

6. An antibody that binds to human CD37 and has a heavy chain comprising the amino acid sequence SEQ ID NO 40 and preferably a light chain comprising the amino acid sequence SEQ ID NO 42.

A DNA molecule comprising a region encoding the variable heavy chain of an antibody of any one of claims 1 to 6.

8. The DNA molecule of claim 7, wherein the variable heavy chain coding region is fused to a region encoding a constant heavy chain of human origin, preferably the human constant heavy chain is IgG1, and preferably the human constant heavy chain has one or more substitutions in the Fc region as defined in claim 4.

9. The DNA molecule of claim 8, wherein said IgG1 is encoded by the sequence set forth in SEQ ID NO. 23, or SEQ ID NO. 39, or SEQ ID NO. 35.