RU2402568C2 - Human anti-cd38-antibodies and their application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: described are isolated human antibodies or humanized antibodies, or their functional fragments, specific with respect to epitope CD38. Presented are variable areas of heavy and light chain of described antibodies. Claimed is pharmaceutical composition, which contains described antibody, or its functional fragment. Claimed is method of treating malfunction or condition, connected with undesirable presence of CD38+ cells, which includes introduction of efficient quantity of claimed pharmaceutical composition to individual. Isolated epitope CD38 is described.

EFFECT: invention allows to mediate lysis of cells, which overexpress CD38, efficiently.

53 cl, 16 dwg, 6 tbl, 11 ex

Description

The present invention claims priority in the following provisional patent applications: No. 60/541911, filed February 6, 2004, No. 60/547584, filed February 26, 2004, No. 60/553948, filed March 18, 2004, and No. 60/599014, filed August 6, 2004, which are incorporated into this description by reference in their entirety.

State of the art

CD38 is a membrane type glycoprotein of the second type, which belongs to the ectoenzyme family due to its enzymatic activity as ADP-ribosyl cyclase and cADP-hydrolase. During ontogenesis, CD38 appears on CD34 + committed stem cells and line-specific committed progenitor cells of lymphoid, erythroid and myeloid cells. It is believed that the expression of CD38 is retained only in lymphoid progenitor cells during the early stages of development of T and B lymphocytes.

The increased regulation of CD38 serves as a marker for the activation of lymphocytes, in particular the differentiation of B-lymphocytes into plasmocytes. The (Co) receptor functions of the CD38 receptor, leading to intracellular signal transduction or intercellular communication through its CD31 ligand, as well as its role as an intracellular regulator of the second messenger, cyclic ADP-ribose (ADP-p), are postulated in a variety of signaling cascades . However, it is necessary to clarify its physiological significance, starting with the knockout of its mouse analogue or human anti-CD38 autoantibodies that do not exhibit harmful properties.

In addition to observing its expression in the hematopoietic system, the researchers noted upregulation of CD38 in various cell lines derived from B-, T- and myeloid / monocytic tumors, including B- or T-cell acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) ), non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). For example, pronounced expression of CD38 is proven in most of all observed patients with MM.

Therefore, overexpression of CD38 in malignant cells provides an advantageous therapeutic target for immunotherapy. Of particular interest is the fact that the most primitive pluripotent stem cells of the hematopoietic system are CD38-negative, and that the magnitude of the cytotoxic effects of antibody-dependent cell-mediated cytotoxicity of ADCC or complement-dependent cytotoxicity of CDC correlates well with the expression levels of the corresponding target.

Current approaches to anti-CD38 therapy can be divided into two groups, which are in vivo and ex vivo approaches. In an in vivo approach, anti-CD38 antibodies are administered to a subject in need of therapy in order to induce antibody-mediated depletion of malignant cells overexpressing CD38. Depletion can be achieved by either ADCC and / or CDC using effector cells or by using anti-CD38 antibodies as targeted functional groups for transporting cytotoxic substances, for example saporin, to target cells, and subsequent internalization. With an ex vivo approach, a cell population, for example, bone marrow cells containing CD38 over-expressing malignant cells, is removed from the subject in need of therapy and contacted with anti-CD38 antibodies. The target cells are either destroyed by cytotoxic substances, for example saporin, as described in the in vivo approach, or removed by contacting the cell population with immobilized anti-CD38 antibodies, thereby removing over-expressing CD38 target cells from the mixture. After this, the depleted cell population is back administered to the patient.

Depending on various properties, antibodies specific for CD38 can be divided into different groups. The binding of certain antibodies to the CD38 molecule (mainly with amino acids 220-300) can trigger activities in the target cell such as Ca ²⁺ release, cytokine release, phosphorylation effects and growth stimulation based on the corresponding specificity of the antibody (Konopleva et al., 1998 ; Ausiello et al., 2000), but no clear correlation was found between the binding site of various known antibodies and their (non-) agonistic properties (Funaro et al., 1990).

Relatively little is known about the effectiveness of published anti-CD38 antibodies. It is known that all known antibodies recognize epitopes (amino acid residues from 220 to 300) located only at the C-terminal region of CD38. To date, no antibodies specific for epitopes at the N-terminal portion of CD38 located in the primary protein sequence far from the active site have been found. However, the inventors of the present invention have found that OKT10, for which clinical testing was performed, has a relatively low affinity and efficacy in the analysis of a chimeric construct containing human Fc regions. In addition, OKT10 is a murine antibody, which makes it unsuitable for administration to humans. A fragment of a human anti-CD38 scFv antibody has recently been described (WO 02/06347). However, this antibody is specific for the selectively expressed CD38 epitope.

Accordingly, in light of the high potential for therapy with anti-CD38 antibodies, there is a great need for human anti-CD38 antibodies with high affinity and high efficiency ADCC and / or CDC-mediated lysis of malignant cells overexpressing CD38.

The present invention satisfies these and other needs by providing fully human and highly effective anti-CD38 antibodies, which are described below.

SUMMARY OF THE INVENTION

An object of the present invention is to provide human and humanized antibodies capable of efficiently mediating the lysis of cells overexpressing CD38.

Another objective of the invention is the provision of antibodies that are safe for administration to humans.

An object of the present invention is also to provide methods for treating a disease and / or conditions associated with increased regulation of CD38 using one or more antibodies of the invention. These and other objectives of the present invention are described more fully below.

In one aspect, the present invention provides an isolated antibody, or a functional fragment of an antibody that contains an antigen binding region that is specific for the CD38 epitope, wherein the antibody or its functional fragment is capable of mediating the lysis of CD38 + target cells (LP-1 (DSMZ: ACC41) and RPMI-8226 (ATCC: CCL-155)) by antibody-dependent cell-mediated cytotoxicity ("ADCC") with an efficiency of at least two to five times that of the chimeric OKT10 antibody having the sequence of SEQ ID NO: 23 and 24 (in about under identical or essentially identical conditions) when using human PBMC peripheral blood mononuclear cells as effectors, and when the ratio of the number of effectors to the number of target cells is in the range from 30: 1 to 50: 1. Such an antibody or functional fragment thereof may comprise an antigen binding region that contains the H-CDR3 region shown in SEQ ID NO: 5, 6, 7 or 8; the antigen binding region may further include an H-CDR2 region shown in SEQ ID NO: 5, 6, 7 or 8; and the antigen binding region may also contain the H-CDR1 region shown in SEQ ID NO: 5, 6, 7 or 8. Such a CD38-specific antibody according to the invention may contain an antigen binding region that contains the L-CDR3 region shown in SEQ ID NO: 13, 14, 15 or 16; the antigen binding region may further include the L-CDR1 region shown in SEQ ID NO: 13, 14, 15, or 16; and the antigen binding region may also contain the L-CDR2 region shown in SEQ ID NO: 13, 14, 15 or 16.

In another aspect, the present invention relates to an isolated antibody or functional fragment of an antibody containing an antigen-binding region that is specific for the CD38 epitope, wherein the antibody or its functional fragment is capable of mediating CD38-transfected CHO cell lysis by CDC with at least a double efficiency exceeding the effectiveness of the chimeric OKT10 (SEQ ID NO: 23 and 24) under identical or substantially identical conditions, as indicated in the previous paragraph. An antibody that meets these criteria may contain an antigen binding region that contains the H-CDR3 region shown in SEQ ID NO: 5, 6, or 7; the antigen binding region may further include an H-CDR2 region shown in SEQ ID NO: 5, 6 or 7; and the antigen binding region may also contain the H-CDR1 region shown in SEQ ID NO: 5, 6 or 7. Such a CD38-specific antibody according to the invention may contain an antigen binding region that contains the L-CDR3 region shown in SEQ ID NO: 13, 14 or 15; the antigen binding region may further include the L-CDR1 region shown in SEQ ID NO: 13, 14 or 15; and the antigen binding region may also contain the L-CDR2 region shown in SEQ ID NO: 13, 14 or 15.

Antibodies (and their functional fragments) according to the invention may contain an antigen-binding region that is specific for the CD38 epitope, and the epitope contains one or more amino acid residues CD38 43-215, as shown in SEQ ID NO: 22. More specifically, the epitope to which the antigen binding region binds may contain one or more amino acid residues found in one or more amino acid sequencing fragments taken from the catalog of amino acid fragments 44-66, 82-94, 142-154, 148-164, 158 -170 and 192-206. For some antibodies, the epitope may be linear, while for others it may be conformational (i.e., discontinuous). An antibody or functional fragment thereof having one or more of these properties may comprise an antigen binding region that contains the H-CDR3 region shown in SEQ ID NO: 5, 6, 7, or 8; the antigen binding region may further include an H-CDR2 region shown in SEQ ID NO: 5, 6, 7 or 8; and the antigen binding region may also contain the H-CDR1 region shown in SEQ ID NO: 5, 6, 7 or 8. Such a CD38-specific antibody according to the invention may contain an antigen binding region that contains the L-CDR3 region shown in SEQ ID NO: 13, 14, 15 or 16; the antigen binding region may further include the L-CDR1 region shown in SEQ ID NO: 13, 14, 15, or 16; and the antigen binding region may also contain the L-CDR2 region shown in SEQ ID NO: 13, 14, 15 or 16.

Peptide variants of the disclosed sequences are also included in the scope of the present invention. Accordingly, the present invention includes anti-CD38 antibodies having a heavy chain of an amino acid sequence having a sequence of at least 60 percent identical in the hypervariable CDR regions to the CDR regions shown in SEQ ID NO: 5, 6, 7 or 8 ; and / or a sequence of at least 80 percent identical in the CDR regions to the CDR regions shown in SEQ ID NO: 5, 6, 7, or 8. Anti-CD38 antibodies having an amino acid light chain are further included in the scope of the present invention. a sequence having a sequence of at least 60 percent identical in the CDR regions to the CDR regions shown in SEQ ID NO: 13, 14, 15 or 16; and / or a sequence of at least 80 percent identical in the CDR regions homologous to the CDR regions shown in SEQ ID NO: 13, 14, 15 or 16.

The antibody of the invention may be IgG (e.g., IgG _I ), while a fragment of the antibody may be, for example, Fab or scFv. A fragment of an antibody according to the invention may, accordingly, be or may contain an antigen binding region that can manifest itself in one or more of the ways described herein.

The present invention also relates to isolated nucleic acid sequences, each of which can encode a binding region with a human antibody or a functional fragment thereof that is specific for the CD38 epitope. Such a nucleic acid sequence can encode an antibody variable heavy chain and includes a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, or 4, or a nucleic acid sequence that hybridizes under stringent conditions with the complementary strand of SEQ ID NO : 1, 2, 3, or 4. The nucleic acid may encode a variable light chain of an isolated antibody or functional fragment thereof and may comprise a sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, or 12, or a sequence nucleic acid that hybridizes under stringent conditions with the complementary strand of SEQ ID NO: 9, 10, 11 or 12.

Nucleic acids according to the invention are suitable for recombinant production. Thus, the invention also relates to vectors and host cells containing the nucleic acid sequence of the invention.

The compositions of the invention can be used for therapeutic or prophylactic use. In this regard, the present invention includes a pharmaceutical composition comprising an antibody of the invention (or a functional fragment of an antibody) and a pharmaceutically acceptable carrier or excipient thereof. In a related aspect, the present invention relates to a method for treating a disorder or condition associated with the undesired presence of CD38 or cells expressing CD38. As indicated or contemplated by the present invention, such a method comprises the steps of administering to a subject in need thereof an effective amount of a pharmaceutical composition that comprises an antibody of the invention.

The present invention also relates to isolated CD38 epitopes, both in linear and in conformational form, and their use for isolating an antibody or its functional fragment; and this antibody or antibody fragment includes an antigen binding region that is specific for said epitope. In this regard, the linear epitope may contain amino acid residues 192-206, while the conformational epitope may contain one or more amino acid residues selected from the group consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170 and 202-224 CD38. The CD38 epitope can be used, for example, to isolate antibodies or their functional fragments (and each of these antibodies or antibody fragments contains an antigen-binding region that is specific for such an epitope) containing the steps of contacting the specified CD38 epitope with an antibody library and isolation of antibodies ( antibodies) or its functional fragment (s).

In another embodiment, the present invention relates to an isolated CD38 epitope, which essentially consists of an amino acid sequence selected from the group consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 CD38. For the purposes of the present invention, such an epitope “essentially consists of” one of the immediately preceding amino acid sequences plus has additional features, provided that the additional features do not substantially affect the original and new characteristics of the epitope.

In yet another embodiment, the present invention relates to an isolated epitope from CD38, which consists of an amino acid sequence selected from the group consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 from CD38.

The present invention also relates to a kit comprising (i) an isolated epitope from CD38 containing one or more segments of the amino acid sequence taken from the list of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192- 206 and 202-224; (ii) an antibody library; and (iii) instructions for using an antibody library to isolate one or more elements of such a library that specifically binds to such an epitope.

Brief Description of the Drawings

On figa presents the nucleic acid sequence of various new variable heavy regions of antibodies.

1b shows the amino acid sequences of various new variable heavy regions of antibodies. The CDR regions of HCDR1, HCDR2, and HCDR3 are indicated from the N- to C-terminus in bold.

Figure 2a shows the nucleic acid sequences of various new variable light regions of antibodies.

Figure 2b shows the amino acid sequences of various new variable light regions of antibodies. The CDR regions of LGDR1, LCDR2, and LCDR3 are indicated from N- to C-end in bold.

Figure 3 shows the amino acid sequences of the variable heavy regions of the various consensus sequences of the main gene of the HuCAL antibody. The CDR regions of HCDR1, HCDR2, and HCDR3 are indicated from the N- to C-terminus in bold.

Figure 4 shows the amino acid sequences of the variable light regions of the various consensus sequences of the main gene of the HuCAL antibody. The CDR regions of LCDR1, LCDR2, and LCDR3 are indicated from the N- to C-terminus in bold.

Figure 5 shows the amino acid sequence of CD38 (primary attachment of SWISS-PROT No. P28907).

Figure 6 shows the nucleotide sequences of the heavy and light chains of the chimeric OKT10.

7 schematically shows a list of epitopes of representatives of antibodies according to the invention.

Figure 8 shows the DNA sequence pMORPH®_h_IgG _l _1 (601-2100 nucleotide pairs (bp)) (SEQ ID NO: 32): The vector is based on plasmid pcDNA3.1 + vectors (Invitrogen). The amino acid sequence of the “extra” VH sequence is shown in bold, while the end reading frames of the leader VH sequence and the constant region gene are printed in normal type. Restriction sites are indicated above the sequence. The priming sites of sequenced primers are underlined.

Figure 9 shows the light chain DNA sequence of the kappa Ig expression vector pMORPH®h_IgK_1 (601-1400 bp) (SEQ ID NO: 33): The vector is based on pcDNA3.1 + vectors (Invitrogen). The amino acid sequence of the “extra” Vk sequence is shown in bold, while the end reading frames of the leader Vk sequence and the constant region gene are printed in normal type. Restriction sites are indicated above the sequence. The priming sites of sequenced primers are underlined.

10 shows the light chain DNA sequence of the HuCAL Ig lambda vector pMORPH®_h_Igλ_1 (601-1400 bp) (SEQ ID NO: 34): The amino acid sequence of the “extra” Vλ sequence is shown in bold, while the reading end frames leader Vλ sequence and constant region gene are printed in regular type. Restriction sites are indicated above the sequence. The priming sites of sequenced primers are underlined.

11 shows the results of a proliferation study: PBMC cells from 6 different healthy donors (indicated by individual dots) were cultured for 3 days in the presence of HuCAL® Mon.AT # 1 antibodies (= MOR03077), Mon.AT # 2 (= MOR03079) and Mon.AT # 3 (= MOR03080), a reference antibody chOKT10, a control agonistic (ag) IB4, an unrelated HuCAL® negative control IgGI (NC), and mouse IgQ2a (Iso) as an isotype-related control for IB4. Standard BrdU tags were used to measure proliferation activity and their incorporation (in RLU, relative light units) was analyzed by ELISA enzyme-linked immunosorbent assay based on chemiluminescence.

12 shows the results of an analysis of IL-6 release: PBMC cells from 4-8 different healthy donors (indicated by single dots) were cultured for 24 hours in the presence of only HuCAL® Mon.AT # 1 antibodies (= MOR03077), Mon.AT # 2 (= MOR03079) and Mon.AT # 3 (= MOR03080), the chOKT10 reference antibody, the control agonist (ag) IB4, the unrelated HuCAL® negative control IgGI (NC) and culture medium (medium). The relative light units (RLU) of IL-6 were analyzed in supernatant culture by the chemiluminescent ELISA method.

13 shows cytotoxicity towards CD34 + / CD38 + precursor cells: PBMC cells from healthy donors, which contain autologous CD34 + / CD38 + precursor cells, were incubated in the presence of HuCAL® Mon.AT # 1 (= MOR03077), Mon.AT # 2 (= MOR03079) and Mon.AT # 3 (= MOR03080), positive control (PC = chOKT10) and unrelated negative control HuCAL®, respectively, for 4 hours. After this, the cell suspension was mixed with conditioned methyl cellulose medium and incubated for 2 weeks. Colony forming units (CFUs) derived from an erythroid colony forming "explosive" unit (BFU-E; column B) and granulocyte / erythroid / macrophage / megakaryocyte stem cells (CFU-GEMM; column B) and granulocyte / macrophage stem cells (CFU-GM ; Column C) was counted and standardized against the control medium (“absent” = medium). Column A shows the total CFU (total number of CFU colonies) for all precursors. Average values from at least 10 different PBMC donors are given. it represents the standard error of the mean.

On Fig shows data related to ADCC in various cell lines:

a: Single measurements (except RPMI8226: average of 4 separate analyzes); the ratio of E: T is 30: 1.

b: Namba et al., 1989.

c: 5 μg / ml used for antibody sequence (except Raji with 0.1 μg / ml).

d: Additional retinoic analysis to stimulate specific lysis of CD38 expression [%] = [(experimental lysis - medium lysis) / (1-average lysis)] × 100.

PC: positive control (= chOKT10).

MM: Multiple myeloma.

CLL: Chronic B-cell leukemia.

ALL: Acute lymphoblastic leukemia.

AML: Acute myeloid leukemia.

DSMZ: German collection of microorganisms and cell cultures GmbH.

ATCC: American Type Culture Collection.

ECACC: European Cell Culture Collection.

MFI: Average fluorescence intensity.

On Fig shows data regarding ADCC for samples with MM:

^a : 2-4 separate studies.

On Fig shows experimental results on average tumor volumes of the xenograft of human myeloma after treatment with MOR03080: group 1: carrier; group 2: MOR03080 as hIgG1 at a dose of 1 mg / kg for 32-68 days every other day; group 3: MOR03080 as hIgG1 at a dose of 5 mg / kg for 32-68 days every other day; group 4: MOR03080 as chIgG2a at a dose of 5 mg / kg for 32-68 days every other day; group 5: MOR03080 as hIgG1 at a dose of 1 mg / kg, for 14-36 days every other day, group 6: without treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of new antibodies that are specific for CD38 or have high affinity for CD38 and are able to bring therapeutic benefits to the subject. Antibodies according to the invention, which may be human or humanized, can be used in many contexts, which are more fully described below.

A “human” antibody or a functional fragment of a human antibody in the present invention is defined as being non-chimeric (eg, not “humanized”) and not obtained (in whole or in part) from species other than non-human. A human antibody or a functional fragment of an antibody can be obtained from a human, or they can be synthetic human antibodies. "Synthetic human antibody" in the present invention is defined as an antibody having a sequence, fully or partially obtained in silico from synthetic sequences based on the analysis of known sequences of human antibodies. The construction of an in silico sequence of a human antibody or fragment thereof can be achieved by analyzing a database of human antibody sequences or antibody fragments and by developing a polypeptide sequence using data derived from it. Another example of a human antibody or a functional fragment of an antibody is an antibody encoded by a nucleic acid isolated from a library of sequences of antibodies of human origin (i.e., such a library that is based on antibodies taken from a human natural source).

A “humanized antibody” or a functional humanized antibody fragment is defined in the present invention as (i) derived from a source of non-human origin (for example, a transgenic mouse carrying a xenogenic immune system), and such an antibody is based on the germline sequence of human cells; or (ii) being chimeric, in which the variable domain is non-human and the constant domain is human, or (iii) is CDR transplanted, in which the regions of the CDRs of the variable domains are non-human, while one or more frameworks of the variable The domains are of human origin, and the constant domain (if any) is of human origin.

In the present description, it is understood that the antibody "binds specifically to", is "specific for / for" or "specifically recognizes" an antigen (here CD38), if such an antibody is capable of distinguishing such an antigen from one or more reference antigens, since the binding specificity is not absolute, but is a relative property. In the most general form (and without reference to specific references), “specific binding” refers to the ability of an antibody to distinguish an antigen of interest from unrelated antigens, as determined, for example, by one of the following methods. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, and peptide scanning. For example, a standard ELISA assay can be performed. You can evaluate the standard development of color (for example, a secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in some wells is evaluated by optical density, for example, at 450 nm. The usual baseline (negative reaction) may be 0.1 OD; the usual positive reaction can be 1 OD. This means that positive and negative reactions can differ by more than 10 times. As a rule, the determination of the binding specificity is carried out using not one reference antigen, but a set of approximately three to five unrelated antigens, such as milk powder, BSA, transferrin, etc.

However, “specific binding” may also refer to the ability of an antibody to distinguish between a target antigen and one or more closely related antigens that are used as reference points, for example, between CD38 and CD157. Additionally, “specific binding” may refer to the ability of an antibody to distinguish different parts of its target antigen, for example, different domains or regions of CD38, such as epitopes in the N-terminal or C-terminal region of CD38, or one or more key amino acid residues or segments [ sequences] amino acid residues of CD38.

Also used in the present invention, the term "immunoglobulin" (Ig) defines a protein belonging to the classes IgG, IgM, IgE, IgA or IgD (or any subclass thereof), and includes all well-known antibodies and their functional fragments. The term “functional fragment” of an antibody / immunoglobulin defines an antibody / immunoglobulin fragment (eg, an IgG variable region) that retains an antigen-binding region. An "antigen binding region" of an antibody is typically detected in one or more hypervariable region (s) of the antibody, for example, in the region of CDR-1, -2, and / or -3; however, variable "frame" regions can also play an important role in antigen binding, for example, by providing a skeleton for CDR regions. Preferably, the "antigen binding region" contains at least 4-103 amino acid residues from variable light (VL) chains, and 5-109 from variable heavy (VH) chains, more preferably 3-107 amino acid residues from VL and 4-111 from VH , and particularly preferably contains complete VL and VH chains (amino acid positions 1-109 in VL and 1-113 in VH; numbering according to WO 97/08320). The preferred class of immunoglobulins for use in the present invention is IgG. "Functional fragments" according to the invention include the domain of the F (ab ') ₂ fragment, Fab fragment, and scFv. F (ab ') ₂ or Fab can be designed to minimize or completely eliminate intermolecular disulfide interactions that arise between the C _HI and C _L domains.

An antibody of the invention can be obtained from a library of recombinant antibodies based on amino acid sequences that were developed in silico and encoded by artificially created nucleic acids. In the silico design, an antibody sequence is obtained, for example, by analyzing a human sequence database and developing a polypeptide sequence using the data obtained in the analysis. Methods for the design and preparation of in silico- generated sequences are described, for example, by Knappik et al., J. Mol. Biol. (2000) 296: 57; Rrebs et al., J. Immunol. Methods (2001) 254: 67; and in US patent No. 6300064, issued in the name of Knappik et al. , references to which in their entirety are included in the present description.

Antibodies according to the invention

Throughout the text of this description there is a reference to the following antibodies according to the invention: "antibody No." or "LACS" or "MOR" 3077, 3079, 3080 and 3100. LAC 3077 is an antibody having a variable heavy region corresponding to the sequence of SEQ ID NO : 1 (DNA) / SEQ ID NO: 5 (protein), and a variable light region corresponding to SEQ ID NO: 9 (DNA) / SEQ ID NO: 13 (protein). LAC 3079 is an antibody having a variable heavy region corresponding to SEQ ID NO: 2 (DNA) / SEQ ID NO: 6 (protein) and a variable light region corresponding to SEQ ID NO: 10 (DNA) / SEQ ID NO: 14 (protein). LAC 3080 is an antibody having a variable heavy region related to SEQ ID NO: 3 (DNA) / SEQ ID NO: 7 (protein) and a variable light region corresponding to SEQ ID NO: 11 (DNA) / SEQ ID NO: 15 (protein). LAC 3100 is an antibody having a variable heavy region corresponding to SEQ ID NO: 4 (DNA) / SEQ ID NO: 8 (protein) and a variable light region corresponding to SEQ ID NO: 12 (DNA) / SEQ ID NO: 16 (protein).

In one aspect, the present invention relates to antibodies having an antigen binding region, capable of specifically binding, or having high affinity for one or more regions of CD38, the amino acid sequence of which is set forth in SEQ ID NO: 22. An antibody is considered to have “high affinity” for an antigen if the affinity value is at least 100 nm (monovalent affinity of the Fab fragment). The antibody or antigen binding region of the invention can preferably bind to CD38 with an affinity of less than 100 nm, more preferably less than about 60 nm and even more preferably less than about 30 nm. Further preferred are antibodies that bind to CD38 with an affinity of less than about 10 nm, and more preferably less than about 3 nm. For example, the affinity of an antibody of the invention for CD38 may be about 10.0 nm or 2.4 nm (monovalent affinity of the Fab fragment).

Table 1 summarizes the overall antibody affinity according to the invention, determined by surface plasmon resonance (Biacore) analysis and FACS-Scatchard analysis.

Table 1
Antibody Affinity Indices Antibody (Fab or IgG1) BIACORE (Fab) K _D  [nM] ^a FACS Scatcgard (IgG1) ^b K _D  [nM] ^a MOR03077 56.0 0.89 MOR03079 2,4 0.60 MOR03080 27.5 0.47 MOR03100 10.0 6.31 Chimeric OKT10 undefined 8.28 ^a : average of at least 2 different definitions of affinity
^b : MM RPMI8226 cell line used in FACS-Scatchards analysis

According to table 1, the affinity of antibodies LAC 3077, 3079, 3080 and 3100 was measured by surface plasmon resonance (Biacore) on immobilized recombinant CD38 and flow cytometry using a CD38-expressing human cell line RPMI8226. Biacore studies were performed on directly immobilized antigen (CD38-Fc fusion protein). The Fab antibody format LAC 3077, 3079, 3080, and 3100 shows a range of monovalent affinity between about 2.4 and 56 nm on the immobilized CD38-FC fusion protein, with the highest affinity shown by LAC 3079, and then Fab 3100, 3080, and 3077 .

To determine the affinity on a cell basis (FACS Scatchard), the IgGI format was used. The right column of Table 1 indicates the binding strength of LAC antibodies in this format. LAC 3080 showed the strongest binding, which is slightly stronger than that of LAC 3079 and LAC 3077.

Another preferred feature of the preferred antibodies of the invention is their specificity for the zone within the N-terminal region of CD38. For example, antibodies of the invention LAC 3077, 3079, 3080, and 3100 can specifically bind to the N-terminal region of CD38.

The type of epitope to which an antibody of the invention binds can be a linear type (i.e., one consecutive stretch of amino acids) or a conformational type (i.e., multiple stretches of amino acids). To determine whether the epitope of a particular antibody is linear or conformational, one skilled in the art can analyze the binding of antibodies to overlapping peptides (e.g., 13-mer peptides with 11 amino acid overlapping) covering different CD38 domains. Using this analysis, the inventors of the present invention found that the LAC 3077, 3080, and 3100 antibodies recognize discontinuous epitopes in the N-terminal region of CD38, while the LAC 3079 epitope can be described as linear (see FIG. 7). In combination with the information presented in the present invention, one skilled in the art will understand how to use one or more of the isolated CD38 epitopes to create antibodies having an antigen binding region that is specific for these epitopes (e.g., the use of synthetic peptides from CD38 epitopes or cells expressing epitopes CD38).

The antibody of the invention is preferably a species antibody that cross-reacts with the human and at least one other species, which may be a rodent species or a non-human primate species. The non-human primate may be Rhesus, baboon and / or cynomolgus. The rodent species may be a mouse, rat, and / or hamster. An antibody that is cross-reactive, for example with at least one species of rodent, compared to known anti-CD38 antibodies, can provide greater flexibility and benefit for conducting in vivo studies with the same antibody in many species.

Preferably, the antibody of the invention is capable of not only binding to CD38, but is also able to mediate the lysis of a cell expressing CD38. More specifically, an antibody of the invention can mediate its therapeutic effect by depleting CD38-positive (e.g., malignant) cells through antibody-effector functions. These functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Table 2 shows the total indicators for determining the EC50 (average dose, causing an effect in 50% of cases) of the antibodies presented in the present invention, both by ADCC and by CDC.

table 2
EC50 anti- tel values Antibody (IgGI) ADCC EC50 [nM] ADCC EC50 [nM] LP-1 RPMI8226 CHO transfectants MOR03077 0.60a 0.08a 0.8c; 0.94d MOR03079 0.09 ^a 0.04 ^a 0.41 ^s MOR03080 0.17 ^b 0.05 ^a 3.2 ^s ; 2.93 ^d MOR03100 1.00 ^b 0.28 ^a 10.9 ^s ; 13.61 ^e Chimeric OKT10 5.23 ^a 4.10 ^a 9.30 ^{s a} : average of at least 2 EC50 definitions
^b : single definition
^c : average of 2 EC50 definitions
^d : average of 3 EC50 definitions
^e : average of 4 EU50 definitions

However, expression of CD38 was detected not only in immune cells of myeloid (e.g., monocytes, granulocytes) and lymphoid origin (e.g., activated B and T cells; plasma cells), but also in the corresponding progenitor cells. Since it is important that antibody-mediated lysis of malignant cells does not act on these cells, the antibodies of the invention are preferably not cytotoxic to progenitor cells.

Along with its catalytic activity as cyclic ADP-ribosyl cyclase and β-hydrolase, CD38 is capable of transducing biological relevance signals (Hoshino et al., 1997; Ausiello et al., 2000). These in vivo functions can be induced, for example, by receptor-ligand interaction or cross-linking with agonistic anti-CD38 antibodies, which leads, for example, to mobilization of calcium, proliferation of lymphocytes and the release of cytokines. Preferably, the antibodies of the invention are non-agonistic antibodies.

Peptide Options

Antibodies of the invention are not limited to the specifically peptide sequences provided by the present invention. Variants of these polypeptides are also carried out in the present invention. With reference to the present description and generally available technologies, one skilled in the art will be able to manufacture, test and use functional variants of the antibodies disclosed in the present invention, given that variants having the ability to mediate the lysis of CD38 + target cells are included in the scope of the present invention. Used in this context, the term "ability to mediate the lysis of CD38 + target cells" means a functional characteristic attributed to the anti-CD38 antibody according to the invention. The ability to mediate the lysis of CD38 + target cells, therefore, includes the ability to mediate the lysis of CD38 + target cells, for example, by ADCC and / or CDC, or using the design of the toxin conjugated to the antibody according to the invention.

A variant may include an antibody having, in comparison with the peptide sequence disclosed in the present invention, at least one altered complementarity determining region (CDR) (hypervariable region) and / or a framework (FR) (variable) domain / position . To better illustrate this concept, a brief description of the structure of the antibody is provided below.

An antibody is composed of two peptide chains, each of which contains one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), which in each case is composed of four FR regions and three intermediate CDR regions. The antigen binding site is formed by one or more CDR regions, while the FR regions provide a structural framework for the CDR regions and therefore play an important role in antigen binding. By altering one or more amino acid residues in the CDR or FR region, one of ordinary skill in the art can produce mutant or diverse antibody sequences that can be selected by antigen, for example, to obtain new or improved properties.

Tables 3a (VH) and 3b (VL) outline the CDR and FR regions for some antibodies of the invention and compare amino acids at a given position with each other and with the corresponding or “main gene” sequence (as described in US Pat. No. 6300064):

One of skill in the art can use the data from Tables 3a and 3b to design peptide variants that are within the scope of the present invention. Preferably, when the options are designed by replacing amino acids within one or more regions of the CDR; an option may also have one or more altered wireframe areas. With regard to comparing new antibodies with each other, probable substitutional residues include, for example, variable light chain residues 4 or 37, and, for example, variable heavy chain residues 13 or 43 from regions LAC 3080 and 3077, since these are variable positions are opposite each other. Replacements can also be made in frame areas. For example, the peptide of the FR domain can be altered if there is a deviation in the residue compared to the sequence of the precursor cell.

As for the comparison of new antibodies with the corresponding sequence or the sequence of the "main gene", the probable residues that are possible to replace include, for example, the remains of the variable light chains 27, 50 or 90 of LAC 3080 compared to VLλ3 and, for example, residues variable heavy chains 33, 52 and 97 of LAC 3080 compared to VH3. On the other hand, the expert can perform a similar analysis by comparing the amino acid sequences disclosed herein to known sequences of the same class of such antibodies, using, for example, the method described by Knappik et al., 2000, and described in patent № 6,300,064, issued in the name of Knappik et al .

In addition, variants can be obtained using one LAC region as a starting point for optimization, by diversifying one or more amino acid residues in the LAC, preferably amino acid residues in one or more CDR regions, and by screening the resulting collection of antibody variants to identify variants with improved properties. Particularly preferred is the introduction of diversity into one or more amino acid residues in the regions of CDR-3 VL, CDR-3 VH, CDR-1 VL and / or CDR-2 VH. Diversity can be achieved by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Vimeks, B., Ge, L., Plckthun, A., Schneider, KC, Wellnhofer, G., and Moroney SE (1994) Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucl. Acids Res. 22, 5600).

Conservative Amino Acid Options

Polypeptide variants can be made that retain the overall molecular structure of the antibody peptide sequence described herein. Given the properties of individual amino acids, the specialist recognizes some rational substitutions. Amino acid substitutions, namely “conservative substitutions”, can be carried out, for example, on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues involved.

For example, (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; (c) positively charged (basic) amino acids include arginine, lysine and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Generally, substitutions can be made within groups (a) to (d). In addition, glycine and proline can be replaced by each other, which is based on their ability to disrupt the [structure] of the α-helix. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are usually more often detected in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are usually more often detected in β-folded structures. Glycine, serine, aspartic acid, asparagine, and proline are usually found at bends [in structure]. Some preferred substitutions can be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L, and I. Given the well-known genetic code and recombinant and synthetic DNA technologies, one can easily construct DNA by encoding conservative amino acid variants. In one specific example, the amino acid at position 3 in SEQ ID NO: 5, 6, 7 and / or 8 can be replaced with Q by E.

Used in the present invention, the term "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical in the two sequences. The term “sequence similarity” indicates the percentage of amino acids that are either identical or result from conservative amino acid substitutions. Preferred polypeptide sequences according to the invention have a sequence identity in the CDR regions of at least 60%, more preferably at least 70% or 80%, even more preferably at least 90% and, most preferably at least 95%. Preferred antibodies also have sequence similarity in the CDR regions of at least 80%, more preferably 90% and most preferably 95%.

DNA molecules according to the invention

The present invention also relates to DNA molecules encoding an antibody of the invention. These sequences include DNA molecules designated in FIGS. 1a and 2a, but not limited to the foregoing.

The DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the scope of the present invention can be described with respect to their physical properties in hybridization. One skilled in the art will easily determine that DNA can be used to identify its complement and its equivalent or homologue using nucleic acid hybridization technology, since DNA has a double helix. It can also be determined that hybridization can occur with a complementarity of less than 100%. However, with the appropriate choice of conditions for distinguishing DNA sequences, hybridization technology based on the structural affinity of their sequences with a specific probe can be used. The following publications provide guidance on these conditions: Sambrook et al., 1989 (Sambrook, J., Fritsch, EF and Matriatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA ) and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K. eds. (1995). Current Protocols in Molecular Biology New York: John Wiley and Sons).

The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which these two sequences will hybridize with each other. Used in the present invention, the term "rigidity" exists in the sense that the conditions are disadvantageous for hybridization. Severe conditions are very disadvantageous for hybridization, and under such conditions only the most structurally related molecules will hybridize with each other. In contrast, non-stringent conditions are favorable for hybridization of molecules exhibiting a lesser degree of structural affinity. Therefore, the stringency of hybridization directly correlates with the structural relationships of the two nucleic acid sequences. The following relationships are useful for [establishing] a correlation between hybridization and structural affinity (where T _m is the melting point of a nucleic acid duplex):

a. T _m = 69.3 + 0.41 (G + C)%;

b. T _m DNA duplex decreases by 1 ° C with an increase of 1% in the number of mismatched base pairs;

c. (T _m ) _μ2 - (T _m ) _μ1 = 18.5 log ₁₀ μ2 / μ1, where μ2 and μ1 represent the ionic strength of two solutions.

Hybridization stiffness is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of substances that break down hydrogen bonds. Hybridization facilitating factors include high DNA concentrations, high ionic strengths, low temperatures, an increased probe size, and the absence of substances that destroy hydrogen bonds. Typically, hybridization is carried out in two stages: the stage of "binding" and the stage of "washing".

First, at the stage of binding, the probe is bound to the target under conditions favorable for hybridization. The stiffness at this stage is usually controlled by changing the temperature. With high stiffness, the temperature usually corresponds to 65-70 ° C if short (<20 bp) oligonucleotide probes are not used. The present hybridization solution contains 6 × sodium chloride and sodium citrate SSC solution, 0.5% SDS sodium dodecyl sulfate, 5 × Denhardt solution and 100 μg of non-specific DNA carrier. See Ausubel et al., Section 2.9, appendix 27 (1994). Of course, many different, but functionally equivalent buffer conditions are known. Where the degree of relationship is lower, a lower temperature can be selected. The low hard binding temperature is between about 25 and 40 ° C. Medium hardness involves a temperature in the range of at least 40 ° C to less than about 65 ° C. High rigidity provides a temperature of at least about 65 ° C.

Secondly, excess probe is removed by washing. It is in this phase that more stringent conditions are usually applied. Therefore, it is this “flushing” stage that is most important in determining kinship through hybridization. Wash solutions typically contain low salt concentrations. In one example, a medium hard solution contains 2X SSC and 0.1% SDS. A high hardness wash solution contains an equivalent amount (in ionic strength) of less than about 0.2X SSC, with a preferred hardness solution containing about 0.1X SSC. The temperatures associated with different stiffness are similar to the above temperatures for "binding". The washing solution is also usually changed repeatedly during washing. For example, typical high stringency washing conditions include washing twice for 30 minutes at 55 ° C and washing three times for 15 minutes at 60 ° C.

Accordingly, the present invention includes nucleic acid molecules that hybridize with the molecules shown in FIGS. 1 and 2a under high stringency binding and washing conditions, in which such nucleic molecules encode an antibody or functional fragment thereof having the properties described in the present invention . Preferred molecules (from the point of view of mRNA) are molecules having sequences of at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homologous or identical to one of the DNA molecules described in the present invention. In one specific example of a variant according to the invention, at position 7 in nucleic acid in SEQ ID NO: 1, 2, 3 and / or 4, C can be replaced with G, thereby changing the CAA codon to GAA.

Functionally Equivalent Options

You can describe another class of DNA variants included in the scope of the present invention, in relation to the product that they encode (see the proteins shown in fig.1b and 2b). These functionally equivalent genes are characterized in that they encode the similar peptide sequences shown in FIGS. 1b and 2b due to the degeneracy of the genetic code. The sequences of SEQ ID NO: 1 and 31 are an example of functionally equivalent variants, since these nucleic acid sequences are different, however, they encode a similar polypeptide, i.e. SEQ ID NO: 5.

It is generally recognized that the variants of the DNA molecules provided by the present invention can be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. Methods for efficiently synthesizing oligonucleotides in the range of from about 20 to about 150 nucleotides are generally available. See Ausubel et al., Section 2.11, appendix 21 (1993). Overlapping oligonucleotides can be synthesized and assembled by the method first described by Khorana et al., In J. Mol Biol. 72: 209-217 (1971); see also Ausubel et al., supra, section 8.2. The construction of synthetic DNAs is preferably carried out with suitable restriction sites created at the 5 ′ and 3 ′ ends of the gene to facilitate cloning into a suitable vector.

As indicated, a method for creating variants is to start with one of the DNAs disclosed in the present invention, and then carry out site-directed mutagenesis. See Ausubel et al., Supra, chapter 8, appendix 37 (1997). In the usual method, the target DNA is cloned into a phage that is a carrier of single-stranded DNA. Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide replacement. A complementary strand is synthesized, and a double-stranded phage is introduced into the host cell. Part of the resulting offspring will carry the desired mutation, which can be confirmed using DNA sequencing. In addition, various methods are available that increase the likelihood that phage offspring will be a desired mutant. These methods are known to those skilled in the art, and kits for creating such mutants are commercially available.

Design and expression of recombinant DNA

The present invention further provides recombinant DNA constructs containing one or more nucleotide sequences of the invention. Recombinant constructs of the invention are used in connection with a vector, such as a plasmid or viral vector, into which a DNA molecule encoding an antibody of the invention is inserted.

The encoded gene can be produced using the techniques described by Sambrook et al., 1989 and Ausubel et al., 1989. Alternatively, DNA sequences can be synthesized chemically, for example using synthesizers. For example, you can refer to the technology described in Oligonucleotide synthesis (1984, Gait, ed., IRL Press, Oxford), the link to which is incorporated into this description in its entirety. Recombinant constructs according to the invention contain expression vectors that are capable of expressing RNA and / or protein products of encoded DNA or several DNAs. The vector may further comprise regulatory sequences including a promoter operably linked to an open reading frame (ORF). The vector may further comprise a selected marker sequence. Specific initiation and bacterial secretion signals may also be necessary for efficient translation of the inserted target gene encoding sequences.

The present invention further provides host cells comprising at least one of the DNAs of the invention. A host cell can be virtually any cell for which expression vectors are acceptable. For example, it may be a host cell of higher eukaryotes, such as a mammalian cell, a host cell of lower eukaryotes, such as a yeast cell, but a prokaryotic cell, such as a bacterial cell, is preferred. The introduction of recombinant constructs into the host cell can be accomplished by transfection with calcium phosphate, diethylaminoethyl DEME, dextran-mediated transfection, electroporation or phage infection.

Bacterial expression

Useful expression vectors for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein along with suitable signals for initiation, translation and termination in the functional reading phase with a functional promoter. The vector will contain one or more phenotype-selectable markers and the start of replication to guarantee the conservation of the vector and, if desired, to ensure amplification in the host cell. Suitable prokaryotic host cells for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genus Pseudomonas, Streptomyces and Stafylococcus.

Bacterial vectors can be vectors, for example, based on phage, plasmids or phagemids. These vectors may contain the selected marker and bacterial origin of replication derived from commercially available plasmids, usually containing elements of the well-known cloning vector pBR322 (ATCC 37017). Subsequently, a suitable host strand is transformed and the host strand grows to an adequate cell density, the selected promoter is repressed / induced by appropriate methods (for example, by temperature or chemical induction) and the cells are cultured for an additional time. Cells are usually harvested by centrifugation, destroyed by physical or chemical means, and the resulting crude extract is stored for further purification.

Some expression vectors in bacterial systems can preferably be selected depending on the intended use of the expressed protein. For example, when it is necessary to produce a large amount of such a protein, for example, to form antibodies or to screen libraries of proteins, vectors that direct the expression of high levels of fusion protein products that are easily purified can be desirable.

Therapeutic methods

Therapeutic methods comprise administering to a subject in need of treatment a therapeutically effective amount of an antibody of the invention. The “therapeutically effective” amount in the present invention is defined as the amount of antibody that is sufficient to deplete the CD38-positive cells in the treatment area of a subject, either as a single dose or in accordance with a multiple dosage regimen, and only or in combination with other agents, which leads to the alleviation of an adverse condition, and at the same time, this amount is toxicologically tolerant. The subject may be a human or non-human mammal (e.g., rabbit, rat, mouse, monkey, or other primate of a lower genus).

The antibody of the invention can be co-administered with known drugs, and in some cases the antibody itself can be modified. For example, an antibody can be conjugated to an immunotoxin or radioisotope to potentially further increase efficacy.

Antibodies according to the invention can be used as a therapeutic or diagnostic tool in various situations where CD38 or its undesirable expression is detected. Particularly suitable disorders and conditions for treating an antibody of the invention are multiple myeloma (MM) and other hematologic diseases such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL) . The antibody of the invention can also be used to treat inflammatory diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).

For the treatment of any of the above disorders, pharmaceutical compositions for use in accordance with the present invention can be formulated by conventional methods using one or more physiologically acceptable carriers or excipients. The antibody according to the invention can be administered by any suitable method, which can be changed depending on the type of disease intended for treatment. Possible routes of administration include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary, and intranasal, and, if desired, the route of administration of the inside of the affected tissue for local immunosuppressive therapy. Additionally, the antibody according to the invention can be administered by pulse infusion, for example, with a decrease in the dose of the antibody. Preferably, the dose is administered by injection, with the most preferred intravenous or subcutaneous administration, partly depending on whether the administration is short-term or continuous. The amount required for administration will depend on various factors, such as clinical symptoms, individual weight, administration of other drugs. The person skilled in the art will determine that the route of administration will vary depending on the disorder or condition intended for treatment.

The determination of a therapeutically effective amount of a new polypeptide according to the present invention will largely depend on the particular characteristics of the patient, the route of administration and the nature of the disease intended for treatment. General practical guidance can be found, for example, in the publications of the International Conference on Harmonization International Conference on Harmonization and in Remington's pharmaceutical sciences, chapters 27 and 28, pages 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa .: Mack Pub. Co., 1990). More specifically, the determination of a therapeutically effective amount will depend on factors such as toxicity and efficacy of the drug. Toxicity can be determined using methods known in the art and set forth in the above references. Efficiency can be determined using the specified practical guidance in conjunction with the methods described below in the examples.

Diagnostic Methods

CD38 is highly expressed in hematologic cells in some cancers; thus, the anti-CD38 antibody of the invention can be used to display or visualize the site of a possible accumulation of malignant cells in a patient. Moreover, the antibody can be labeled for detection using radioisotopes, affinity tags (such as biotin, avidin, etc.), fluorescent tags, paramagnetic atoms, etc. The execution order of such labeling methods is known in the art. A review of the clinical use of antibodies for diagnostic imaging is provided by Grossman, H.B., Urol. Clin. North Amer. 13: 465-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20: 693-700 (1985)) and Khaw, B.A. et al., Science 209: 295-297 (1980).

The detection of the foci of such detectable antibodies can be, for example, an indicator of the localization of tumor development. In one embodiment, this study is carried out by taking tissue or blood samples, and incubating such samples in the presence of detectable antibody labels. In a preferred embodiment, this technology is carried out in a non-invasive manner using magnetic imaging, fluorography, etc. Such a diagnostic study can be used to monitor the success of treatment of diseases in which the presence or absence of CD38-positive cells is an essential indicator. The present invention also contemplates, as described below, the use of anti-CD38 antibodies for diagnosis in an ex vivo setup.

Therapeutic and diagnostic compositions

Antibodies according to the invention can be formed according to known methods for the manufacture of pharmaceutically useful compositions in which the antibody according to the invention (including any functional fragment thereof) is combined with a pharmaceutically acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's pharmaceutical sciences (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa .: Mack Pub. Co., 1990). To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more antibodies of the invention, together with a suitable amount of carrier.

The formulation of the compositions can be formed in such a way as to specify a controlled release of the active compound. Controlled release formulations can be prepared using polymers to complex or absorb the anti-CD38 antibody. Controlled delivery can be accomplished by selecting the appropriate macromolecules (e.g., polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate) and the concentration of the macromolecules, as well as the inclusion method to achieve controlled release. Another possible way to control the duration of action with release control drugs is to incorporate the anti-CD38 antibody into particles of a polymer material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these agents into polymer particles, these materials can be captured in prepared microcapsules, for example, by coacervation or surface polymerization methods, for example, hydroxymethyl cellulose or gelatin microcapsules and, accordingly, into poly (methyl methacrylate) microcapsules, or into colloidal drug delivery systems for example, in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences (1980).

The compounds may be formulated for parenteral administration by injection, for example, a bolus injection, or by continuous infusion. The composition for administration may be in a unit dosage form, for example, in ampoules, or in the form of multi-dose containers with the addition of a preservative. The compositions may be in such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and may contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

If desired, the compositions may be a package or dispenser, which may contain one or more unit dosage forms containing the active ingredient. A package, such as a blister pack, may contain, for example, a metal or plastic film. The packaging or dispenser may be supplemented with instructions for administration.

The present invention will be further understood by the following working examples, intended to illustrate the present invention, and thus, do not limit its scope.

EXAMPLES

Cell lines

The following cell lines were obtained from the European Cell Culture Collection (ECACC), the German Microorganism Collection (DSMZ) or the American Type Culture Collection (ATCC): hybridoma cell line producing CD38 murine IgGI monoclonal antibody OKT10 (ECACC, # 87021903), Jurkat cells ( DSMZ, ACC282), LP-1 (DSMZ, ACC41), RPMI8226 (ATCC, CCL-155), HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CRL-61) and Raji (ATCC, CGL-86 )

Cells and culture conditions

All cells were cultured under standard conditions at 37 ° C and 5% CO ₂ in a humidified incubator. Cell lines LP-1, RPMI8226, Jurkat and Raji were cultured in RPMI 1640 medium (Pan biotech GmbH, # P04-16500) with the addition of 10% fetal calf serum FCS (Pan biotech GmbH, # P30-3302), 50 units / ml penicillin , 50 μg / ml streptomycin (Gibco, # 15140-122) and 2 mM glutamine (Gibco, # 25030-024) and, in the case of Jurkat and Raji cells, with the addition of 10 mM Hepes (Pan biotech GmbH, # P05-01100 ) and 1 mM sodium pyruvate (Pan biotech GmbH, # P04-43100).

CHO-K1 and HEK293 were grown in the Dulbecco-modified Eagle DMEM medium (Gibco, # 10938-025) supplemented with 2 mM mm glutamine and 10% FCS. Stable CD38 CHO-K1 transfectants were maintained in the presence of G418 (PAA GmbH, Pll-012), while the addition of 1 mM sodium pyruvate was mandatory for HEK293. After transient transfection of HEK293, 10% FCS was replaced with ultra low IgG FCS (Invitrogen, # 16250-078). The OKT10 cell line was cultured in IDMEM (Gibco, # 1980-022) supplemented with 2 mM glutamine and 20% FCS.

Preparation of a suspension culture of peripheral blood cells

All blood samples were taken after informed consent. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using a Histopaque®-1077 instrument (Sigma) according to the manufacturer's instructions. These cell suspensions were purified from red blood cells by incubation in ACK lysis buffer (ACK Lysis Buffer) (0.15 M NH ₄ Cl, 10 mM KHCO ₃ , 0.1 M EDTA) for 5 minutes at room temperature (CT) or commercially (Bioscience , # 00-4333). Cells were washed twice with PBS (phosphate buffered saline) and then further processed for flow cytometry or ADCC (see below).

Flow cytometry ("FACS")

All staining was performed in 96-well culture plates with a round bottom (Nalge Nunc) with 2 × 10 ⁵ cells per well. Cells were incubated with Fab or IgG antibodies at indicated concentrations in 50 μl of FACS buffer (PBS, 3% FCS, 0.02% NaN ₃ ) for 40 minutes at 4 ° C. Cells were washed twice and then incubated with R-phycoerythrin (PE) conjugated to goat anti-human or goat anti-mouse IgG (H + L) F (ab ') ₂ (Jackson Immuno Research), diluted 1: 200 FACS buffer for 30 minutes at 4 ° C. Cells were washed again, resuspended in 0.3 ml FACS buffer and then analyzed by flow cytometry in a FACSCalibur instrument (Becton Dickinson, San Diego, CA).

For FACS-based Scatchard analysis, RPMI8226 cells were stained in 12 different dilutions (1: 2 ⁿ ), starting with a final concentration of 12.5 μg / ml (IgG). At least two independent measurements were used for each concentration and K _D values were extrapolated from the average fluorescence intensity according to Chamow et al. (1994).

Surface plasmon resonance

Kinetic constants k _on and k _{off were} determined by successive dilutions of the corresponding Fab bound to the covalently immobilized fusion protein CD38-Fc using BIAcore 3000 instruments (Biacore, Uppsala, Sweden). For covalent antigenic immobilization, the standard EDC-NHS amine chemical coupling method was used. For direct attachment of a CD38 Fc fusion protein, the CM5 sensor chips (Biacore) coated ~ 600-700 RU in 10 mM acetate buffer, pH 4.5. An appropriate amount of HSA (human serum albumin) was used for the reference cell stream. Kinetic measurements were performed in PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.76 mM KH ₂ PO ₄ , pH 7.4) at a flow rate of 20 μl / min using a Fab concentration in the range from 1.5 to 500 nM. The administration time for each concentration was 1 minute, followed by 2 minute dissociation phases. For regeneration, 5 μl of 10 mM HCl was used. All sensograms were selected by location using BIA 3.1 assessment software (Biacore).

EXAMPLE 1

Creating Antibodies from HuCAL Libraries

To create therapeutic antibodies against CD38, selection was made based on the MorphoSys HuCAL GOLD phage display library. HuCAL GOLD® is a Fab library based on the HuCAL® concept (Knappik et al., 2000; Krebs et al., 2001), where all six regions of the CDR are different, and where CysDisplay ^™ technology is ^used to connect Fab fragments to the surface phage (Löhning, 2001).

A. Preparation of phagemid, amplification of phage and purification

The HuCAL GOLD® phagemid library was amplified in 2 × TY medium containing 34 μg / ml chloramphenicol and 1% glucose (2 × TY-CG). After infection of the phage helper (VCSM13) in 0.5 OD600 (30 minutes at 37 ° C without a shaker; 30 minutes at 37 ° C with a shaker at 250 rpm), the cells were besieged (4120 × g; 5 minutes; at 4 ° C), resuspended in 2 × TY / 34 μg / ml chloramphenicol / 50 μg / ml kanamycin and grown overnight at 22 ° C. The phages were precipitated from the supernatant on polyethylene glycol (PEG), resuspended in PBS / 20% glycerol and stored at -80 ° C. The phage amplification between the two stages of panning was carried out as follows: the mid-log phase of mid-log growth of TG1 cells was infected with eluted phages and placed on Luria-Bertani LB agar plates, supplemented with 1% glucose and 34 μg / ml chloramphenicol (LB-CG). After overnight incubation at 30 ° C, the colonies were removed mechanically, the OD600 was adjusted to 0.5, and a phage helper was added as described above.

B. Pencings with HuCAL GOLD®

For the selection of antibodies from HuCAL GOLD® phage libraries, they were divided into three pools corresponding to different control VH genes (pool 1: VH1 / 5λκ, pool 2: VH3 / λκ, pool 3: VH2 / 4 / 6λκ). These pools were individually subjected to 3 stages of whole cell panning on CD38-expressing CHO-K1 cells, followed by pH dilution and a post-adsorption step on CD38-negative CHO-K1 cells to deplete unrelated phage antibodies. Finally, the remaining phage antibodies were used to infect E. coli TG1 cells. After centrifugation, the bacterial pellet was resuspended in 2 × TY medium, placed on agar plates and incubated overnight at 30 ° C. Selected clones were mechanically removed from the plates, phages were released and amplified. The second and third stage of selection was carried out similarly to the initial stage.

Fab coding inclusions of the selected HuCAL GOLD® phage were subcloned into the pMORPH®x9_Fab_FS expression vector (Rauchenberger et al., 2003) to facilitate rapid expression of soluble Fab. The DNA of the selected clones was processed using Xbal and EcoRI, thereby cutting off the Fab coding insert (ompA-VLCL and phoA-Fd), and cloned into the Xbal / EcoRI secant vector pMORPH® × 9_Fab_FS. The Fab expressed in this vector carries two C-terminal tags (FLAGT and Strep-tag® II) for detection and purification.

EXAMPLE 2

Biological samples

Antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity were measured according to a published protocol based on flow cytometry analysis (Naundorf et al., 2002) as follows:

ADCC:

To measure ADCC, target cells (T) were adjusted to 2.0E + 05 cells / ml and labeled with calcein AM (Molecular Probes, C-3099), 100 ng / ml, in RPMI 1640 medium (Pan biotech GmbH) for 2 minutes at room temperature. Residual calcein was removed in 3 washing steps in RPMI 1640 medium. At the same time, PBMC cells were prepared as a source of effector cells (E) (natural killer cells), they were adjusted to 1.0E + 07 and mixed with labeled target cells to obtain E ratio : T of 50: 1 or less, depending on the conditions of the analysis. The cells were washed once and the cell mixture was resuspended in 200 μl of RPMI 1640 medium containing the corresponding antibody in various dilutions. The plate was incubated for 4 hours under standard conditions at 37 ° C and 5% CO ₂ in a humidified incubator. Prior to FACS analysis, cells were labeled with propidium iodide (PI) and analyzed by flow cytometry (Becton-Dickinson). Counting was performed between points 50,000 and 150,000 in each sample.

The following equation allows you to get the degree of increase in lysis activity [in%]:

where ED ^A = dead cell points (calcein + PI stained cells), and

EL ^A = living cell points (calcein stained cells).

CDC:

To measure CDC, transfectants of CD38 CHO-K1 in an amount of 5.0E + 04 were added to a microliter well plate (Nunc), together with human serum (Sigma, # S-1764) in a 1: 4 dilution and the corresponding antibody. All reagents and cells were diluted with RPMI 1640 medium (Pan biotech GmbH ), with the addition of 10% FCS. The reaction mixture was incubated for 2 hours under standard conditions at 37 ° C and 5% CO ₂ in a humidified incubator. As a negative control group, both heat-inactivated complement and CD38 transfectants without antibodies were used. Cells were labeled with PI and subjected to FACS analysis.

A total of 5,000 points and the number of dead cells were calculated at various antibody concentrations used to determine the EC50 values. The following equation gives the degree of increase in lysis activity [in%]:

where ED ^C = dead cell points (cells stained with PI), and

EL ^C = points of living cells (unpainted cells).

Cytotoxicity values from a total of 12 different dilutions of the antibody (1: 2 ⁿ ) in triplicate were used in ADCC and in duplicate in CDC for each antibody to obtain EC50 values using standard software (PRISM®, Graph Pad Software).

EXAMPLE 3

Creation of a stable CD38 transfectant and CD38 Fc fusion proteins

To create a CD38 protein for panning and screening, two different expression systems must be established. One strategy involves creating a CD38-Fc fusion protein that is purified from supernatants after transient transfection of HEK293 cells. The second strategy encompasses the development of a stable CHO-KL cell line for a high degree of surface expression of CD38 for use in the selection of phage antibodies by panning of whole cells.

As the initial stage, Jurkat cells (DSMZ ACC282) were used to create cDNA (Invitrogen), followed by amplification of full-length CD38 coding sequences using primers complementary to the first 7 and last 9 codons of CD38, respectively (primer MTE001 and MTE002rev; table 4 ) CD38 insert sequence analysis confirmed published by Jackson et al. (1990) the amino acid sequence, except at position 49, in which glutamine was detected instead of tyrosine, as described by Nata et al. (1997). To introduce restriction sites of endonucleases and cloning into various derivatives of the pcDNA3.1 expression vector (Stratagene), the purified PCR product served as a template for reamplification of the full gene (primers MTE006 and MTE007rev, table 4) or part of the gene (primers MTE004 and MTE009rev, table 4). In the latter case, a fragment encoding the extracellular domain (amino acids 45-300) was amplified and cloned in the reading frame between the human Vkarra leader sequence and the Fc-gamma 1 template sequence. This vector serves as an expression vector for creating a soluble CD38-Fc fusion protein. Another pcDNA3.1 derivative without leading sequence was used to insert the unprocessed CD38 gene. In this case, the stop codon in front of the Fc coding region and the missing leader sequence led to surface expression of CD38. HEK293 cells were transfected with a vector of Fc protein fusion to create a soluble Fc-CD38 fusion protein, and in the case of an unprocessed derivative, CHO-K1 cells were transfected to create a stable cell line expressing CD38.

EXAMPLE 4

Cloning, expression and purification of IgG1 HuCAL®

To express unprocessed IgG, variable fragments of the heavy (VH) and light chain (VL) domains were subcloned from Fab expression vectors into suitable pMORPH®_hIg vectors (see FIGS. 8-10). The restriction endonuclease pairs Blp I / Mfe I (to obtain an insert) and Blp I / Eco RI (to obtain a vector) were used to subclone a fragment of the VH domain into pMORPH®_hIgG1. The pairs of enzymes Eco RV / HpaI (lambda insert) and EcoRV / BsiWI (kappa insert) were used to subclone fragments of the VL domain into the corresponding vectors pMORPH®_hIgκ_l or pMORPH®_h_Igλ_1. The resulting IgG constructs were expressed in HEK293 cells (ATCC CRL-1573) by transient transfection using standard DNA calcium phosphate coprecipitation technology. IgG was purified from cell culture supernatants by affinity chromatography on a Protein A Sepharose column. The further process involved buffer exchange by gel filtration and sterile filtration of purified IgG. Quality control yielded a purity of> 90% upon reducing SDS-PAGE polyacrylamide gel polyacrylamide gel and> 90% monomeric IgG, as determined by analytical size exclusion chromatography. The endotoxin content in the material was determined as a result of a kinetic reaction with a LAL amoebocyte lysate (Cambrex European Endotoxin Testing Service, Belgium).

EXAMPLE 5

Creation and production of chimeric OKT10 (chOKT10; SEQ ID NO: 23 and 24)

To construct chOKT10, the mouse VH and VL regions were amplified by PCR using cDNA prepared from OKT10 mouse hybridoma cell line (ECACC # 87021903). A set of primers was used according to the publications (Dattamajumdar et al., 1996; Zhou et al., 1994). PCR products were used for topo-cloning (Invitrogen; pCRII vector), and single colonies were subjected to sequence analysis (reverse primer M13), which revealed two different kappa light chain sequences and one heavy chain sequence. According to sequence alignment (EMBL database of nucleotide sequences) and published data (Krebber et al., 1997), one of the kappa sequences belongs to the intrinsic spectrum of the fused tumor cell partner X63Ag8.653 and, therefore, does not belong to the OKT10 antibody. Thus, only a new kappa sequence and a single VH fragment was used for further cloning. Both fragments were reamplified to complement the restriction sites of endonucleases, followed by cloning into the corresponding expression vectors-IgG1 pMORPH®. The sequences of the heavy chain (SEQ ID NO: 23) and light chain (SEQ ID NO: 24) are shown in Fig.6. HEK293 cells were transfected and the supernatant analyzed by FACS for binding of the chimeric OKT10 antibody to the Raji cell line, overexpressing CD38 (ATCC).

EXAMPLE 6

Epitope mapping

Materials and methods:

Antibodies:

The following anti-CD38-IgG were used to map the epitope:

MOR # Lot # Format Sequence [mg / ml] / Volume [μl] MOR03077 2CHE106_030602 Human IgG1 0.44 / 1500 MOR03079 2APO31 Human IgG1 0.38 / 500 MOR03080 030116_4CUE16 Human IgG1 2.28 / 200 MOR03100 030612_6SBA6 Human IgG1 0.39 / 500 Chimeric OKT10 * 030603_2CHE111 Human IgG1 0.83 / 500 * chimeric OCT10 consisting of human Fc and variable murine regions

The sequence of CD38:

The amino acid (aa) sequence (positions 44-300) is based on human CD38 taken from the published sequence from the primary complement of SWISS-PROT under the number P28907. When constructing the peptide at position 49, amino acid Q was used (instead of T).

PepSpot Protein Spot Analysis:

Antigen peptides were synthesized on the cellulose membrane in stages, to obtain a specific location (peptide matrix), and covalently linked to the cellulose membrane. Binding assays were performed directly on the peptide matrix. Typically, the antigen peptide matrix was incubated with blocking buffer for several hours to reduce non-specific antibody binding. After incubation with the primary (antigen-peptide binding) antibody in blocking buffer, incubation was performed with a secondary antibody labeled with peroxidase (POD), which selectively binds the primary antibody. A short-term washing was performed in (Tween) -Tris-buffer solution (TBS) immediately after incubation of the antigen peptide matrix with a secondary antibody, followed by the first chemiluminescent experiment, which was carried out in order to obtain preliminary results that determine which antigen peptides bind to the primary antibody. The subsequent several stages of washing with buffer (T-TBS and TBS-buffer) were intended to reduce false-positive binding (non-specific binding of the antibody directly to the cellulose membrane). After these washing steps, a final chemiluminescence analysis was performed. These data were analyzed using image systems showing signal intensity (Boehringer Light units, BLU) as separate measurements for each peptide. To evaluate the non-specific binding of secondary antibodies (against human IgG), these antibodies were incubated as a first step with a peptide matrix in the absence of primary antibodies. If the primary antibody did not show binding to peptides at all, it could be directly labeled with POD, which increased the sensitivity of the system (as was done for MOR3077). In this case, conventional chemical bonding methods via free amino groups were carried out. The antigen was scanned with 13-dimensional peptides (11 overlapping amino acids). The result was a matrix of 123 peptides. Binding assays were performed directly on the matrix. Peptide-bound antibodies MOR03077, MOR03079, MOR03080, MOR03100, and chimeric OKT10 were detected using a secondary antibody labeled with peroxidase (goat peroxidase conjugated anti-human IgG antibody specific for the gamma chain isolated from the affinity of the Ald29 antibody, Sigma; . Mapping was performed with a chemiluminescent substrate in combination with an image system. Additionally, direct POD labeling of MOR03077 was performed to increase the sensitivity of the system.

2. Summary and conclusions

All five antibodies showed different profiles in the analysis of protein spots. A schematic overview is presented in FIG. 7, which illustrates various amino acid sequences of recognized CD38. The epitope for MOR03079 and the chimeric OKT10 can well be considered linear. The epitope for MOR03079 can be postulated on the amino acid segment 192-206 (VSRRFAEAACDVVHV) CD38, while the sequence between amino acids 284 and 298 (FLQCVKNPEDSSCTS) for the chimeric OKT10 is recognized as predominant. Recent results confirm published data for parenteral mouse OKT10 (Hoshino et al., 1997), according to which this epitope is postulated between amino acids 280-298. At the same time, to more accurately determine the epitope and determine key amino acids (the main antigen-antibody interaction sites), the shortening of the VSRRFAEAACDVVHV and FLQCVKNPEDSSCTS peptides and the alanine scan of these two peptides should be provided.

The epitopes for MOR03080 and MOR03100 may well be considered discontinuous, as several peptides were recognized covering various sites of protein sites. These peptides contain amino acids 82-94 and 158-170 of MOR03080 and 82-94, 142-154, 158-170, 188-200 and 280-296 of MOR03100. However, some overlapping between the two epitopes can be postulated, since two different sites located within amino acids 82-94 (CQSVWDAFKGAFI; peptide # 20) and 158-170 (TWCGEFNTSKINY; peptide # 58) are recognized by both antibodies.

The epitope for MOR03077 can be considered as clearly different from the last two epitopes, and it can be described as a discontinuous epitope divided into many segments. The epitope includes amino acids 44-66, 110-122, 148-164, 186-200 and 202-224.

EXAMPLE 7

Analysis of the release / proliferation of IL-6

1. Materials and methods

The proliferation and release analysis of IL-6 was carried out according to Ausiello et al., (2000) with the following modifications: PBMC cells from various healthy donors (with their consent) were purified by density gradient centrifugation using a Histopaque cell separation system according to the supplier’s instructions ( Sigma), and cultured under standard conditions (5% CO ₂ , 37 ° C) in RPMI 1640 medium supplemented with 10% FCS and glutamine (“complete RPMI 1640 medium”). The following antibodies were used for both analyzes: monoclonal anti-CD38-IgGl MOR03077, MOR03079 and MOR03080 HuCAL®, agonistic mouse monoclonal antibody IgG2a (IB4; Malavasi et al., 1984), unrelated IgG1 HuCAL® antibody, control paired isotypesG (IgG1 anti-trinitrophenol, hapten-specific antibody; catalog: # 555571, clone G155-178; Becton Dickinson) or control medium. To analyze the release of IL-6, 1.0E + 06 PBMC cells in 0.5 ml of complete RPMI 1640 medium were incubated for 24 hours in a 15 ml culture tube (Falcon) in the presence of 20 μg / ml antibody. Cell culture supernatants were collected and analyzed for IL-6 release using the Quantikine kit according to the manufacturer's protocol (R&D systems). For proliferation assay, 2.0E + 05 PBMC cells were incubated for 3 days in a 96-well flat-bottom plate (Nunc) in the presence of 20 μg / ml antibody. Each analysis was performed in duplicate. After 4 days, BrdU was added to each well, and before cell fixation and DNA denaturation, the cells were further incubated for 24 hours at 37 ° C according to the supplier's protocol (Roche). BrdU incorporation was measured by anti-BrdU peroxidase-linked antibody in a chemiluminescent setup.

2. Summary and conclusions

Proliferation Analysis

In addition to its catalytic activity as cyclase and hydrolase of cyclic ADP-ribose, CD38 is able to transmit biological relevance signals (Hoshino et al., 1997; Ausiello et al., 2000). These functions can be induced in vivo, for example, by receptor-ligand interactions or cross-linking with anti-CD38 antibodies. Such signals lead, for example, to mobilization of calcium, proliferation of lymphocytes and the release of cytokines. However, this signaling depends not only on the antigenic epitope, but may also vary among different donors (Ausiello et al., 2000). In light of immunotherapy, non-agonistic antibodies are preferred over agonistic antibodies. Therefore, HuCAL® anti-CD38 antibodies (monoclonal antibodies MOR03077; MOR03079, MOR03080) had additional differences in the proliferation and release analysis of IL-6 (an important MM growth factor) compared to the chOKT10 reference antibody and IB4 agonistic anti-CD38 monoclonal antibody.

As shown in FIG. 11 and FIG. 12, the anti-CD38 antibodies of Mon.AT # 1, 2, and 3 HuCAL, as well as the chOKT10 reference antibody and the corresponding negative control, did not or only caused weak proliferation induction and did not cause IL- 6 compared with the IB4 agonist antibody.

EXAMPLE 8

Clonogenicity Analysis

1. Materials and methods

PBMC cells containing autologous CD34 + / CD38 + progenitor cells were isolated from healthy individuals (with their consent) by density gradient centrifugation using a Histopaque cell separation system according to the supplier’s instructions (Sigma) and incubated with various anti-CD38- antibodies IgGI HuCAL® (Mon.AT MOR03077, MOR03079 and MOR03080) and positive control (PC) with chOKT10 in an amount of 10 μg / ml. Culture medium and unrelated IgGl HuCAL® served as initial control. Each ADCC assay used 4.0E + 05 RBMC cells, which were incubated for 4 hours at 37 ° C in RPMI 1640 medium supplemented with 10% FCS. For clonogenicity analysis, 2.50 ml of “complete” methylcellulose (CellSystems) was injected into 2.5E + 05 cells from an ADCC assay and incubated to develop colonies for at least 14 days in a controlled environment (37 ° C; 5% CO ₂ ) The colonies were analyzed by two independent operators and grouped into BFU-E + CFU-GEMM (erythroid colony-forming "explosive" units and granulocyte / erythroid / macrophage / megakaryocyte stem cells) and CFU-GM (granulocyte / macrophage stem cells).

2. Summary and conclusions

Since the expression of CD38 was detected not only in myeloid immune cells (e.g., monocytes, granulocytes) and lymphoid (e.g., activated B- and T-cells; plasma cells) origin, but also in the corresponding progenitor cells (CD34 + / CD38 +) It seems important that these cells are not damaged by antibody-mediated lysis. In this regard, a clonogenicity analysis was used to study these effects with respect to CD34 + / CD38 + precursor cells.

Healthy donor PBMC cells were incubated with HuCAL® anti-CD38 antibodies (Mon.AT # 1, Mon.AT # 2 and Mon.AT # 3) or several control groups (unrelated HuCAL® antibody, culture medium and chOKT10 reference antibody as a positive control) according to the standard ADCC protocol, followed by additional incubation in conditioned methylcellulose for colony development. As shown in FIG. 13, no significant decrease in colony forming units was detected for all HuCAL® anti-CD38 antibodies compared to an unrelated antibody or a reference antibody.

EXAMPLE 9

ADCC assays with different cell lines and primary multiple myeloma cells

1. Materials and methods

Isolation and ADCC samples in patients with MM: bone marrow punctate was obtained from patients with multiple myeloma (with their consent). Malignant cells were purified according to standard protocol using anti-CD138 magnetic beads (Milteny Biotec) after density gradient centrifugation (Sigma). ADCC analysis was performed as described above.

2. Summary and conclusions

For ADCC analysis, several cell lines derived from various malignant neoplasms were used to display the cytotoxic effect of HuCAL® anti-CD38 antibodies in a wider range of cell lines, including different origins and levels of expression of CD38. As shown in FIG. 14, all cells underwent lysis in ADCC at a constant antibody concentration (5 μg / ml) and an E: T ratio of 30: 1. Cytotoxicity through ADCC was also detected in several samples in patients with multiple myeloma. All HuCAL® anti-CD38 antibodies were capable of dose-dependent lysis of MM cells, and EC50 values ranged from 0.006 to 0.249 nm (FIG. 15).

EXAMPLE 10

Cross-reactivity analysis by FACS and immunohistochemistry (IHC)

1. Materials and methods

Tonsil Immunohistochemistry: For ILC analysis, anti-CD38 HuCAL® monoclonal antibodies and an unrelated negative control antibody were converted to the dHLX bivalent format (Plückthun & Pack, 1997). Cryosections of 5 μm from lymph nodes taken from cynomolgus macaque, rhesus and human macaques (obtained from the archive of the Institute of Pathology of the University of Graz, Austria) were performed on a Leica CM3050 cryostat. Sections were air dried for 30 minutes to 1 hour, fixed in ice-cold methanol for 10 minutes and washed with PBS. To detect the dHLX format, a mouse anti-His antibody (Dianova) was used in combination with the Envison kit (DAKO). Only Envison kit was used to detect murine anti-CD38 antibodies (e.g., reference monoclonal mouse OKT10).

FACS analysis of lymphocytes: EDTA-treated blood samples were obtained from healthy people (with their consent), from rhesus monkeys and cynomolgus and subjected to density gradient centrifugation using a Histopaque cell separation system according to the instructions of the supplier (Sigma). For FACS analysis, interphase cells with primary antibodies (anti-CD38 HuCAL® and monoclonal antibodies (Mon.AT) were incubated as a negative control, such as murine IgG2a or Fab format, with a positive control in the form of a mouse antibody OKT10 and a control for related isotypes ) followed by incubation with anti-M2-Flag (Sigma; Fab format only) and phycoerythrin (PE) labeled anti-mouse conjugate (Jackson Research). FACS analysis was performed on a population of lymphocytes giving a signal above a threshold value.

2. Summary and conclusions

Anti-CD38 HuCAL® was analyzed for interspecific cross-reactivity. Although all monoclonal anti-CD38 antibodies were capable of detecting human CD38 on lymphocytes in FACS and IHC analyzes, only MOR03080, together with the OKT10 positive control, showed additional reactivity for cynomolgus and rhesus CD38 macaques (see Table 5: cross-reactivity analysis).

Table 5 Lymphocytes (FACS) and lymph nodes (IHC) from: Antibody Person Macaque cynomolgus Macaque rhesus Mon.AT # 1 ++ - - Mon.AT # 2 ++ - - Mon.AT # 3 ++ ++ ++ PC ++ ++ ++ NC - - - ++: highly positive coloration; -: lack of staining; NC: negative control; RS: positive control (= reference Mon.AT cynomolgus)

EXAMPLE 11

Treatment with MOR03080 xenografts of human myeloma in mice (using RPMI8226 cell line)

1. Setting the subcutaneous mouse model

A subcutaneous murine model of human myeloma tumor cell line RPMI8226 in female CB-17-SCID mice was performed according to Aurigon Life Science GmbH (Tutzing, Germany) as follows: on day -1, 0 and 1, polyclonal anti-asialo-GM1 antibodies were intravenously administered (ASGM) (WAKO-Chemicals), depleting xenoreactive NK cells in SCID mice, to deactivate any residual specific immune reactivity in CB-17-SCID mice. On day 0, RPMI8226 tumor cells were inoculated subcutaneously in an amount of either 5 × 10 ⁶ or 1 × 10 ⁷ in 50 μl of PBS in the right flank of both ASGM-treated mice (as described above) and not (each group consisted of five mice ) In all 4 inoculated groups, the tumor developed in a similar way without significant differences, which was detected with or without the introduction of anti-asial GM1 antibodies or by inoculating a different number of cells. Tumors showed slow growth, with a tendency to slow down or change in size over several days. Two tumors detected a change in size throughout the study period, and one tumor after a peak volume of 321 mm ^{3 was} even regarded as completely gone. To study the treatment on this tumor model, it is necessary to include a large number of animals with a vaccinated tumor per group.

2. Treatment with MOR03080

2.1 Purpose of the study

This study was performed according to Aurigon Life Science GmbH (Tutzing, Germany) to compare the antitumor efficacy of the intraperitoneal antibodies (anti-CD38 HuCAL®) used with the carrier efficacy (PBS). The human antibody hMOR03080 (IgGl isotype) was tested with various amounts and treatment regimens. Additionally tested the chimeric antibody chMOR03080 (IgG2a isotype: a chimeric antibody containing the variable regions of MOR03080 and constant mouse regions was constructed by the method similar to that described in Example 5 for chimeric OKT10 (mouse VH / VL and human constant regions). The RPMI8226 malignant cell line was selected in as a model and subcutaneously inoculated to female SCID mice as described above, the final parameters of the study were body weight (bw), tumor volume and clinical symptoms.

2.2 Antibodies and Carrier

The ready-to-use Aurigon Pharmaceutical Antibodies at concentrations of 2.13 mg / ml (MOR03080 hIgG1) and 1.73 mg / ml (MOR03080 chIgG2a were stored at -80 ° C until use. Antibodies were thawed and diluted with PBS to the appropriate final concentration. Aurigon carrier (PBS) was stored at 4 ° C until use.

2.3 Characterization of animals

Type: mouse

Load: C.B.-17-SCID (C.B-Igh-lb / IcrTac) by Fox chase

Quantity and gender: 75 females

Supplier: Taconic M&B, Bomholtvej 10, DK-8680 Ry

Health Status: SPF

Prescribed weight: approximately 18 grams

Adaptation: 9 days

2.4 Tumor cell line

Tumor cells (cell line RPMI8226) were grown and transferred to Aurigon Life Science GmbH, where the cells were separated and grown for another cycle. In Aurigon, cells were prepared for injection on the day of inoculation. The culture medium used for cell reproduction was RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine and penstrap. No unexpected manifestations of growth rate or behavior were observed in the cells.

For inoculation, tumor cells were suspended in PBS and adjusted to a final concentration of 1 × 10 ⁷ cells / in 50 μl of PBS. The tumor cell suspension was thoroughly mixed before injection.

2.5 order of experiment

On day 0, 1 × 10 ⁷ RPMI8226 tumor cells were subcutaneously inoculated into the right dorsal region of 75 SCID mice. The first group was formed from 15 randomized animals (group 5) immediately after inoculation. This group was administered 1 mg / kg body weight hIgG1-MOR03080 every other day between 14 and 36 days. Of the remaining 60 animals on day 31, 4 groups of ten animals in each group were formed (the tumor volume was about 92 mm ³ ). Groups 1-4 were formed according to comparable tumor sizes and standard deviations. Additionally, a group of 5 animals was selected (group 6), in which relatively small tumor volumes (tumor volume of about 50 mm ³ ) were revealed for comparison with the group that underwent preliminary treatment (in all but three mice, the tumor volume was less than 10 mm ³ , one about 22 mm ³ , one about 44 mm ³ and one about 119 mm ³ ). Groups 1-4 every other day, starting from 32 days to 68 days, were administered either PBS (vehicle; group 1), 1 mg / kg body weight hIgG1-MOR03080 (group 2) or 5 mg / kg bw hIgG1-MOR03080 (group 3), or 5 mg / kg bw chIgG2a-MOR03080 (group 4). Group 6 did not receive any treatment (see Table 6). Tumor volumes, body weight, and clinical symptoms were measured twice a week until the end of the study.

Table 6 Group Number of animals Type of introduction Substance Mode The administered dose (mcg / kg) The applied volume (µl / kg) one 10 vb Carrier (PBS) Every second day between 32 and 68 in the afternoon - 10 2 10 vb MOR03080 human IgG1 Every second day between 32 and 68 in the afternoon one 10 3 10 vb MOR03080 human IgG1 Every second day between 32 and 68 in the afternoon 5 10 four 10 vb MOR03080 chimeric IgG2a Every second day between 32 and 68 in the afternoon 5 10 5 fifteen vb MOR03080 human IgG1 Every second day between 14 and 36 in the afternoon one 10 6 5 - - - - -

2.6 Results

Clinical observations and mortality

No specific clinical manifestations or mortality associated with the tumor or injected substances were detected. In group 3 (5 mg / kg hIgG1), four animals died during blood sampling (one mouse on day 3, one on day 34; two on day 52). In group 4 (1 mg / kg muIgG2a), the only animal died during blood sampling (on day 34). All other animals that died during the study were specifically euthanized due to the size of the tumor.

Body weight change

No effect was found on the change in mass associated with the drug, compared with group 1 (carrier). Blood collection was significantly affected by blood sampling in group 3 (5 mg / kg muIgG2a) and in group 4 (5 mg / kg muIgG2a). Despite such gaps, the average value of the weight gain in all groups was constant.

Tumor development (see Fig. 16)

In group 1 (carrier), tumor growth occurred at the expected rate with slow progression. Due to the fact that this cell line has strongly expressed standard deviations, values for the largest and smallest tumors were excluded from further statistical analysis. Tumor growth in animals in group 1 was comparable to tumor growth in group 6 (without treatment), despite the fact that in this group the initial average tumor volume was 31 days less. Thus, treatment could have a small effect on tumor growth rate. In group 1, two mice had to be sacrificed before 83 days due to the size of the tumor, and an additional one mouse before 87 days, so the average value of the tumor volume after 80 days was not representative. In group 6, one mouse had to be sacrificed before day 80 due to the size of the tumor, two mice before day 83, and additionally before day 87, so the average tumor volume after day 76 was not representative.

In group 2, which was administered 1 mg / kg hIgG1 body weight, one animal was excluded from further analysis due to the fact that the tumor grew into muscle tissue, and this is usually the reason for the increase in the tumor growth rate. Compared with control group 1 (carrier), the average tumor size began to vary significantly, starting from day 45 until the end of the study. After treatment, no increase in tumor growth was detected (day 68).

Animals in group 3 (5 mg / kg bw hIgG1) showed a marked decrease in tumor growth compared to group 1 (carrier), which became statistically significant from day 38 to day 83. The average tumor volume again began to grow significantly approximately two weeks after the end of treatment. One in ten tumors disappeared on day 45 and did not begin to re-grow until 19 days after the end of treatment

The best indicators in all treated groups, with an initial tumor volume of 92 mm ³ , were found in group 4 (5 mg / kg bw muIgG2a), where a clear regression of the average tumor volume was manifested, and in 4 animals even the tumor disappeared before the end observation period. The differences in the mean volume of a group 1 tumor (carrier) were very significant, from day 38 until the end of the study.

Initial administration of 1 mg / kg bw hIgG1 between 14 and 36 days (group 5) had an early and long-lasting effect on tumor development; one animal was excluded from further analysis because the tumor grew into muscle tissue. On day 31, only five animals had a measurable tumor at the inoculation site, compared with the rest of the inoculated animals, of which only 2 out of 60 did not respond to inoculation of the tumor. Tumor progression was delayed by approximately 31 days (when comparing 52 days from control group 1 with 83 days from group 5). In approximately 50% of the animals, no tumor was detected at the inoculation site at the end of the study.

2.7 Conclusions

No specific tumor or substance-related clinical or mortality data were detected compared with group 1 (control).

No effect was found on the change in mass associated with the drug.

Tumor growth from RPMI8226 tumor cells after treatment was reduced as follows, in order of effectiveness: 1 mg / kg hIgG1, from 14 to 36 days every other day (group 5)> 5 mg / kg muIgG2a, from 32 to 68 days every other day (group 4 )> 5 mg / kg hIgG1, from 32 to 68 days a day (group 3)> 1 mg / kg hIgG1, from 32 to 68 days a day (group 2). In groups 2-4, the average tumor volumes increased again after treatment in a variable range.

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Claims (53)

1. The isolated human antibody, or a humanized antibody, or a functional fragment thereof specific for the CD38 epitope (SEQ ID NO: 22), wherein said antibody, or its functional fragment, is able to mediate the lysis of CD38 + target cells by ADCC (antibody- dependent cytotoxicity), with an efficiency of at least five times that of chimeric OCT 10 (SEQ ID NOs: 23 and 24) under the same or substantially the same conditions when using human PBMC cells as effector cells, where indicated cl CD38 + target chains are selected from the group consisting of LP-1 (DSMZ: ACC41) and RPMI-8226 (ATCC: CCL-155), and where the ratio of effector cells to target cells is from about 30: 1 to about 50: 1 and where the isolated human antibody, its functional fragment, is specific for the CD38 epitope, wherein said epitope contains one or more amino acid residues from amino acid residues 1-215 of CD38 (SEQ ID NO: 22); preferably the epitope contains one or more amino acid residues contained in one or more of the amino acid segments taken from the list of amino acid segments 44-66, 82-94, 142-154, 148-164, 158-170 and 192-206 of CD38. 2. The selected antibody or its functional fragment according to claim 1, which is a human antibody or its functional fragment. 3. The selected antibody or its functional fragment according to claim 1 or 2, containing the H-CDR3 region, the H-CDR2 region and the H-CDR1 region, which essentially correspond to SEQ ID NO: 5, 6, 7 or 8; and comprising an L-CDR3 region, an L-CDR2 region, and an L-CDR1 region, which essentially correspond to SEQ ID NO: 13, 14, 15, or 16. 4. The selected antibody or its functional fragment according to claim 3, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 5; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 13. 5. The selected antibody or its functional fragment according to claim 4, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 5, and the variable region of the light chain, presented as SEQ ID NO: 13. 6. The selected antibody or its functional fragment according to claim 3, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 6; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 14. 7. The selected antibody or its functional fragment according to claim 6, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 6, and the variable region of the light chain, presented as SEQ ID NO: 14. 8. The selected antibody or its functional fragment according to claim 3, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 7; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 15. 9. The selected antibody or its functional fragment of claim 8, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 7, and the variable region of the light chain, presented as SEQ ID NO: 15. 10. The selected antibody or its functional fragment according to claim 3, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 8; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are represented as SEQ ID NO: 16. 11. The selected antibody or its functional fragment of claim 10, where the specified antibody or its functional fragment contains the variable region of the heavy chain represented by SEQ ID NO: 8, and the variable region of the light chain represented by SEQ ID NO: 16. 12. The selected antibody or its functional fragment according to claim 1 or 2, containing the variable region of the heavy chain, presented as SEQ ID NO: 5, 6, 7 or 8. 13. The selected antibody or its functional fragment according to claim 1 or 2, containing the variable region of the light chain, presented as SEQ ID NO: 13, 14, 15 or 16. 14. The selected antibody or its functional fragment according to claim 1 or 2, which is a Fab or scFv. 15. The selected antibody according to claim 1 or 2, which is an IgG. 16. The isolated antibody of claim 15, wherein said IgG is IgGl. 17. The isolated human antibody, or a humanized antibody or its functional fragment, is specific for the CD38 epitope (SEQ ID NO: 22), while this antibody or its functional fragment is capable of mediating the death of a CD38-transfected CHO cell by CDC (complement dependent cytotoxicity) , with an efficacy of at least two times that of chimeric OCT 10 (SEQ ID NOs: 23 and 24) under identical or substantially identical conditions, wherein said antibody or its functional fragment is specific for ep stamp CD38, wherein said epitope comprises one or more amino acid residues from amino acid residues 1-215 CD38 (SEQ ID NO: 22); preferably said epitope contains one or more amino acid residues contained in one or more of the amino acid segments taken from the list of amino acid segments 44-66, 82-94, 142-154, 148-164, 158-170 and 192-206 of CD38. 18. The selected antibody or its functional fragment according to 17, which is a human antibody or its functional fragment. 19. The selected antibody or its functional fragment according to 17 or 18, containing the H-CDR3 region, the H-CDR2 region and the H-CDR1 region, which essentially correspond to SEQ ID NO: 5, 6, 7 or 8; and comprising an L-CDR3 region, an L-CDR2 region, and an L-CDR1 region, which essentially correspond to SEQ ID NO: 13, 14, 15, or 16. 20. The selected antibody or its functional fragment according to claim 19, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 5; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 13. 21. The selected antibody or its functional fragment according to claim 20, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 5, and the variable region of the light chain, presented as SEQ ID NO: 13. 22. The selected antibody or its functional fragment according to claim 19, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 6; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 14. 23. The selected antibody or its functional fragment according to item 22, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 6, and the variable region of the light chain, presented as SEQ ID NO: 14. 24. The selected antibody or its functional fragment according to claim 19, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 7; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are presented as SEQ ID NO: 15. 25. The selected antibody or its functional fragment according to paragraph 24, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 7, and the variable region of the light chain, presented as SEQ ID NO: 15. 26. The selected antibody or its functional fragment according to claim 19, where the specified antibody or its functional fragment contains an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 8; and where the specified antibody or its functional fragment contains the L-CDR1 region, the L-CDR2 region and the L-CDR3 region, which are represented as SEQ ID NO: 16. 27. The selected antibody or its functional fragment according to p, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 8, and the variable region of the light chain, presented as SEQ ID NO: 16. 28. The selected antibody or its functional fragment according to 17 or 18, containing a variable heavy chain, presented as SEQ ID NO: 5, 6, 7 or 8. 29. The selected antibody or its functional fragment according to 17 or 18, containing a variable light chain, presented as SEQ ID NO: 13, 14, 15 or 16. 30. The isolated antibody or fragment thereof according to 17 or 18, which is a Fab or scFv. 31. The isolated antibody or fragment thereof according to 17 or 18, which is an IgG. 32. The isolated antibody of claim 31, wherein said IgG is IgGl. 33. The selected antibody or its functional fragment specific for the CD38 epitope (SEQ ID NO: 22), containing the H-CDR1 region, the H-CDR2 region and the H-CDR3 region, which are represented as SEQ ID NO: 5, 6, 7 or 8; and wherein said antibody or fragment thereof comprises an L-CDR1 region, an L-CDR2 region, and an L-CDR3 region, which are represented as SEQ ID NO: 13, 14, 15, or 16. 34. The selected antibody or fragment thereof according to claim 33, wherein said antibody or fragment thereof comprises an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 5; and wherein said antibody or fragment thereof comprises an L-CDR1 region, an L-CDR2 region and an L-CDR3 region, which are represented as SEQ ID NO: 13. 35. The selected antibody or its functional fragment according to clause 34, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 5, and the variable region of the light chain, presented as SEQ ID NO: 13. 36. The selected antibody or fragment thereof according to claim 33, wherein said antibody or fragment thereof comprises an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 6; and wherein said antibody or fragment thereof comprises an L-CDR1 region, an L-CDR2 region, and an L-CDR3 region, which are represented as SEQ ID NO: 14. 37. The selected antibody or its functional fragment according to clause 36, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 6, and the variable region of the light chain, presented as SEQ ID NO: 14. 38. The isolated antibody or fragment thereof according to claim 33, wherein said antibody or fragment thereof comprises an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 7; and wherein said antibody or fragment thereof comprises an L-CDR1 region, an L-CDR2 region and an L-CDR3 region, which are represented as SEQ ID NO: 15. 39. The selected antibody or its functional fragment according to § 38, where the specified antibody or its functional fragment contains the variable region of the heavy chain, presented as SEQ ID NO: 7, and the variable region of the light chain, presented as SEQ ID NO: 15. 40. The isolated antibody or fragment thereof according to claim 33, wherein said antibody or fragment thereof comprises an H-CDR1 region, an H-CDR2 region and an H-CDR3 region, which are represented as SEQ ID NO: 8; and wherein said antibody or fragment thereof comprises an L-CDR1 region, an L-CDR2 region, and an L-CDR3 region, which are represented as SEQ ID NO: 16. 41. The selected antibody or its functional fragment according to claim 40, wherein said antibody or its functional fragment contains the variable region of the heavy chain represented by SEQ ID NO: 8 and the variable region of the light chain represented by SEQ ID NO: 16. 42. The isolated antibody or functional fragment according to claim 33, which is a Fab or scFv. 43. The isolated antibody of claim 33, which is IgG. 44. The isolated antibody of claim 43, wherein said IgG is IgGl. 45. The variable region of the heavy chain of the isolated antibody or its functional fragment according to any one of claims 1-44, encoded by (i) a nucleic acid sequence containing SEQ ID NO: 1, 2, 3 or 4; or (ii) a nucleic acid sequence that hybridizes under stringent conditions to the complementary strand of SEQ ID NO: 1, 2, 3 or 4, wherein said antibody or functional fragment thereof is specific for the CD38 epitope, or (iii) a nucleic acid sequence that 85% identical to the sequence of SEQ ID NO: 1, 2, 3 or 4, where the specified antibody or its functional fragment is specific for the CD38 epitope. 46. The variable region of the heavy chain according to item 45, which is encoded by a nucleic acid sequence containing SEQ ID NO: 1, 2, 3 or 4. 47. The variable region of the light chain of the isolated antibody or its functional fragment according to any one of claims 1-44, encoded by (i) a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 9, 10, 11 or 12; or (ii) nucleic acid sequences that hybridize under stringent conditions to the complementary strand of SEQ ID NO: 9, 10, 11, or 12, wherein said antibody or functional fragment thereof is specific for the CD38 epitope; or (iii) a nucleic acid sequence that is 85% identical to SEQ ID NO: 9, 10, 11 or 12, wherein said antibody or functional fragment thereof is specific for the CD38 epitope. 48. The variable region of the light chain according to clause 47, which is encoded by a nucleic acid sequence containing SEQ ID NO: 9, 10, 11 or 12. 49. A pharmaceutical composition for anti-CD38 therapy, comprising an effective amount of an antibody or functional fragment thereof according to any one of claims 1 to 44 and a pharmaceutically acceptable carrier or excipient. 50. A method of treating a disorder or condition associated with the undesirable presence of CD38 + cells, comprising administering to the individual an effective amount of a pharmaceutical composition according to claim 49. 51. The method of claim 50, wherein said disorder or condition is a hematologic disease, multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, and acute lymphocytic leukemia. 52. The method of claim 50, wherein said disorder or condition is an inflammatory disease selected from rheumatoid arthritis and systemic lupus erythematosus. 53. An isolated CD38 epitope essentially consisting of an amino acid sequence selected from the group consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 of CD38; or an isolated CD38 epitope consisting of an amino acid sequence selected from the group consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 CD38; or a kit containing an isolated CD38 epitope containing one or more amino acid fragments from the list 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224, and an antibody library and instructions for application.

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