CN1976950B - Anti-CD38 human antibodies and uses therefor. - Google Patents

Abstract

The present invention provides recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for CD38, which plays an integral role in various disorders or conditions. These antibodies, accordingly, can be used to treat, for example, hematological malignancies such as multiple myeloma. Antibodies of the invention also can be used in the diagnostics field, as well as for investigating the role of CD38 in the progression of disorders associated with malignancies. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use. The invention also provides isolated novel epitopes of CD38 and methods of use therefore.

Description

Anti-CD38 human antibody and use thereof

This application claims U.S. Provisional Application 60/541,911 filed February 6, 2004, U.S. Provisional Application 60/547,584, filed February 26, 2004, U.S. Provisional Application 60/553,948, filed March 18, 2004, and 2004 Priority to U.S. Provisional Application 60/599,014, filed August 6, the contents of which are hereby incorporated by reference in their entirety. the

Background of the invention

CD38 is a type II membrane glycoprotein that belongs to the family of extracellular enzymes due to its enzymatic activities of ADP ribosyl cyclase and cADP hydrolase. During ontogeny, CD38 is present on CD34+ committed stem cells as well as lineage-committed progenitors of lymphocytes, erythroid and myeloid cells. CD38 is thought to be expressed only in the lymphoid lineage during the early stages of T and B cell development. the

Upregulation of CD38 may serve as a marker of lymphocyte activation—particularly B cell differentiation along the plasmacytoid pathway. The (co-)receptor function of CD38 is presumed to cause intracellular signaling or intercellular communication through its ligand CD31, and at the same time act as an intracellular regulator of the second messenger loop ADPr in various signaling cascades. However, since knockout of the analog in mice or anti-CD38 autoantibodies in humans appears harmless, its physiological importance remains to be elucidated. the

In addition to observing its expression in the hematopoietic system, the researchers found that CD38 is upregulated in various cell lines derived from B-cell tumors, T-cell tumors, and myeloid/monocytic tumors, including B- or T- Acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). For example, strong expression of CD38 was observed in most of all patient samples in MM. the

Thus, overexpression of CD38 on malignant cells provides an attractive therapeutic target for immunotherapy. It is particularly noteworthy that most early pluripotent stem cells in the hematopoietic system are CD38 negative, and the degree of cytotoxic effect produced by ADCC or CDC is well correlated with the expression level of each target. the

Existing approaches to anti-CD38 therapy can be divided into two categories: in vivo and in vitro approaches. In an in vivo approach, an anti-CD38 antibody is administered to an individual in need of such treatment to produce an anti-CD38 antibody-mediated reduction in CD38-overexpressing malignant cells. Reduction can be achieved by antibody-mediated ACDD and/or CDC produced by effector cells, or by using anti-CD38 antibodies as targeting moieties to deliver cytotoxic substances such as saporin to target cells for subsequent internalization. In an in vitro method, a cell population containing CD38-overexpressing malignant cells, such as bone marrow cells, is removed from an individual in need of treatment and contacted with an anti-CD38 antibody. Target cells can be destroyed with cytotoxic substances such as saporin (as described in the in vivo method), or removed by contacting the cell population with immobilized anti-CD38 antibodies, thereby removing target cells overexpressing CD38 from the mixture. The cell population from which the target cells have been removed is then returned to the patient. the

Antibodies specific to CD38 can be divided into different types according to different characteristics. Binding of some antibodies to CD38 molecules (mainly amino acids 220-300) can elicit activities within target cells such as Ca2+ release, cytokine release, phosphorylation events, and growth stimulation, depending on the specificity of each antibody (Konopleva et al., 1998 Ausiello et al., 2000), but no exact relationship was found between the binding sites of various known antibodies and their (non-)competitive properties (Funaro et al., 1990). the

Relatively little is known about the efficacy of published anti-CD38 antibodies. It is now known that all known antibodies appear to specifically recognize an epitope in the C-terminal portion of CD38 (amino acid residues 220-300). So far, no antibodies specific for epitopes located in the N-terminal portion of CD38, far from the active site in the primary sequence of the protein, are known. However, we found in clinical trials that when OKT10 is a chimeric construct containing a human Fc part, it has relatively low affinity and efficacy. Furthermore, OKT10 is a murine antibody and therefore not suitable for administration to humans. Human anti-CD38 scFv antibody fragments have recently been described (WO 02/06347). However, this antibody is specific for selectively expressed CD38 epitopes. the

Therefore, considering the great therapeutic potential of anti-CD38 antibodies, there is an urgent need for human anti-CD38 antibodies with high affinity and high efficacy in killing CD38-overexpressing malignant cells mediated by ADCC and/or CDC. the

The present invention fulfills these needs and others by providing fully human and highly potent anti-CD38 antibodies, as described below. the

Summary of the invention

One object of the present invention is to provide human and humanized antibodies that can effectively mediate killing of CD38 overexpressing cells. the

Another object of the present invention is to provide antibodies that can be safely administered to humans. the

Yet another object of the present invention is to provide methods of treating diseases and/or disorders associated with up-regulation of CD38 by using one or more antibodies of the present invention. These and other objects of the invention are described in more detail below. the

In one aspect, the invention provides an isolated antibody or functional antibody fragment comprising an antigen binding region specific for an epitope of CD38, wherein, when human PBMC cells are used as effector cells and when the ratio of effector cells to target cells is about When 30:1 to 50:1, under the same or substantially the same conditions, the antibody or functional fragment thereof is capable of passing antibody-dependent cellular cytotoxicity ("ADCC") better than those having SEQ ID NO: 23 and 24 The chimeric OKT10 was at least 2-5 fold more potent in mediating CD38+ target cell killing (LP-1 (DSMZ: ACC41) and RPMI-8226 (ATCC: CCL-155)). This antibody or functional fragment thereof may contain an antigen binding region comprising the H-CDR3 region shown in SEQ ID NO: 5, 6, 7 or 8; the antigen binding region may also comprise SEQ ID NO: 5 , the H-CDR2 region shown in 6, 7 or 8; and the antigen-binding region may also comprise the H-CDR1 region shown in SEQ ID NO: 5, 6, 7 or 8. The CD38-specific antibody of the present invention may contain an antigen-binding region, the antigen-binding region comprising the L-CDR3 region shown in SEQ ID NO: 13, 14, 15 or 16; the antigen-binding region may also comprise SEQ ID NO: The L-CDR1 region shown in 13, 14, 15 or 16; the antigen binding region may also comprise the L-CDR2 region shown in SEQ ID NO: 13, 14, 15 or 16. the

In another aspect, the present invention provides an isolated antibody or functional antibody fragment comprising an antigen-binding region specific for an epitope of CD38, wherein said antibody or its The functional fragment was able to mediate killing of CD38-transfected CHO cells by CDC with at least 2-fold greater potency than chimeric OKT10 (SEQ ID NO: 23 and 24). The antibody satisfying these conditions may contain an antigen-binding region comprising the H-CDR3 region shown in SEQ ID NO: 5, 6 or 7; the antigen-binding region may also comprise SEQ ID NO: 5, 6 or 7 and the antigen-binding region may also comprise the H-CDR1 region shown in SEQ ID NO: 5, 6 or 7. The CD38-specific antibody of the present invention may contain an antigen-binding region, and the antigen-binding region includes the L-CDR3 region shown in SEQ ID NO: 13, 14 or 15; the antigen-binding region may also include SEQ ID NO: 13, 14 or the L-CDR1 region shown in 15; and the antigen-binding region may also comprise the L-CDR2 shown in region SEQ ID NO: 13, 14 or 15. the

Antibodies of the present invention (and functional fragments thereof) may contain an antigen-binding region specific to an epitope of CD38, wherein the epitope contains one or more of amino acid residues 43-215 of CD38, as shown in SEQ ID NO: ID NO: 22. More specifically, the epitope bound by the antigen-binding region may contain one or more amino acid stretches selected from amino acid stretches 44-66, 82-94, 142-154, 148-164, 158-170, and 192-206 One or more amino acid residues contained in (amino acid stretch). For some antibodies the epitope may be linear and for others the epitope may be conformational (ie discontinuous). An antibody or functional fragment thereof having one or more of these properties may contain an antigen binding region comprising the H-CDR3 region shown in SEQ ID NO: 5, 6, 7 or 8; the antigen binding region may also be comprising the H-CDR2 region shown in SEQ ID NO: 5, 6, 7 or 8; and the antigen-binding region may also comprise the H-CDR1 region shown in SEQ ID NO: 5, 6, 7 or 8. The CD38-specific antibody of the present invention may contain an antigen-binding region, the antigen-binding region comprising the L-CDR3 region shown in SEQ ID NO: 13, 14, 15 or 16; the antigen-binding region may also comprise SEQ ID NO: 13, The L-CDR1 region shown in 14, 15 or 16; and the antigen-binding region may also comprise the L-CDR2 region shown in SEQ ID NO: 13, 14, 15 or 16. the

Peptide variants of the sequences disclosed herein are also included within the scope of the present invention. Accordingly, the present invention includes anti-CD38 antibodies comprising the heavy chain amino acid sequence: a sequence whose CDR region has at least 60% sequence identity with the CDR region shown in SEQ ID NO: 5, 6, 7 or 8; and/or its CDR A sequence having at least 80% sequence homology to the CDR region shown in SEQ ID NO: 5, 6, 7 or 8. Also included are anti-CD38 antibodies comprising the following light chain amino acid sequences: its CDR region has at least 60% sequence identity to the CDR region shown in SEQ ID NO: 13, 14, 15 or 16; ID NO: a sequence having at least 80% sequence homology to the CDR region shown in 13, 14, 15 or 16. the

For example, antibodies of the invention may be IgG (eg, IgG ₁ ), and antibody fragments may be Fab or scFv. Accordingly, antibody fragments of the invention may be, or may contain, an antigen binding region having one or more of the characteristics described herein.

The invention also relates to isolated nucleic acid sequences, each encoding an antigen-binding region of a human antibody or a functional fragment thereof, the antigen-binding region being specific for an epitope of CD38. This nucleic acid sequence can encode the variable heavy chain of an antibody, and contains a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 or 4, or under highly stringent conditions with SEQ ID NO: 1, 2, 3 Or 4 nucleic acid sequences that hybridize to the complementary strand. The nucleic acid may encode the variable light chain of an isolated antibody or a functional fragment thereof, and may contain a sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 or 12, or under highly stringent conditions with SEQ ID NO: 9 , 10, 11 or 12 complementary strand hybridized nucleic acid sequence. the

The nucleic acids of the invention are suitable for recombinant production. Accordingly, the invention also relates to vectors and host cells comprising the nucleic acid sequences of the invention. the

The compositions of the invention may be used for therapeutic or prophylactic use. Therefore, the present invention includes a pharmaceutical composition comprising the antibody (or functional antibody fragment) of the present invention and a pharmaceutically acceptable carrier or excipient. In a related aspect, the invention provides methods of treating diseases or conditions associated with the undesirable presence of CD38 or CD38 expressing cells. The method comprises administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising an antibody described or contemplated herein. the

The present invention also relates to isolated epitopes of CD38, in linear or conformational form, and their use in isolated antibodies or functional fragments thereof comprising an antigen binding region specific for said epitopes. In this regard, a linear epitope may comprise amino acid residues 192-206, whereas a conformational epitope may comprise amino acids 44-66, 82-94, 142-154, 148-164, 158-170 selected from CD38 and one or more amino acid residues of 202-224. For example, an epitope of CD38 can be used to isolate antibodies or functional fragments thereof (each antibody or antibody fragment comprising an antigen binding region specific for such an epitope) comprising contacting the epitope of CD38 with an antibody library and The step of isolating said one or more antibodies or functional fragments thereof. the

In another embodiment, the invention provides an isolated epitope of CD38 consisting essentially of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192- The amino acid sequence of 206 and 202-224 constitutes. In the present invention, such an epitope "consists essentially of" one of the above-mentioned amino acid sequences plus other characteristics, provided that the other characteristics do not substantially affect the basic characteristics and novelty of the epitope. the

In yet another embodiment, the invention provides an isolated epitope of CD38 consisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 amino acid sequence composition. the

The present invention also provides a kit comprising (i) one or more amino acid segments selected from 44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224 (ii) an antibody library; and (iii) instructions for using the antibody library to isolate one or more members of the library that specifically bind to the epitope. the

Brief description of the drawings

Figure 1a provides the nucleic acid sequences of various novel antibody heavy chain variable regions;

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

Figure 2a provides the nucleic acid sequences of various novel antibody light chain variable regions;

Figure 2b provides the amino acid sequences of the light chain variable regions of various novel antibodies. CDR regions LCDR1, LCDR2 and LCDR3 are indicated in bold from N- to C-terminus;

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

Figure 4 provides the amino acid sequence of the light chain variable region of the HuCAL antibody main gene sequence based on various consensus sequences. CDR regions LCDR1, LCDR2 and LCDR3 are indicated in bold from N- to C-terminus;

Figure 5 provides the amino acid sequence of CD38 (SWISS-PROT main accession number P28907);

Figure 6 provides the nucleotide sequences of the heavy and light chains of chimeric OKT10;

Figure 7 provides a schematic diagram of the epitopes of representative antibodies of the invention;

_h_Igκ_1的DNA序列(bp601-1400)(SEQ ID NO：33)：该载体基于pcDNA3.1+载体(Invitrogen)。Vκ-填充序列的氨基酸序列用黑体表示，而Vκ-前导序列最后一个阅读框及恒定区基因用非黑体表示。限制性位点表示在序列上方。测序引物的引导位点用下划线表示；  Figure 8 provides DNA sequence of _h_IgG1_1 (bp601-2100) (SEQ ID NO: 32). The vector is based on the pcDNA3.1+ vector (Invitrogen): the amino acid sequence of the VH-stuffer sequence is in bold, while the last reading frame of the VH-ladder sequence and the constant region genes are in non-bold. Restriction sites are indicated above the sequence. The guide sites of the sequencing primers are underlined; Figure 9 provides the Igκ light chain expression vector

DNA sequence of _h_Igκ_1 (bp601-1400) (SEQ ID NO: 33): This vector is based on the pcDNA3.1+ vector (Invitrogen). The amino acid sequence of the Vκ-stuffer sequence is in bold, while the last reading frame of the Vκ-leader sequence and the constant region genes are in non-bold. Restriction sites are indicated above the sequence. The guide sites of the sequencing primers are underlined;

_h_Igλ_1的DNA序列(bp601-1400)(SEQ ID NO：34)：Vλ-填充序列的氨基酸序列用黑体表示，而Vλ-梯序列最后一个阅读框及恒定区基因用非黑体表示。限制性位点表示在序列上方。测序引物的引导位点用下划线表示；  Figure 10 provides the HuCAL Igλ light chain vector

DNA sequence of _h_Igλ_1 (bp601-1400) (SEQ ID NO: 34): The amino acid sequence of the Vλ-stuffer sequence is in bold, while the last reading frame of the Vλ-ladder sequence and the constant region gene are in non-bold. Restriction sites are indicated above the sequence. The guide sites of the sequencing primers are underlined;

抗体Mab#1(＝MOR03077)、Mab#2(＝MOR03079)、Mab#3(＝MOR03080)、参考抗体chOKT10、激动性(ag.)对照IB4、无关

阴性对照IgG1(NC)和作为IB4的匹配同种型对照的鼠IgG2a(Iso)的情况下培养3天。用BrdU标记的标准物来测量增殖活性，并通过基于化学发光的ELISA来分析其掺入(RLU＝相对光单位)；  Figure 11 provides the results of the proliferation assay: PBMCs (indicated by individual dots) from 6 different healthy donors in the presence of

Antibody Mab#1 (=MOR03077), Mab#2 (=MOR03079), Mab#3 (=MOR03080), reference antibody chOKT10, agonistic (ag.) control IB4, irrelevant

Incubate for 3 days with negative control IgGl (NC) and murine IgG2a (Iso) as a matched isotype control for IB4. Proliferative activity was measured with BrdU-labeled standards and their incorporation was analyzed by chemiluminescence-based ELISA (RLU = Relative Light Units);

抗体Mab#1(＝MOR03077)、 Mab#2(＝MOR03079)、Mab#3(＝MOR03080)、参考抗体chOKT10、激动性(ag.)对照IB4、无关

阴性对照(NC)和单纯培养基(培养基)的情况下培养24小时。通过基于化学发光的ELISA从培养物上清分析IL-6的量，用相对光单位(RLU)表示；  Figure 12 provides the results of the IL-6 release assay: PBMCs (indicated by separate dots) from 4-8 different healthy donors were mixed in the presence of

Antibody Mab#1 (=MOR03077), Mab#2 (=MOR03079), Mab#3 (=MOR03080), reference antibody chOKT10, agonistic (ag.) control IB4, irrelevant

Incubate for 24 hours in the case of negative control (NC) and medium alone (medium). The amount of IL-6 expressed in relative light units (RLU) was analyzed from culture supernatants by chemiluminescence-based ELISA;

抗体Mab#1(＝MOR03077)、Mab#2(＝MOR03079)、Mab#3(＝MOR03080)、阳性对照(PC＝chOKT10)和无关

阴性对照的情况下培养4小时。之后将细胞悬液与润湿的甲基纤维素基质混合并培育2周。计数来自红系暴发集落形成单位(BFU-E；B栏)和粒细胞/红系细胞/巨噬细胞/巨核细胞干细胞(CFU-GEMM；B栏)以及粒细胞/巨噬细胞干细胞(CFU-GM；C栏)的集落形成单位(CFU)，并根据培养基对照(“无”＝培养基)标准化。A栏表示所有祖细胞的CFU总数(总CFU)。给出了至少10个不同PBMC供体的平均值。误差棒表示与平均值的标准误差；  Figure 13 provides data on cytotoxicity to CD34+/CD38+ progenitor cells: PBMC from healthy donors with autologous CD34+/CD38+ progenitor cells were separated in the presence of

Antibody Mab#1 (=MOR03077), Mab#2 (=MOR03079), Mab#3 (=MOR03080), positive control (PC=chOKT10) and irrelevant

Incubate for 4 hours in the case of a negative control. The cell suspension was then mixed with a moistened methylcellulose matrix and incubated for 2 weeks. Counts were derived from erythroid burst colony-forming units (BFU-E; column B) and granulocyte/erythroid/macrophage/megakaryocyte stem cells (CFU-GEMM; column B) and granulocyte/macrophage stem cells (CFU- Colony forming units (CFU) in GM; column C) and normalized to medium control ("none" = medium). Column A represents the total number of CFU of all progenitor cells (total CFU). Average values of at least 10 different PBMC donors are given. Error bars represent standard error from the mean;

Figure 14 provides ADCC data for different cell lines:

a: Single measurement (except RPMI8226, which is the average of 4 measurements); E:T ratio: 30:1

b: Namba et al., 1989

c: The antibody concentration is 5 μg/ml (except for Raji, which is 0.1 μg/ml)

d: adding retinoic acid to determine the stimulation of specific killing of CD38 expression [%]=[(expected killing value-intermediate killing value)/(1-intermediate killing value)]×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 Animal Cell Collection Center

MFI: mean fluorescence intensity;

Figure 15 provides the ADCC data for the MM samples:

^a : 2-4 separate analyzes;

Figure 16 provides the experimental results of mean tumor volume after treatment of human myeloma xenografts with MOR03080: Group 1: vehicle; Group 2: MOR03080, 1 mg/kghIgG1 given every other day on days 32-68; Group 3: MOR03080, 5mg/kg hIgG1 was given every other day on days 32-68; Group 4: MOR03080, 5 mg/kg chIgG2a was given every other day on days 32-68; Group 5: MOR03080, 1 mg/kg hIgG1 was given every other day on days 14-36; Group 6 : Not processed. the

Detailed description of the invention

The present invention is based on the discovery of novel antibodies which are specific for CD38 or have high affinity for CD38 and which may be of therapeutic benefit to an individual. The antibodies of the present invention can be human antibodies or humanized antibodies, and can be used in various aspects of the present invention, which will be described in more detail in this specification. the

A "human" antibody or functional human antibody fragment is defined herein as an antibody that is not chimeric (eg, not "humanized") and is not from a species (in whole or in part) that is not human. A human antibody or functional antibody fragment can be derived from a human or can be a synthetic human antibody. A "synthetic human antibody" is defined herein as an antibody having a sequence that is partially or wholly derived from a computer-synthesized sequence based on analysis of known human antibody sequences. For example, in silico design of human antibody sequences or fragments thereof can be accomplished by analyzing databases of human antibody or antibody fragment sequences and using the data obtained therefrom to design polypeptide sequences. Another example of a human antibody or functional antibody fragment is a human antibody or functional antibody fragment encoded by nucleic acid isolated from a library of antibody sequences of human origin (ie, a library based on antibodies from natural human origin). the

A "humanized antibody" or functional humanized antibody fragment is defined herein as one of the following: (i) an antibody derived from a non-human source (e.g., a transgenic mouse with a xenogeneic immune system) that is based on a human embryo (germline) sequence; or (ii) a chimeric antibody whose variable region is derived from a non-human source and whose constant region is derived from a human source, or (iii) a CDR-grafted antibody whose CDRs of the variable region are derived from a non-human source and whose variable One or more frameworks of the regions are of human origin, and the constant regions, if any, are of human origin. the

Because binding specificity is not absolute, but a relative property, in this specification, if an antibody can distinguish an antigen (here CD38) from one or more reference antigens, the antibody "specifically binds in", "specific for" or "specifically recognizes" the antigen. In its most general form (when no stated reference is mentioned), "specifically binds" refers to the ability of an antibody to distinguish an antigen of interest from an unrelated antigen, as determined, for example, by one of the methods below. Such methods include, but are not limited to, Western blot assays, ELISA assays, RIA assays, ECL assays, IRMA assays, and peptide scanning. For example, standard ELISA assays can be performed. Scoring can be done by standard chromogenic reactions (eg, secondary antibody and horseradish peroxide, and tetramethylbenzidine and hydrogen peroxide). Responses in certain wells are scored by, for example, optical density at 450 nm. A typical background (=negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. This means that the difference between positive/negative can be greater than 10-fold. Usually, determination of binding specificity is performed not only with a single reference antigen, but with a set of about 3-5 unrelated antigens such as milk powder, BSA, transferrin, etc. the

However, "binding specificity" also refers to the ability of an antibody to distinguish a target antigen from one or more closely related antigens (eg, CD38 and CD157) that are used as reference points. In addition, "binding specificity" may include the ability of an antibody to distinguish between different parts of its target antigen, such as different domains or regions of CD38, such as epitopes in the N-terminal or C-terminal regions of CD38, or to distinguish CD38 amino acid residues. The ability of one or more key amino acid residues or amino acid stretches of the base. the

Additionally, "immunoglobulin (Ig)" is defined herein as a protein belonging to the classes IgG, IgM, IgE, IgA or IgD (or any subclass thereof), including all conventionally known antibodies and functional fragments thereof. A "functional fragment" of an antibody/immunoglobulin is thus defined herein as a fragment of the antibody/immunoglobulin that retains the antigen binding region (eg, the variable region of IgG). The "antigen-binding region" of an antibody is typically found in one or more of the hypervariable regions of the antibody, i.e., the CDR-1, -2 and/or -3 regions; however, variable "framework" regions may also be found in antigenic Play an important role in binding, such as providing scaffolds for CDRs. Preferably, the "antigen binding region" comprises at least amino acid residues 4-103 of the variable light (VL) chain and amino acid residues 5-109 of the variable heavy (VH) chain, more preferably amino acid residues 3-109 of the VL. 107 and amino acid residues 4-111 of VH, particularly preferably comprising the complete VL and VH chains (amino acids 1-109 of VL and amino acids 1-113 of VH; see WO 97/08320 for numbering). The preferred immunoglobulin class for use in the present invention is IgG. "Functional fragments" of the present invention include F(ab') ₂ fragments, Fab fragments and domains of scFv. The F(ab') ₂ or Fab can be designed to minimize or completely eliminate the intermolecular disulfide interactions that exist between the _CH1 and _CL domains.

Antibodies of the invention may be derived from recombinant antibody libraries based on amino acid sequences designed in silico and encoded by synthetically produced nucleic acids. For example, in silico design of antibody sequences can be accomplished by analyzing databases of human sequences and using the data obtained therefrom to design polypeptide sequences. Methods for designing and obtaining in silico-generated sequences are found, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001) 254:67; and Knappik et al. 6,300,064, which is incorporated herein by reference in its entirety.

Antibodies of the invention

In this specification, the following representative antibodies of the present invention are referred to: "Antibody Number" or "LAC" or "MOR" 3077, 3079, 3080 and 3100. LAC3077 is indicated to have a heavy chain variable region corresponding to SEQ ID NO: 1 (DNA)/SEQ ID NO: 5 (protein) and a light chain corresponding to SEQ ID NO: 9 (DNA)/SEQ ID NO: 13 (protein) Variable region antibodies. LAC3079 represents a heavy chain variable region corresponding to SEQ ID NO: 2 (DNA)/SEQ ID NO: 6 (protein) and a light chain corresponding to SEQ ID NO: 10 (DNA)/SEQ ID NO: 14 (protein). chain variable region antibodies. LAC3080 represents a heavy chain variable region corresponding to SEQ ID NO: 3 (DNA)/SEQ ID NO: 7 (protein) and a light chain corresponding to SEQ ID NO: 11 (DNA)/SEQ ID NO: 15 (protein). chain variable region antibodies. LAC3100 represents a heavy chain variable region corresponding to SEQ ID NO: 4 (DNA)/SEQ ID NO: 8 (protein) and a light chain corresponding to SEQ ID NO: 12 (DNA)/SEQ ID NO: 16 (protein). Variable region antibodies. the

In one aspect, the present invention provides an antibody with an antigen-binding region, the antibody-binding region can specifically bind to CD38 or have high affinity for one or more regions of CD38, and the amino acid sequence of CD38 is shown in SEQ ID NO: 22. An antibody is said to have "high affinity" for the antigen if the measured affinity is at least 100 nM (monovalent affinity for a Fab fragment). An antibody or antigen binding region of the invention preferably binds CD38 with an affinity of less than about 100 nM, more preferably less than about 60 nM, most preferably less than about 30 nM. Still more preferred are antibodies that bind CD38 with an affinity of less than about 10 nM, more preferably less than about 3 nM. For example, the antibodies of the invention have an affinity for CD38 of about 10.0 nM or 2.4 nM (monovalent affinity for Fab fragments). the

Table 1 summarizes the affinities of representative antibodies of the invention as determined by surface plasmon resonance (Biacore) and FACS Scatchard analysis:

Table 1: Antibody Affinities

Antibody (Fab or IgG1) BIACORE(Fab)K _D [nM] ^a FACS Scatchard(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 undecided 8.28

^a : mean of at least 2 different affinity determinations

^b : RPMI8226MM cell line used for FACS-Scatchard

Referring to Table 1, the affinities of LAC3077, 3079, 3080 and 3100 were measured on immobilized recombinant CD38 by surface plasmon resonance (Biacore) and by flow cytometry using the human RPMI8226 cell line expressing CD38. Biacore studies were performed on directly immobilized antigen (CD38-Fc fusion protein). The Fab forms of LAC3077, 3079, 3080 and 3100 showed monovalent affinities between about 2.4 and 56 nM on immobilized CD38-Fc fusion protein, with LAC3079 showing the highest affinity, followed by Fab3100, 3080 and 3077. the

The IgG1 format was used in a cell-based affinity assay (FACS Scatchard). The right column of Table 1 shows the binding strength of this form of LAC. LAC3080 showed the strongest binding, which was slightly stronger than LAC3079 and 3077. the

Another preferred feature of preferred antibodies of the invention is that they are specific for a region within the N-terminal region of CD38. For example, LAC3077, 3079, 3080 and 3100 of the present invention can specifically bind to the N-terminal region of CD38. the

The type of epitope to which an antibody of the invention binds can be linear (ie, a stretch of consecutive amino acids) or conformational (ie, a stretch of amino acids). To determine whether the epitope of a particular antibody is linear or conformational, the skilled artisan can analyze the antibody for binding to overlapping peptides covering different regions of CD38 (eg, binding of a 13-mer peptide with an 11 amino acid overlap). Using this analysis, the inventors found that LAC3077, 3080 and 3100 recognize discontinuous epitopes in the N-terminal region of CD38, whereas the epitope of LAC3079 can be described as linear (see Figure 7). Combining the knowledge provided by this specification, one of ordinary skill in the art will know how to use one or more isolated epitopes of CD38 to generate antibodies with an antigen-binding region specific for the epitopes (e.g., using an epitope of CD38 or CD38 cell-expressed epitopes). the

Antibodies of the invention are preferably species cross-reactive with humans and at least one other species, which may be a rodent or a non-human primate. The non-human primate may be a rhesus monkey, a baboon and/or a macaque. The rodent may be a mouse, a rat and/or a hamster. Antibodies that cross-react with at least one rodent provide, for example, flexibility and benefits over known anti-CD38 antibodies for in vivo studies in multiple species with the same antibody. the

Preferably, the antibody of the present invention can not only bind to CD38, but also mediate the killing of CD38-expressing cells. More specifically, the antibodies of the invention are capable of exerting their therapeutic efficacy by reducing CD38-positive (eg, malignant) cells through antibody-effector functions. These functions include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). the

Table 2 summarizes the ADCC and CDC EC50 values of representative antibodies of the invention:

Table 2: EC50 values of antibodies

^a : mean of at least 2 EC50 measurements

^b : one measurement

^c : mean of 2 EC50 measurements

^d : mean of 3 EC50 measurements

^e : mean of 4 EC50 measurements

However, CD38 was found not only to be expressed on immune cells of the myeloid (eg monocytes and granulocytes) and lymphoid lineages (eg activated B and T cells, plasma cells), but also on the respective precursor cells. Since those cells are not affected by antibody-mediated killing of malignant cells (which is important), the antibodies of the invention are not cytotoxic to precursor cells. the

In addition to its catalytic activity as a cyclic ADP-ribose cyclase and hydrolase, CD38 also displays biologically relevant signal transduction capabilities (Hoshino et al., 1997; Ausiello et al., 2000). Those functions can be induced in vivo by, for example, receptor-ligand interactions or by cross-linking with agonistic anti-CD38 antibodies, resulting in, for example, calcium mobilization, lymphocyte proliferation and cytokine release. Preferably, the antibodies of the invention are non-agonistic antibodies. the

peptide variant

The antibodies of the present invention are not limited to the specific peptide sequences provided in this specification. The invention may include variants of these polypeptides. With reference to the disclosure of the present invention and routinely available techniques and references, a skilled artisan will be able to make, test and use functional variants of the antibodies disclosed herein, and will be able to understand those that have the ability to mediate CD38+ target cell killing. Variations of capabilities are within the scope of the invention. Herein, "the ability to mediate the killing of CD38+ target cells" refers to the functional properties of the anti-CD38 antibody of the present invention. Thus, the ability to mediate killing of CD38+ target cells includes the ability to mediate killing of CD38+ target cells by eg ADCC and/or CDC or by a toxic construct conjugated to an antibody of the invention. the

Variants may include, for example, antibodies having at least one altered complementarity determining region (CDR) (hypervariable) and/or framework (FR) (variable) determining region/site relative to the peptide sequence disclosed herein . To better illustrate this concept, a brief description of the antibody structure is given below.

Antibodies are composed of two peptide chains, each containing 1 (light chain) or 3 (heavy chain) constant regions and variable regions (VL, VH), the latter consisting of 4 FR regions and 3 centrally located CDRs constitute. The antigen binding site is composed of one or more CDRs, and the FR region provides a structural framework for the CDRs, thus playing an important role in antigen binding. By altering one or more amino acid residues in the CDR or FR regions, the skilled artisan can conveniently generate mutated or altered antibody sequences which, for example, can be screened for new or improved properties using antigens. the

Tables 3a (VH) and 3b (VL) describe the CDR and FR regions of certain antibodies of the invention, and compare specific CDR and FR regions to each other and to the corresponding consensus or "master gene" sequence (as described in U.S. Patent 6,300,064). Amino acid at position:

The skilled artisan can use the data in Tables 3a and 3b to design peptide variants within the scope of the present invention. Variants are preferably constructed by altering amino acids within one or more CDR regions, and variants may also have one or more altered framework regions. Candidate residues that may be altered include, for example, residues 4 or 37 of the variable light chain of LAC3080 and 3077 and residues 13 or 43 of the variable heavy chain, based on comparisons of the new antibody with other antibodies, because these positions change with each other. Alterations can also occur in framework regions. For example, peptide FR domains may be altered, in which case residues are changed compared to the embryonic line sequence. the

Candidate residues that may be altered include, for example, residues 27, 50, or 90 of the variable light chain of LAC3080 compared to VLλ3, and, for example, the corresponding Residues 33, 52 and 97 of the variable heavy chain of LAC3080 compared to VH3. Alternatively, the skilled artisan can perform the same analysis by comparing the amino acid sequences disclosed in the present invention with known sequences of such antibodies of the same type, for example using Knappik et al. (2000) and U.S. Patent 6,300,064 to Knappik et al. analyzed by the method described above. the

In addition, variants can be obtained using one LAC as a starting point by altering one or more amino acid residues in the LAC, preferably within one or more CDRs, and screening the resulting collection of variants for Variants with improved properties are obtained. Particularly preferred is the diversification of one or more amino acid residues in CDR-3 of VL, CDR-3 of VH, CDR-1 of VL and/or CDR-2 of VH. Diversification can be achieved by using the trinucleotide mutagenesis (TRIM) technique (Vimek S, 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 .) A collection of synthetic DNA molecules to accomplish this.

conservative amino acid variant

Polypeptide variants can be prepared to retain the overall molecular structure of the antibody peptide sequences described herein. Given the properties of the individual amino acids, some reasonable substitutions will be known to the skilled artisan. For example, amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic properties of the residues concerned, "conservative substitutions". the

For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; ( b) polar 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 including aspartic acid and glutamic acid. Substitutions can generally be made within groups (a)-(d). In addition, since both glycine and proline can break the α-helix, they can replace each other. Similarly, certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine are more common in α-helices, while val Amino acid, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are more commonly found in β-sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions may be made within the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Using the known genetic code and recombinant and synthetic DNA techniques, the skilled artisan can conveniently construct DNA encoding conservative amino acid variants. In a specific embodiment, amino acid position 3 in SEQ ID NO: 5, 6, 7 and/or 8 may be changed from Q to E. the

In this specification, "sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the two sequences. "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. Preferably, the sequence identity of the CDR regions of the polypeptide sequences of the present invention is at least 60%, more preferably at least 70% or 80%, even more preferably at least 90%, most preferably at least 95%. Preferred antibody CDR regions have a sequence similarity of at least 80%, more preferably 90%, most preferably 95%.

DNA molecule of the present invention

The invention also relates to DNA molecules encoding the antibodies of the invention. These sequences include, but are not limited to, the DNA molecules shown in Figures 1a and 2a. the

The DNA molecules of the present invention are not limited to the sequences disclosed in this specification, but also include variants thereof. The DNA variants of the invention can be described in terms of their hybridization physical properties. Those skilled in the art will know that since DNA is double-stranded, DNA can be used to identify its complement, its equivalents or homologues using nucleic acid hybridization techniques. It will also be known that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural relationship to specific probes. For teachings on this condition, see Sambrook et al., 1989 (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA) and Ausubel et al., 1995 (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Sedman, J.G., Smith, J.A. and Struhl, K. eds. (1995), Current Protocols in Molecular Biology. NewYork: John Wiley and Sons). the

Structural similarity between two polypeptide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences hybridize to each other. In this specification, the term "stringency" refers to the degree to which conditions do not favor hybridization. Stringent conditions are extremely unfavorable for hybridization, and only the most structurally related molecules can hybridize to each other under such conditions. Conversely, non-stringent conditions favor hybridization of less structurally related molecules to each other. Thus, hybridization stringency is directly related to the structural relationship of the two nucleic acid sequences. The following relationship is useful for correlated hybridization and correlation (where _Tm is the melting temperature of a nucleic acid duplex):

aT _m =69.3+0.41(G+C)%

b. For every 1% increase in the number of mismatched base pairs, the T _m of the duplex DNA will decrease by 1°C

c.(T _m ) _μ2 -(T _m ) _μ1 ＝18.5log ₁₀ μ2/μ1

where μ1 and μ2 are the ionic strengths of the two solutions. the

Hybridization stringency is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of agents capable of breaking hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, longer probe size, and the absence of reagents capable of breaking hydrogen bonds. Hybridization is usually performed in two phases: a "binding" phase and a "washing" phase. the

First, during the binding phase, the probe binds to the target under conditions favorable for hybridization. Stringency is usually controlled at this stage by varying the temperature. Unless short (<20nt) oligonucleotide probes are used, the temperature for high stringency is usually between 65°C and 70°C. A typical hybridization solution contains 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100μg non-specific carrier DNA. See Ausubel et al., Section 2.9, Suppl. 27 (1994). Of course, many different but functionally identical buffer conditions are known. Lower temperatures may be chosen when the degree of correlation is low. Low stringency binding temperatures are between 25°C and 40°C. A temperature of moderate stringency is at least about 40°C to less than about 65°C. A high stringency temperature is at least about 65°C. the

Second, excess probes are removed by washing. This stage usually uses more stringent conditions. Therefore, the "washing" phase is the most important phase for determining relatedness by hybridization. The wash solution is usually of low salt concentration. A typical medium stringency solution contains 2X SSC and 0.1% SDS. High stringency wash solutions contain less than about 0.2X SSC (on an ionic strength basis), with preferred stringent solutions containing about 0.1X SSC. Temperatures associated with various stringencies are as discussed above for "Binding". During the wash period several wash solution changes are usually required. For example, typical high stringency wash conditions include two washes at 55°C for 30 minutes each and three washes at 60°C for 15 minutes each. the

Accordingly, the present invention includes nucleic acid molecules that hybridize under high stringency binding and washing conditions to the molecules shown in Figures 1a and 2a, wherein the nucleic acid molecules encode antibodies or functional fragments thereof having the properties described herein. Preferred molecules (in terms of mRNA) have at least 75% or 80% (preferably at least 85%, more preferably at least 90%, most preferably at least 95%) homology or sequence to one of the DNA molecules according to the invention identical molecules. In a specific example of a variant according to the invention, the nucleic acid at position 7 of SEQ ID NO: 1, 2, 3 and/or 4 may be changed from C to G, thereby changing the codon from CAA to GAA. the

Functionally Equivalent Variants

Another class of DNA variants of the present invention can be described in terms of their encoded products (see listed peptides in Figures 1b and 2b). These functionally equivalent genes are characterized in that, due to the degeneracy of the genetic code, they encode the same peptide sequences as listed in Figures 1b and 2b. SEQ ID NO: 1 and 31 are examples of functionally equivalent variants, ie they differ in nucleic acid sequence but encode the same polypeptide, ie SEQ ID NO: 5. the

Variants of the DNA molecules provided by the present invention are known to be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. There are many methods for efficiently synthesizing oligonucleotides ranging in length from 20 to about 150 nucleotides. See Ausubel et al., Section 2.11, Suppl. 21 (1993). Overlapping oligonucleotides can be synthesized and assembled using methods first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. The synthetic DNA is preferably designed with convenient restriction sites at the 5' and 3' ends of the gene to facilitate cloning into a suitable vector. the

As described above, the method for generating variants is to start with a DNA according to the invention, followed by site-directed mutagenesis. See Ausubel et al., supra, Chapter 8, Suppl. 37 (1997). In a typical approach, target DNA is cloned into a single-stranded DNA phage vector. Single-stranded DNA is isolated and hybridized to oligonucleotides containing the desired nucleotide changes. The complementary strand is synthesized and the double-stranded phage is introduced into the host. Some of the resulting progeny will have the desired mutation, as can be determined by DNA sequencing. In addition, various methods exist to increase the likelihood that the progeny phage will become the desired mutant. These methods are well known to those skilled in the art and commercially available kits can be used to generate such mutants. the

Recombinant DNA constructs and expression

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

The encoding gene can be prepared using the techniques described by Sambrook et al. (1989) and Ausubel et al. (1989). Alternatively, the DNA sequence can be chemically synthesized using, for example, a synthesizer. See, eg, the techniques described in OLIGONUCLEOTIDE SYNTHESIS (1984, Ed. Gait, IRL Press, Oxford), which is incorporated herein by reference in its entirety. The recombinant constructs of the present invention comprise expression vectors capable of expressing protein products encoded by RNA and/or DNA. The vector also contains regulatory sequences, including a promoter operably linked to an open reading frame (ORF). The vector also contains a selectable marker sequence. Specific initiation and bacterial secretion signals may also be required for efficient translation of inserted target gene coding sequences. the

The invention also provides host cells comprising at least one DNA of the invention. The host cell can be virtually any cell for which an expression vector is available. For example, it may be a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, but preferably a prokaryotic cell such as a bacterial cell. Recombinant constructs can be introduced into host cells by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection. the

bacterial expression

Bacterial expression vectors are constructed by inserting the structural DNA sequence encoding the desired protein together with appropriate translation initiation and termination signals into an operable reading phase with a functional promoter. The vector will contain one or more phenotypically selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various bacteria of the genera Pseudomonas, Streptomyces, and Staphylococcus. the

Bacterial vectors may be, for example, phage, plasmid or phagemid based. These vectors may contain selectable markers and bacterial origins of replication from commercially available plasmids which generally contain elements of the well known cloning vector pBR322 (ATCC37017). After transformation of a suitable host strain and growth of the host strain to a suitable cell density, the selected promoter is suppressed/induced by a suitable method (eg, temperature change or chemical induction) and the cells are cultured for an additional period of time. Cells are usually harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. the

In bacterial systems, a number of expression vectors can be advantageously selected according to the use of the expressed protein. For example, when large quantities of such proteins are to be produced for antibody production or for screening peptide libraries, vectors capable of expressing high levels of easily purified fusion protein products are required, for example. the

treatment method

Methods of treatment comprise administering to a subject in need thereof a therapeutically effective amount of an antibody contemplated by the invention. A "therapeutically effective" amount is defined herein as that amount of the antibody sufficient to reduce CD38-positive cells in a subject's treatment area—administered alone or in combination with other agents, in a single or multiple dose regimen May relieve adverse symptoms, but is toxicologically tolerable. A subject can be a human or a non-human animal (eg, rabbit, rat, mouse, monkey, or a lower primate thereof).

Antibodies of the present invention can be administered with known drugs, and in some cases the antibodies themselves can be modified. For example, antibodies can be conjugated with immunotoxins or radioisotopes to further enhance their efficacy. the

The antibodies of the present invention are useful as therapeutic or diagnostic tools in various diseases in which CD38 is unfavorably expressed or found. Diseases and conditions that are particularly suitable for treatment with the antibodies of the invention are multiple myeloma (MM) and other blood disorders such as chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML ) and acute lymphoblastic leukemia (ALL). Antibodies of the invention can also be used to treat inflammatory diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). the

To treat the above-mentioned diseases, the pharmaceutical composition used according to the present invention can be prepared by conventional methods with one or more pharmaceutically acceptable carriers or excipients. Antibodies of the invention may be administered by any suitable means, which may vary depending on the type of disease being treated. Possible routes of administration include parenteral (eg intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous), intrapulmonary and intranasal, and if necessary local immunosuppressive therapy, intralesional administration. Furthermore, antibodies of the invention may be administered by pulse infusion, eg, in decreasing doses of the antibody. Preferably, dosing is by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is short term or chronic. The amount to be administered depends on various factors such as clinical symptoms, body weight of the individual, whether other drugs are administered, and the like. The skilled artisan will appreciate that the route of administration varies depending on the disease or condition being treated. the

Determining a therapeutically effective amount of a novel polypeptide according to the invention will largely depend on the characteristics of the particular patient, the route of administration and the nature of the disease being treated. General teaching can be found in, for example, publications of the International Conference on Harmonisation and Chapters 27 and 28 of REMINGTON'S PHARMACEUTICAL SCIENCES, pp. 484-528 (p. 18th edition, edited by Alfonso R. Gennaro, 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 by methods known in the art and found in the above references. Efficacy can be determined in the same way and as described in the Examples below. the

diagnosis method

In certain malignancies, CD38 is highly expressed in blood cells; therefore, anti-CD38 antibodies of the invention can be used to image or visualize sites in a patient where malignant cells may accumulate. In this regard, antibodies may be detectably labeled through the use of radioactive isotopes, affinity labels (eg, biotin, avidin, etc.), fluorescent labels, paramagnetic atoms, and the like. Methods for such labeling are well known in the art. The clinical application of antibodies in diagnostic imaging can refer to Grossman, H.B., Urol. Clin. North Amer. 13:465-474 (1986), Unger, E.C. et al., Invest.Radiol. , B.A. et al., Science 209:295-297 (1980). the

Detection of foci with such detectably labeled antibodies can indicate, for example, the site of tumor development. In one embodiment, such detection is accomplished by taking samples from tissue or blood and incubating these samples in the presence of detectably labeled antibodies. In preferred embodiments, this technique is accomplished in a non-invasive manner using magnetic imaging, autofluorescence, and the like. This diagnostic test can be used to monitor the success of disease treatment, where the presence or absence of CD38 positive cells is a corresponding indicator. The invention also contemplates the use of the anti-CD38 antibodies of the invention for in vitro diagnostics. the

Therapeutic and Diagnostic Compositions

The antibody of the present invention can be prepared into a pharmaceutically usable composition according to known methods, wherein the antibody of the present invention (including any functional fragment thereof) is combined with a pharmaceutically acceptable carrier to form a mixture. Suitable carriers and their preparation are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., Ed. Alfonso R. Gennaro, Easton, Pa.: MackPub. Co., 1990) describe. To formulate a pharmaceutically acceptable composition suitable for effective administration, such composition will contain an effective amount of one or more antibodies of the present invention and an appropriate amount of carrier vehicle. the

The formulations may be prepared by suitable methods for controlled release of the active compound. Polymers can be used to complex or absorb the anti-CD38 antibody to obtain a controlled release formulation. By selecting the appropriate macromolecules (such as polyester, polyamino acid, polyethylene, pyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules and Incorporation of controlled release methods for controlled delivery. Another possible approach to control the duration of action by controlling the formulation is to incorporate anti-CD38 antibodies into polymeric materials such as polyesters, polyamino acids, hydrogels, polylactic acid, or ethylene-vinyl acetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, these materials can be encapsulated in microcapsules or colloidal drug delivery systems or macroemulsions, for example, microcapsules can be prepared by coacervation techniques or by interfacial polymerization (e.g. hydroxymethylcellulose, respectively Sutin or gelatin microcapsules and poly(methyl methacrylate) microcapsules), colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules. These techniques are described in Remington's Pharmaceutical Sciences (1980). the

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

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. For example, the package may comprise metal or plastic foil, such as a blister pack. Instructions for use may also be included in the packaging or dispensing device. the

The present invention can be further understood by reference to the following working examples, which are intended to illustrate the invention and therefore do not constitute any limitation thereto. the

Example

cell line

The following cell lines were obtained from the European Collection of Cell Cultures (ECACC), the German Collection of Microorganisms (DSMZ), or the American Type Culture Collection (ATCC): Hybridoma cell line producing the CD38 mouse IgG1 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, CCL-86). the

Cells and Culture Conditions

All cells were cultured in a humidified incubator under standard conditions of 37 °C and 5% _CO2 . The cell lines LP-1, RPMI8226, Jurkat and Raji were treated with 10% FCS (PAN biotech GmbH, #P30-3302), 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco, #15140-122) and 2 mM gluten. Aminoamide (Gibco, #25030-024) was cultured in RPMI1640 (Pan biotech GmbH, #P04-16500), while Jurkat cells and Raji cells were also supplemented with 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 DMEM (Gibco, #10938-025) supplemented with 2 mM glutamine and 10% FCS. Stable CD38CHO-K1 transfectants were maintained in the presence of G418 (PAA GmbH, P11-012), while HEK293 also required the addition of 1 mM sodium pyruvate. 10% FCS was replaced with very low IgG FCS (Invitrogen, #16250-078) after brief transfection of HEK293. The cell line OKT10 was cultured in IDMEM (Gibco, #31980-022) supplemented with 2 mM glutamine and 20% FCS. the

Preparation of single cell suspensions from peripheral blood

-1077(Sigma)根据制造商的说明从健康供体分离外周血单核细胞(PBMC)。在ACK裂解缓冲液(0.15MNH₄Cl、10mM KHCO₃、0.1M EDTA)中于室温培育5分钟或用市售产品(Bioscience，#00-4333)除去这些细胞悬液中的红血细胞。细胞用PBS洗涤两次，然后进一步处理用于流式细胞术或ADCC(见下文)。  All blood samples were obtained with consent. use

-1077 (Sigma) Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors according to the manufacturer's instructions. Red blood cells in these cell suspensions were removed by incubating in ACK lysis buffer (0.15M NH ₄ Cl, 10 mM KHCO ₃ , 0.1 M EDTA) at room temperature for 5 minutes or using commercially available products (Bioscience, #00-4333). Cells were washed twice with PBS before further processing for flow cytometry or ADCC (see below).

Flow Cytometry (“FACS”)

All stainings were performed in 96-well round bottom culture plates (Nalge Nunc) containing 2 x ¹⁰⁵ cells per well. Cells were incubated with indicated concentrations of Fab or IgG antibodies in 50 μl FACS buffer (PBS, 3% FCS, 0.02% NaN ₃ ) at 4° C. for 40 minutes. Cells were washed twice and then treated with goat anti-human or goat anti-mouse IgG(H+L)F(ab') ₂ (Jackson Immune Research) at 4°C for 30 minutes. Cells were washed again, resuspended in 0.3 ml FACS buffer, and analyzed by flow cytometry in a FACSCalibur (Becton Dickinson, San Diego, CA).

For FACS-based Scatchard analysis, 12 different dilutions (1 :2 ⁿ ) starting from a final concentration of 12.5 μg/ml (IgG) were stained. At least two independent measurements were made for each concentration and KD values were extrapolated from median fluorescence intensities following the method of Chamow et al. (1994).

surface plasmon resonance

The kinetic constants kon and koff were determined using a BIAcore3000 device (Biacore, Uppsala, Sweden) using serial dilutions of various Fabs bound to a covalently immobilized CD38-Fc fusion protein. For covalent antigen immobilization, standard EDC-NHS amine coupling chemistry was used. For direct coupling of CD38Fc-fusion proteins, CM5 sensor (senor) chips (Biacore) were coated with approximately 600-700 RU in 10 mM acetate buffer (pH 4.5). For reference flow cells, various amounts of HSA (human serum albumin) were used. Kinetic measurements were performed in PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO0 ₄ , 1.76 mM KH ₂ PO ₄ pH 7.4) at a flow rate of 20 μl/min with Fab concentrations ranging from 1.5-500 nM. The injection time for each concentration was 1 minute, followed by 2 minutes of dissociation. Regenerate using 5 μl of 10 mM HCl. All sensograms were locally fitted with BIA evaluation software 3.1 (Biacore).

Example 1: Generation of antibodies from the HuCAL library

是一种基于

概念(Knappik等，2000；Krebs等，2001)的Fab文库，其中所有6个CDR都是多样化的，且它采用CysDisplay^TM技术将Fab片段连接到噬菌体表面(L

hning，2001)。  To generate therapeutic antibodies against CD38, selection was performed with the MorphoSys HuCAL GOLD phage display library. HuCAL

is a based on

Concept (Knappik et al., 2000; Krebs et al., 2001) Fab library in which all 6 CDRs are diversified, and it uses CysDisplay ^TM technology to attach Fab fragments to the phage surface (L

hning, 2001).

A. Phagemid collection, phage amplification and purification

噬菌粒文库在含有34μg/ml氯霉素和1％葡萄糖的2×TY培养基(2×TY-CG)内扩增。在OD600为0.5时感染辅助噬菌体(VCSM13)(37℃不振荡30分钟；37℃以250rpm振荡30分钟)，然后将细胞甩下(4120g；5min；4℃)，重悬于2×TY/34μg/ml氯霉素/50μg/ml卡那霉素，并于22℃生长过夜。从上清液用PEG沉淀噬菌体，重悬于PBS/20％甘油中，并于-80℃储存。如下进行两轮淘选(panning)之间的噬菌体扩增：中期-对数期TG1细胞用洗脱的噬菌体感染并涂布到添加有1％葡萄糖和34μg/ml氯霉素的LB-琼脂(LB-CG)上。在30℃培育过夜之后，刮下集落，将OD600调节到0.5并如上所述加入辅助噬菌体。  HuCAL

Phagemid libraries were amplified in 2×TY medium (2×TY-CG) containing 34 μg/ml chloramphenicol and 1% glucose. Infect helper phage (VCSM13) at OD600 of 0.5 (37°C without shaking for 30 minutes; 37°C at 250 rpm for 30 minutes), then shake off the cells (4120g; 5min; 4°C) and resuspend in 2×TY/34μg /ml chloramphenicol/50 μg/ml kanamycin and grown overnight at 22°C. Phage were precipitated with PEG from the supernatant, resuspended in PBS/20% glycerol, and stored at -80°C. Phage amplification between two rounds of panning was performed as follows: Meta-log phase TG1 cells were infected with eluted phage and plated onto LB-agar supplemented with 1% glucose and 34 μg/ml chloramphenicol ( LB-CG). After overnight incubation at 30°C, colonies were scraped, the OD600 adjusted to 0.5 and helper phage added as described above.

B. Use HuCAL panning

For selection, HuCAL The antibody-phage was divided into 3 fractions (1st fraction: VH1/5λκ, 2nd fraction: VH3λκ, 3rd fraction: VH2/4/6λκ). These aliquots were each subjected to 3 rounds of whole-cell panning on CD38-expressing CHO-K1 cells, followed by pH-elution and a post-adsorption step on CD38-negative CHO-K1 cells to remove irrelevant antibody-phage. Finally, E. coli TG1 cells were infected with the remaining antibody phages. After centrifugation, the bacterial pellet was resuspended in 2×TY medium, spread on agar plates and incubated overnight at 30°C. Selected clones were then scraped from the plate, and the phage collected and amplified. Do the second and third rounds of selection as you did the first round.

噬菌体的Fab编码插入片段亚克隆入表达载体 x9_Fab_FS(Rauchenberger等，2003)以便于快速表达可溶性Fab。所选克隆的DNA用XbaI和EcoRI消化从而切开Fab编码插入片段(ompA-VLCL和phoA-Fd)，并克隆入XbaI/EcoRI切割载体 

x9_Fab_FS中。该载体中表达的Fab带有两个C-末端标签(FLAG^TMhII)以供检测和纯化。  Select HuCAL

Subcloning of Fab-encoding inserts from phage into expression vectors x9_Fab_FS (Rauchenberger et al., 2003) to facilitate rapid expression of soluble Fab. DNA from selected clones was digested with XbaI and EcoRI to cut the Fab-encoding inserts (ompA-VLCL and phoA-Fd) and cloned into XbaI/EcoRI cut vectors

x9_Fab_FS. The Fab expressed in this vector carries two C-terminal tags (FLAG ^™ h II) for detection and purification.

Example 2: Bioassay

Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity were measured following published methods 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 100 ng/ml Calcein AM (Molecular Probes, C-3099) in RPMI1640 medium (Pan biotech GmbH) at room temperature 2 minutes. Wash 3 times in RPMMI1640 medium to remove residual calcein. At the same time, prepare PBMC as a source of (natural killer) effector cells (E), adjust to 1.0E+07 and mix with labeled target cells, so that the final E:T ratio is 50:1 or lower according to the assay conditions. Cells were washed once and the cell mixture was resuspended in 200 μl of RPMI1640 medium containing various antibodies at different dilutions. Plates were incubated for 4 hours in a humidified incubator under standard conditions of 37 °C and 5% _CO2 . Cells were labeled with propidium iodide (PI) and analyzed by flow cytometry (Becton-Dickinson) prior to FACS analysis. Each assay counts between 50.000 and 150.000 events. Killing activity [%] was calculated by the following formula:

where ^EDA = dead cell event (calcein + PI stained cells), and

^ELA = live cell events (calcein stained cells)

CDC:

To measure CDC, 5.0E+04CD38CHO-K1 transfectants were added to microtiter well plates (Nunc) along with 1:4 dilutions of human serum (Sigma, #S-1764) and various antibodies. All reagents and cells were diluted in RPMI1640 medium (Pan biotech GmbH) supplemented with 10% FCS. The reaction mixture was incubated for 2 h in a humidified incubator under standard conditions of 37 °C and 5% _CO2 . Negative controls were heat inactivated complement or CD38-transfectants without antibody. Cells were labeled with PI and subjected to FACS analysis.

A total of 5000 events were counted and the number of dead cells at different antibody concentrations was used to determine EC50 values. Use the following formula to calculate the lethal activity [%]:

where ^EDC = dead cell event (PI stained cells), and

^ELC = viable cell events (unstained)

For each antibody, a total of 12 different antibody dilutions (1: ²ⁿ ) had cytotoxicity values in triplicate for ADCC and in duplicate for CDC to allow analysis with standard analysis software ( Graph Pad Software) to obtain EC-50 values.

Example 3: Generation of stable CD38-transfectants and CD38Fc fusion proteins

In order to produce CD38 protein for panning and selection, two different expression systems had to be established. The first method involves the generation of CD38-Fc fusion protein, which is purified from the supernatant after transient transfection of HEK293 cells. The second approach involves generating stable CHO-K1-cell lines for selection of antibody-phages by whole-cell panning with high CD38 surface expression. the

The initial step was to use Jurkat cells (DSMZ ACC282) to generate cDNA (Invitrogen), followed by amplification of intact CD38 using primers complementary to the first 7 and last 9 codons of CD38 (primers MTE001 and MTE002rev; Table 4) coding sequence. Sequence analysis of the CD38 insert sequence confirmed that the 49th position of the amino acid sequence disclosed by Jackson et al. (1990) is glutamine, which is different from the tyrosine described by Nata et al. (1997). For the introduction of restriction endonuclease sites and cloning into different derivatives of the expression vector pcDNA3.1 (Stratagene), the purified PCR product was used as a template to reamplify its complete gene (primers MTE006 and MTE007rev, Table 4) or Some genes (primers MTE004 and MTE009rev, Table 4). In the latter case, a fragment encoding the extracellular domain (amino acids 45-300) was amplified and cloned into a framework between the human VK ladder sequence and the human Fc-γ1 sequence. This vector is an expression vector for the production of a soluble CD38-Fc fusion protein. Another pcDNA3.1 derivative without the leader sequence was used to insert into the full-length CD38 gene. At this time, the stop codon and the missing leader sequence before the Fc coding region allow CD38 to be expressed on the surface. HEK293 cells were transiently transfected with Fc fusion protein vectors to generate soluble CD38-Fc fusion proteins and, if full-length derivatives, CHO-K1 cells to generate stable CD38 expressing cell lines.

Table 4:

^* A stop codon (TGA) is generated in the sense orientation.

IgG1的克隆、表达和纯化：  Example 4:

Cloning, expression and purification of IgG1:

_hIg载体中(见图8-10)。采用限制性内切核酸酶对BlpI/MfeI(制备插入片段)和BlpI/EcoRI(制备载体)将VH结构域片段亚克隆入

_hIgG1。用内切酶对EcoRV/HpaI(λ插入片段)和EcoRV/BsiWI(κ插入片段)将VL结构域片段亚克隆入各自的载体 

_hIgκ_1或_h_Igλ_1中。采用标准磷酸钙-DNA共沉淀技术短暂转染以使所得到的IgG构建体在HEK293细胞(ATCC CRL-1573)中表达。用蛋白质A琼脂糖凝胶柱通过亲和层析从细胞培养物的上清中纯化IgG。接下来的处理包括通过凝胶过滤进行缓冲液交换和无菌过滤纯化的IgG。质量控制显示纯度>90％(通过还原性SDS-PAGE测定)、单体IgG>90％(通过分析型大小排阻层析测定)。该物质的内毒素含量通过基于动力学LAL的测定法((Cambrex European Endotoxin Testing Service，比利时)确定。  To express full-length IgG, variable domain fragments of the heavy (VH) and light (VL) chains were subcloned from Fab expression vectors into suitable

_hIg vector (see Figure 8-10). Subcloning the VH domain fragments into

_hIgG1. Subcloning of VL domain fragments into respective vectors using endonuclease pairs EcoRV/HpaI (λ insert) and EcoRV/BsiWI (κ insert)

_hIgκ_1 or _h_Igλ_1. The resulting IgG constructs were expressed in HEK293 cells (ATCC CRL-1573) by transient transfection using standard calcium phosphate-DNA co-precipitation techniques. IgG was purified from cell culture supernatants by affinity chromatography using protein A Sepharose columns. Subsequent processing involves buffer exchange and sterile filtration of the purified IgG by gel filtration. Quality control showed >90% purity (determined by reducing SDS-PAGE), >90% monomeric IgG (determined by analytical size exclusion chromatography). The endotoxin content of the material was determined by a kinetic LAL-based assay (Cambrex European Endotoxin Testing Service, Belgium).

IgG1-表达载体。重链(SEQ ID NO：23)和轻链(SEQ ID NO：24)的序列示于图6。短暂转染HEK293细胞并用FACS分析上清中的与CD38过表达Raji细胞系(ATCC)结合的嵌合OKT10抗体。  Example 5: Generation and preparation of chimeric OKT10 (chOKT10; SEQ ID NO: 23 and 24) To construct chOKT10, mouse VH was amplified by PCR using cDNA prepared from the murine OKT10 hybridoma cell line (ECACC #87021903) and VL area. A published set of primers was used (Dattamajumdar et al., 1996; Zhou et al., 1994). PCR products were used for Topo-cloning (Invitrogen; pCRII-vector) and sequence analysis (M13 reverse primer) of a single clone revealed two different kappa light chain sequences and one heavy chain sequence. According to the sequence alignment (EMBL nucleotide sequence database) and the literature (Krebber et al., 1997), one of the kappa sequences belongs to the internal component of the tumor cell fusion partner X63Ag8.653 and thus does not belong to the OKT10 antibody. Therefore only a new kappa sequence and a VH fragment were further cloned. Both fragments were reamplified for insertion of restriction endonuclease sites and then cloned into their respective

IgG1-expression vector. 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 transiently transfected and supernatants were analyzed by FACS for chimeric OKT10 antibody binding to CD38 overexpressing Raji cell line (ATCC).

Example 6: Epitope Mapping

1. Materials and methods:

Antibody:

The following anti-CD38 IgGs were used for epitope mapping:

MOR# LOT# form Concentration [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 OKT10 is composed of human Fc and mouse variable regions.

CD38 sequence:

The amino acid (aa) sequence (positions 44-300) is based on human CD38 obtained from the published sequence of SWISS-PROT master accession number P28907. A Q (instead of T) at amino acid position 49 was used for peptide design. the

PepSpot analysis:

Antigenic peptides are analyzed on cellulose membranes in a stepwise manner to obtain defined arrangements (peptide arrays) and covalently bind the antigenic peptides to the cellulose membrane. Binding assays were performed directly on peptide arrays. Antigen peptide arrays are usually incubated with blocking buffer for several hours to reduce non-specific binding of antibodies. Incubate with primary (antigen peptide-conjugated) antibody in blocking buffer, followed by incubation with peroxidase (POD)-labeled secondary antibody that selectively binds to the primary antibody. A brief wash with T(Tween)-TBS buffer directly after the incubation of the antigen peptide array with the secondary antibody, followed by a first chemiluminescent assay gives a first overview of which antigen peptides bind the primary antibody. This was followed by several buffer washes (T-TBS buffer and TBS buffer) to remove false positive binding (non-specific antibodies bound to the cellulose membrane). These washing steps were followed by a final chemiluminescence analysis. Data were analyzed as a single measurement for each peptide with an imaging system (Boehringer Light units, BLU) showing signal intensity. To assess non-specific binding of secondary antibodies (anti-human IgG), these antibodies were incubated with the peptide arrays as in the first step without primary antibodies. If the primary antibody does not show any binding to the peptide it can be directly labeled with POD, which increases the sensitivity of the system (as was done for MOR3077). In this case, conventional coupling chemistry is performed via free amino acids. Antigens were scanned with 13-mer peptides (11 amino acid overlap). This yielded an array of 123 peptides. Binding assays were performed directly on the array. Peptide-bound antibodies MOR03077, MOR03079, MOR03080 were detected with a peroxidase-conjugated secondary antibody (peroxidase-conjugated goat anti-human IgG, gamma chain-specific, affinity-isolated antibody; Sigma-Aldrich, A6029) , MOR03100 and chimeric OKT10. Mapping is performed using chemiluminescent substances and imaging systems. In addition, MOR03077 was directly labeled with POD to increase the sensitivity of the system. the

2. Summary and conclusion:

All 5 antibodies showed different profiles in PepSpot analysis. Figure 7 gives a schematic diagram in which the different amino acid sequences of CD38 can be seen. It can be clearly seen that the epitopes of MOR03079 and chimeric OKT10 are linear. The epitope of MOR03079 was putatively located between amino acids 192-206 of CD38 (VSRRFAEAACDVVHV), while the epitope of chimeric OKT10 was mainly located between amino acids 284 and 298 (FLQCVKNPEDSSCTS). The latter result corroborates published data on parental murine OKT10 (Hoshino et al., 1997), which puts its epitope between amino acids 280-298. However, for more precise epitope determination and determination of key amino acids (main antigen-antibody interaction sites), a simplified form of the peptides VSRRFAEAACDVVHV and FLQCVKNPEDS SCTS and alanine scanning of both can be used. the

Since some peptides cover different protein positions, the epitopes of MOR03080 and MOR03100 are undoubtedly non-contiguous. These peptides included amino acids 82-94 and amino acids 158-170 of MOR03080 and amino acids 82-94, 142-154, 158-170, 188-200 and 280-296 of MOR03100. However, some overlap between these two epitopes can be assumed, as both antibodies recognize two of different sites. the

The epitope of MOR03077 is obviously different from the latter two, and can also be described as a multisegmented discontinuous epitope. Its epitope includes amino acids 44-66, 110-122, 148-164, 186-200 and 202-224. the

Embodiment 7: IL-6-release/proliferation test

1. Materials and methods:

IgG1抗体，匹配的同种型对照(鼠IgG2a：抗三硝基苯酚、半抗原特异性抗体；编号：555571，G155-178克隆；BectonDickinson)或培养基对照。为了进行IL-6释放试验，在存在20μg/ml抗体时将0.5ml完全RPMI1640培养基中的1.0E+06PBMC在15ml培养试管(Falcon)中培育24小时。收获细胞培养物上清并用Quantikine试剂盒按照制造商(R&D systems)的方法分析IL-6释放。为了进行增殖试验，在存在20μg/ml抗体时将2.0E+05PBMC在96孔平底平板(Nunc)内培育3天。每种试验进行两次。4天后在每个孔中加入BrdU，将细胞在37℃再培育24小时，然后固定细胞并按照供应商(Roche)的方法使DNA变性。在基于化学发光的装置中通过抗BrdU过氧化物酶偶联抗体测量BrdU的掺入。  Proliferation and IL-6 release assays were performed according to the method of Ausiello et al. (2000) with the following modifications: PBMCs from healthy donors (after obtaining consent) were purified by density gradient centrifugation using the Histopaque cell separation system according to the supplier's (Sigma) instructions, And cells were cultured in RPMI1640 medium ("complete RPMI1640") supplemented with 10% FCS and glutamine under standard conditions (5% _CO2 , 37°C). The following antibodies were used for both assays: Anti-CD38 IgG1 monoclonal antibodies MOR03077, MOR03079, and MOR03080, agonistic murine IgG2a monoclonal antibody (IB4; Malavasi et al., 1984), unrelated

IgG1 antibody, matched isotype control (mouse IgG2a: anti-trinitrophenol, hapten-specific antibody; Cat: 555571, clone G155-178; Becton Dickinson) or media control. For the IL-6 release assay, 1.0E+06 PBMCs in 0.5 ml complete RPMI1640 medium were incubated in 15 ml culture tubes (Falcon) for 24 hours in the presence of 20 μg/ml antibody. Cell culture supernatants were harvested and analyzed for IL-6 release using the Quantikine kit following the manufacturer's (R&D systems) protocol. For proliferation assays, 2.0E+05 PBMCs were incubated in 96-well flat bottom plates (Nunc) for 3 days in the presence of 20 μg/ml antibody. Each test was performed twice. After 4 days BrdU was added to each well and the cells were incubated for a further 24 hours at 37°C before being fixed and DNA denatured according to the supplier's (Roche) protocol. BrdU incorporation was measured by an anti-BrdU peroxidase-conjugated antibody in a chemiluminescence-based device.

2. Summary and conclusion:

Proliferation test:

In addition to its catalytic activity as a cyclic ADP-ribose cyclase and hydrolase, CD38 displays biologically relevant signal transduction capabilities (Hoshino et al., 1997; Ausiello et al., 2000). Those functions can be induced in vivo by, for example, receptor-ligand interactions or by cross-linking with anti-CD38 antibodies. Those signaling events result in, for example, calcium mobilization, lymphocyte proliferation and cytokine release. However, this signaling occurs not only depending on the antigenic epitope, but also varies from donor to donor (Ausiello et al., 2000). From the viewpoint of immunotherapy, non-agonistic antibodies are more preferable than agonistic antibodies. Therefore, further characterization in proliferation assays and IL-6 (an important MM growth factor) release assay Anti-CD38 antibodies (Mab MOR03077, MOR03079, MOR03080), which were compared with reference antibody chOKTI0 and agonistic anti-CD38 monoclonal antibody IB4.

As shown in Figure 11 and Figure 12, HuCAL anti-CD38 antibody Mab#1, 2 and 3 as well as the reference antibody chOKT10 and the corresponding negative control showed no or only weak induction of proliferation compared to the agonistic antibody IB4, and no IL- 6 release. the

Embodiment 8: clone generation test

1. Materials and methods:

IgG1抗CD38抗体(Mab MOR03077、MOR03079和MOR03080)及阳性对照(PC)chOKT10一起培育。培养基和无关HuCAL

IgG1作为背景对照。每次ADCC试验包括将4.0E+05PBMC在添加有10％FCS的RPMI1640培养基中于37℃培育4小时。为了进行克隆生成试验，在2.50ml“完全”甲基纤维素(CellSystems)上接种2.5E+05来自ADCC试验的细胞并在受控环境(37℃；5％CO₂)下培育至少14天以形成集落。集落由两名独立的操作员来分析并分成BFU-E+CFU-GEMM(红系暴发集落形成单位和粒细胞/红系细胞/巨噬细胞/巨核细胞干细胞)和CFU-GM(粒细胞/巨噬细胞干细胞)。  PBMCs containing autologous CD34+/CD38+ precursor cells were isolated from healthy individuals (after consent was obtained) by density gradient centrifugation using the Histopaque cell isolation system according to the supplier's (Sigma) instructions, and different HuCAL at 10 μg/ml

IgGl anti-CD38 antibodies (Mabs MOR03077, MOR03079 and MOR03080) were incubated with positive control (PC) chOKT10. Medium and irrelevant HuCAL

IgG1 served as a background control. Each ADCC experiment consisted of incubating 4.0E+05 PBMCs in RPMI1640 medium supplemented with 10% FCS at 37°C for 4 hours. For clonogenic assays, 2.5E+05 cells from ADCC assays were seeded on 2.50 ml "complete" methylcellulose (CellSystems) and incubated for at least 14 days in a controlled environment (37°C; 5% CO ₂ ). Form colonies. Colonies were analyzed by two independent operators and divided into BFU-E+CFU-GEMM (erythroid outbreak colony forming unit and granulocyte/erythroid/macrophage/megakaryocyte stem cells) and CFU-GM (granulocyte/ macrophage stem cells).

2. Summary and conclusion:

抗CD38抗体(Mab#1、Mab#2和Mab#3)或一些对照(无关

抗体、培养基和作为阳性对照的参考抗体chOKT10)一起培育，然后在润湿的甲基纤维素上继续培育以产生集落。如图13所示，相比无关抗体或参考抗体，所有抗CD38抗体的集落形成单位未显著减少。  Since CD38 expression is found not only on immune cells of the myeloid (e.g. monocytes, granulocytes) and lymphoid lineage (e.g. activated B and T cells; plasma cells), but also on the respective precursor cells (CD34+/CD38+) Expression of CD38 was found on cells, so it is important that those cells are not affected by antibody-mediated killing. Therefore, clonogenic assays were used to analyze the effect on CD34+/CD38+ precursors. PBMC from healthy donors were combined with

Anti-CD38 antibodies (Mab#1, Mab#2 and Mab#3) or some controls (irrelevant

Antibody, culture medium and reference antibody chOKT10) were incubated together as a positive control, and then continued to grow on wet methylcellulose to generate colonies. As shown in Figure 13, compared to an irrelevant antibody or a reference antibody, all Colony forming units were not significantly reduced by anti-CD38 antibody.

Example 9: ADCC assay with different cell lines and primary multiple myeloma cells

1. Materials and methods:

Isolation and ADCC of MM patient samples: Bone marrow aspirates were obtained from multiple myeloma patients (after consent was obtained). Malignant cells were purified by standard methods with anti-CD38 magnetic beads (Milteny Biotec) after density gradient centrifugation (Sigma). ADCC assays were performed as described above. the

2. Summary and conclusion:

抗CD38抗体对多种细胞系，包括不同来源和不同CD38表达水平的细胞系的细胞毒效应。如图14所示，在恒定抗体浓度(5μg/ml)及E:T比例为30:1时所有细胞都被ADCC杀伤。患者的一些多发性骨髓瘤样品也显示出经ADCC产生的细胞毒性。所有

抗CD38抗体能够剂量依赖性地杀伤MM细胞，其EC50值在0.006和0.249nM之间(图15)。  Several cell lines derived from different malignancies were used in ADCC to show

Cytotoxic effects of anti-CD38 antibodies on a variety of cell lines, including cell lines of different origin and different CD38 expression levels. As shown in Figure 14, all cells were killed by ADCC at a constant antibody concentration (5 μg/ml) and an E:T ratio of 30:1. Some multiple myeloma samples from patients also showed cytotoxicity via ADCC. all

Anti-CD38 antibody was able to kill MM cells in a dose-dependent manner, with EC50 values between 0.006 and 0.249 nM (Figure 15).

Example 10: Cross-reactivity analysis by FACS and immunohistochemistry (IHC)

1. Materials and methods:

抗CD38Mab和无关阴性对照抗体转变成二价dHLX形式(Plückthun&Pack，1997)。用Leica CM3050恒冷切片机切得来自短尾猴、恒河猴和人(从奥地利格拉茨大学病理学研究所的档案中找到)淋巴结的5μm冷冻切片。将切片空气干燥30分钟至1小时，在冰冷的甲醇中固定10分钟并用PBS洗涤。为检测dHLX形式，组合使用小鼠抗His抗体(Dianova)及Envision试剂盒(DAKO)。为了检测抗CD38小鼠抗体(例如参考小鼠单克隆OKT10)，仅使用Envison试剂盒。  IHC of tonsils: For IHC, the

Anti-CD38 Mab and irrelevant negative control antibodies were converted to the bivalent dHLX form (Plückthun & Pack, 1997). 5 [mu]m cryosections of lymph nodes from macaques, rhesus monkeys and humans (retrieved from the archives of the Institute of Pathology, University of Graz, Austria) were cut with a Leica CM3050 cryostat. Sections were air dried for 30 min to 1 h, fixed in ice-cold methanol for 10 min and washed with PBS. For detection of the dHLX form, a mouse anti-His antibody (Dianova) was used in combination with the Envision kit (DAKO). For detection of anti-CD38 mouse antibodies (eg reference mouse monoclonal OKT10), only the Envison kit was used.

FACS analysis of lymphocytes: EDTA-treated blood samples were obtained from healthy humans (after consent was given), rhesus monkeys and cynomolgus monkeys and subjected to density gradient centrifugation using a Histopaque cell separation system according to the supplier's (Sigma) instructions. For FACS analysis, interphase cells and primary antibody (HuCAL Anti-CD38 and negative control Mabs were incubated as murine IgG2a or Fab format, positive control murine antibody OKT10 and matched isotype control) and then labeled with anti-M2Flag (Sigma; for Fab format only) and phycoerythrin (PE) Anti-mouse conjugates (Jackson Research) were incubated together. FACS analysis was performed on a gated lymphocyte population.

2. Summary and conclusion:

Analyze HuCAL Anti-CD38 to understand interspecies CD38 cross-reactivity. All anti-CD38 Mabs were able to detect human CD38 on lymphocytes in FACS and IHC, only MOR03080 and the positive control OKT10 had additional reactivity with cynomolgus and rhesus monkey CD38 (Table 5: Cross-reactivity analysis).

table 5:

++: Strong positive staining; -: No staining; NC: Negative control; PC: Positive control (i.e. reference cMAb)

Example 11: Treatment of human myeloma xenografts in mice (using RPMI8226 cell line) with MOR03080

1. Establish subcutaneous mouse model:

A subcutaneous mouse model of human myeloma-derived tumor cell line RPMI8226 was established in female CB-17-SCID mice by Aurigon Life Science GmbH (Tutzing, Germany) as follows: on days -1, 0 and 1, Anti-asialo GM1 polyclonal antibody (ASGM) (WAKO-Chemicals), which depletes xenoreactive NK cells in SCID mice, was administered intravenously to eliminate any residual specific immunity in CB-17-SCID mice reaction. On day 0, 5 x ¹⁰⁶ or 1 x ¹⁰⁷ RPMI8226 tumor cells in 50 μl of PBS were subcutaneously inoculated into the right flank of mice treated or untreated with ASGM (as described above) (5 mice per group) . Tumor development was similar for all 4 vaccination groups, no significant differences were found with or without anti-asialo GM1 antibody treatment or vaccination with different cell numbers. The tumor appears to grow slowly for several days with a tendency to stagnate or oscillate in size. Two tumors oscillated in size throughout the study period, and one tumor even completely disappeared from a peak volume of 321 ^mm3 . Treatment studies with this tumor model should have a relatively large number of tumor-inoculated animals per group.

2. Treat with MOR03080:

2.1 Research purpose

This study was performed by Aurigon LifeScience GmbH (Tutzing, Germany) to compare antibodies administered intraperitoneally ( Anti-tumor efficacy of anti-CD38) versus vehicle treatment (PBS). The human antibody hMOR03080 (isotype IgGl) was tested with different amounts and treatment regimens. In addition, the chimeric antibody chMOR03080 (isotype IgG2a: chimeric OKT10 (mouse VH/VL and human constant region) constructed in a similar manner to Example 5, containing the MOR03080 variable region and the mouse constant region, was also tested. conjugated antibody). The RPMI8226 cancer cell line was chosen as a model and inoculated subcutaneously into female SCID mice as described above. Study endpoints were judged based on body weight (bw), tumor volume, and clinical symptoms.

2.2 Antibodies and carriers

Antibodies were provided to Aurigon in ready-to-use form at concentrations of 2.13 mg/ml (MOR03080hIgG1) and 1.73 mg/ml (MOR03080chIgG2a) and stored at -80°C until use. Antibodies were thawed and diluted with PBS to their respective final concentrations. Vehicle (PBS) was supplied to Aurigon in a ready-to-use form and stored at 4°C until use. the

2.3 Animal specifications

species: mouse

Species: Fox chase C.B-17-scid (C.B-Igh-1b/IcrTac)

Number and sex: 75, female

Supplier: Taconic M&B, Bomholtvej10, DK-8680Ry

Health status: SPF

Weight: about 18g

Acclimatization: 9 days

2.4 Tumor cell lines

Tumor cells (RPMI8226 cell line) were grown and sent to Aurigon Life Science GmbH where the cells were split and grown for another cell cycle in Aurigon. Aurigon prepares the cells for injection on the day of inoculation. The medium used to propagate the cells was RPMI1640 supplemented with 5% FCS, 2mM L-glutamine and PenStrep. Cells showed no undesired growth rate or behavior. the

For inoculation, tumor cells were suspended in PBS and adjusted to a final concentration of 1×10 ⁷ cells/50 μl with PBS. Mix the tumor cell suspension well before injection.

2.5 Test process

On day 0, 1×10 ⁷ RPMI8226 tumor cells were subcutaneously inoculated into the right dorsal rib of 75 SCID mice. A first group of 15 animals (group 5) was randomly selected directly after inoculation. This group was treated with 1 mg/kg b.w.hIgG1-MOR03080 every other day on days 14-36. All other 60 animals were divided into 4 groups of 10 animals on day 31 (tumor volume approximately 92 mm ³ ). Groups 1-4 consisted of animals with comparable mean tumor sizes and standard deviations. An additional group (group 6) of 5 animals showing relatively small tumor volume (tumor volume approximately 50 mm ³ ) was selected for comparison with pretreated group 5 (all but 3 mice had tumor volumes less than 10mm ³ , one about 22mm ³ , one about 44mm ³ , and one about 119mm ³ ). Groups 1-4 received PBS (vehicle; Group 1), 1 mg/kg b.w.hlgG1-MOR03080 (Group 2), 5 mg/kg bwhlgG1-MOR03080 (Group 3) or 5 mg/kg b.w. .w.chIgG2a-MOR03080 (group 4) treatment. Group 6 received no treatment (see Table 6). Tumor volume, body weight and clinical signs were measured twice weekly until the end of the study.

Table 6:

Group number of animals Administration form substance plan Therapeutic dose [mg/kg] Dosing volume [μl/kg] 1 10 ip Carrier (PBS) Every other day on days 32-68 - 10 2 10 ip MOR03080 Human IgG1 Every other day on days 32-68 1 10 3 10 ip MOR03080 Human IgG1 Every other day on days 32-68 5 10 4 10 ip MOR03080 Chimeric IgG2a Every other day on days 32-68 5 10 5 15 ip MOR03080 Human IgG1 Every other day on days 14-36 1 10 6 5 - - - - -

2.6 Results

Clinical Findings and Fatalities

No clinical findings or fatalities related to specific tumors or substances were observed. In group 3 (hlgG 15 mg/kg), 4 animals died at the time of blood sampling (1 on day 3, 1 on day 34; 2 on day 52). In group 4 (muIgG2a 1 mg/kg), 4 animals died at the time of blood sampling (day 34). All other animals that died during the study were euthanized due to tumor size (excessive). the

weight gain

No drug-related interference with body weight gain was observed compared to group 1 (vehicle). In groups 3 (hIgG 15 mg/kg) and 4 (muIgG2a 5 mg/kg), body weight was significantly affected by blood sample collection. Apart from this effect, mean body weight gain was continuous across all groups. the

Tumor development (see Figure 16)

In group 1 (vehicle), tumor growth was found to progress slowly at the expected rate. Since this cell line had defined standard deviation values, the largest and smallest tumors were excluded for further statistical analysis. Tumor growth in Group 1 animals was comparable to that in Group 6 (untreated), although Group 6 consisted of animals with smaller mean tumor volumes at day 31. Therefore, the treatment had only a slight effect on the rate of tumor growth. In group 1, 2 mice were euthanized before day 83 due to tumor size and 1 before day 87, so the mean value of tumor volume was no longer representative after day 80. In group 6, 1 mouse was euthanized due to tumor size before day 80, 2 before day 83 and 1 before day 87, so the mean value of tumor volume was after day 76 No longer representative. the

In group 2 treated with hIgG1 at 1 mg/kg b.w., 1 animal was excluded from further analysis due to tumor growth into muscle tissue (which would normally increase tumor growth rate). Compared to control group 1 (vehicle), mean tumor size was significantly different from day 45 onwards until the end of the study. No accelerated tumor growth was observed after the end of treatment (day 68).

Animals of group 3 (5 mg/kg b.w. hIgG1 ) showed a significant decrease in tumor growth compared to group 1 (vehicle), statistically significant from day 38 until day 83. The average tumor volume began to re-grow strongly about 2 weeks after the end of treatment. One in 10 tumors disappeared by day 45 and did not grow back until day 19 after the end of treatment. the

The best performance of all treatment groups starting from a tumor volume of 92 ^mm was seen in group 4 (5mg/kgb.w.muIgG2a) where the mean tumor volume was significantly reduced and tumors in 4 animals were even higher at the end of the observation period. disappear. The difference in mean tumor volume from Group 1 (vehicle) was highly significant from day 38 until the end of the study.

An early study (Group 5) with 1 mg/kg b.w.hIgG1 on days 14-36 showed early and long-term effects on tumor development. One animal was excluded from further analysis due to tumor growth into muscle tissue. At day 31, only 5 animals had measurable tumors at the inoculation site compared to the other vaccinated animals, and only 2 of 60 animals were non-responsive to tumor inoculation. Tumor progression was delayed by about 31 days (compare control group 1 at day 52 to group 5 at day 83). About 50% of the animals showed no tumor at the inoculation site at the end of the study. the

2.7 Conclusion

Compared to Group 1 (control), no clinical findings or fatalities related to specific tumors or substances were observed. the

No drug-related interference with weight gain was observed. the

The tumor growth of RPMI8226 tumor cells decreased in the following order after treatment: hIgG11mg/kg, treated every other day for 14-36 days (group 5) > muIgG2a5 mg/kg, treated every other day for 32-68 days (group 4) > hIgG15mg/kg , treatment every other day for 32-68 days (group 3) > hIgG1 1 mg/kg treatment every other day for 32-68 days (group 2). In Groups 2-4, the mean tumor volume increased again to a variable degree after the end of treatment.

References:

Ausiello C.M., Urbani F., Lande R., la Sala A., Di Carlo B.Baj G. Surico N., Hilgers J., Deaglio S., Funaro A., Malavasi F. (2000) Functional topography of discrete domains ofhuman CD38.Tissue Antigens.2000Dec;56(6):539-47.

Chamow, S.M., Zhang, D.Z., Tan, X.Y, Mathre, S.M., Marsters, S.A., Peers, D.H., Byrn, R.A., Ashknazi, A., Junghans, R.P (1994). Humanized, bispecific immunoadhesin-antibody that targets CD3+ effectors to kill HIV-1-infected cells.J Immunol.1994Nov1;153(9):4268-80

Dattamajumdar, A.K., Jacobsen, D.P., Hood, L.E., Osman, G.E. (1996). Rapid cloning of rearranged mouse immunoglobulin variable genes. Immunogenetics 43, 141-151

Funaro, A., Spagnoli, G.C., Ausiello, C.M., Alessio, M., Roggero, S., Delia, D., Zaccolo, M., and Malavasi, F. (1990) Involvement of the multilineage CD38molecule in a unique pathway of Cell activation and proliferation. J. Immunol. 145, 2390-2396.

Hoshino S., Kukimoto I., Kontani K., Inoue S., Kanda Y., Malavasi F., Katada T. (1997) Mapping of the catalytic and epitopic sites of human CD38/NAD+glycohydrolase to functional domain in the carylbox terminus.J Immunol.158(2):741-7.

Jackson D.G., Bell J.I.(1990) Isolation of a cDNA encoding the human CD38(T10)molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation.J Immunol.141-5(7):

Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B. (2000). Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol296, 57-86.

Konopleva M., Estrov Z., Zhao S., Andreeff M., Mehta K. (1998) Ligation of cell surface CD38 protein with agonistic monoclonal antibody induces a cell growth signal in myeloidleukemia cells. J Immunol. 161-8: .

Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bossard, H.R., Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J.Imm.Meth.201, 35-55. 

Krebs, B., Rauchenberger, R., Reiffert, S., Rothe, C., Tesar, M., Thomassen, E., Cao, M., Dreier, T., Fischer, D., Hoss, A., Inge, L., Knappik, A., Marget, M., Pack, P., Meng, X.Q., Schier, R., Sohlemann, P., Winter, J., Wolle, J., and Kretzschmar, T. ( 2001). High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254, 67-84.

C.(2001).Novel methods for displaying(poly)peptides/proteins on bacteriophageparticles via disulfide bonds.WO 01/05950.

C.(2001).Novel methods for displaying(poly)peptides/proteins on bacteriophageparticles via disulfide bonds.WO 01/05950.

Malavasi, F., Caligaris-Cappio, F., Milanese, C., Dellabona, P., Richiardi, P., Carbonara, A.O. (1984). Characterization of a murine monoclonal antibody specific for human earlylymphohemapoietic cells. Hum. Immunol. 9:9-20

Namba, M., Otsuki, T., Mori, M., Togawa, A., Wada, H., Sugihara, T., Yawata, Y., Kimoto, T. (1989).Establishment of five human myeloma cell lines .In Vitro Cell Dev.Biol.25:723.

Nata K., Takamura T., Karasawa T., Kumagai T., Hashioka W., Tohgo A., Yonekura H., Takasawa S., Nakamura S., Okamoto H. (1997). Human gene encoding CD38 (ADP- ribosylcyclase/cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing. Gene186(2): 285-92.

Naundorf, S., Preithner, S., Mayer, P., Lippold, S., Wolf, A., Hanakam, F., Fichtner, I., Kufer, P., Raum, T., Riethmüller, G., Baeuerle, P.A., Dreier, T. (2002). Int. J. Cancer 100, 101-110.

Plückthun A, and Pack P. (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3 (2): 83-105.

Rauchenberger R., Borges E., Thomassen-Wolf E., Rom E., Adar R., Yaniv Y., Malka M., Chumakov I., Kotzer S., Resnitzky D., Knappik A., Reiffert S., Prassler J., Jury K., Waldherr D., Bauer S., Kretzschmar T., Yayon A., Rothe C. (2003). Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J 7 Biol (40): 38194-205.

Zhou, H., Fisher, R.J., Papas, T.S. (1994). Optimization of primer sequences for .

Claims (20)

1. isolating people's antibody or its functional fragment; It comprises the antigen binding domain of the epi-position that is specific to the CD38 of its aminoacid sequence shown in SEQID NO:22, and wherein said antibody or its functional fragment comprise and be in three variable heavy chain complementary determining regions (H-CDRs) and three the variable light chain complementary determining regions (L-CDRs) that are selected from following variable heavy chain and variable light chain centering:

(i) SEQ ID NO:5 and SEQ ID NO:13;

(ii) SEQ ID NO:6 and SEQ ID NO:14;

(iii) SEQ ID NO:7 and SEQ ID NO:15; With

(iv) SEQ ID NO:8 and SEQ ID NO:16.

2. isolated antibody as claimed in claim 1 or its functional fragment, it comprises L-CDR1, L-CDR2 and the L-CDR3 district shown in the H-CDR1 shown in the SEQ IDNO:5, H-CDR2 and H-CDR3 district and the SEQ ID NO:13.

3. isolated antibody as claimed in claim 2 or its functional fragment, it comprises the variable light chain shown in variable heavy chain shown in the SEQ IDNO:5 and the SEQ ID NO:13.

4. isolated antibody as claimed in claim 1 or its functional fragment, it comprises L-CDR1, L-CDR2 and the L-CDR3 district shown in the H-CDR1 shown in the SEQ IDNO:6, H-CDR2 and H-CDR3 district and the SEQ ID NO:14.

5. isolated antibody as claimed in claim 4 or its functional fragment, it comprises the variable light chain shown in variable heavy chain shown in the SEQ IDNO:6 and the SEQ ID NO:14.

6. isolated antibody as claimed in claim 1 or its functional fragment, it comprises L-CDR1, L-CDR2 and the L-CDR3 district shown in the H-CDR1 shown in the SEQ IDNO:7, H-CDR2 and H-CDR3 district and the SEQ ID NO:15.

7. isolated antibody as claimed in claim 6 or its functional fragment, it comprises the variable light chain shown in variable heavy chain shown in the SEQ IDNO:7 and the SEQ ID NO:15.

8. isolated antibody as claimed in claim 1 or its functional fragment, it comprises L-CDR1, L-CDR2 and the L-CDR3 district shown in the H-CDR1 shown in the SEQ IDNO:8, H-CDR2 and H-CDR3 district and the SEQ ID NO:16.

9. isolated antibody as claimed in claim 8 or its functional fragment, it comprises the variable light chain shown in variable heavy chain shown in the SEQ IDNO:8 and the SEQ ID NO:16.

10. isolated antibody as claimed in claim 1 or its functional fragment, it is right that it comprises the heavy chain amino acid sequence and the light-chain amino acid sequence that are selected from down group:

(i) SEQ ID NO:5 and SEQ ID NO:13;

(ii) SEQ ID NO:6 and SEQ ID NO:14;

(iii) SEQ ID NO:7 and SEQ ID NO:15; With

(iv) SEQ ID NO:8 and SEQ ID NO:16

11. isolated antibody as claimed in claim 1, it is IgG.

12. isolated antibody as claimed in claim 11, it is IgG1.

13. isolating functional fragment as claimed in claim 1, it is Fab or scFv antibody fragment.

14. the variable heavy chain of coding isolated antibody or its functional fragment and the nucleotide sequence of variable light chain are right, it comprises, and to be selected from following nucleotide sequence right: the sequence of SEQ ID NO:1/SEQ ID NO:9, SEQID NO:2/SEQ ID NO:10, SEQ ID NO:3/SEQ ID NO:11 and SEQ ID NO:4/SEQ ID NO:12.

15. contain the carrier of nucleotide sequence as claimed in claim 14.

16. contain the isolated cell of carrier as claimed in claim 15.

17. isolated cell as claimed in claim 16, wherein, said cell is a bacterial cell.

18. isolated cell as claimed in claim 16, wherein, said cell is a mammalian cell.

19. pharmaceutical composition, said pharmaceutical composition contain just like each described antibody or its functional fragment among the claim 1-13, and pharmaceutically acceptable carrier or vehicle.

20. like each described people's antibody among the claim 1-13, wherein, said people's antibody is synthetic people antibody.

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