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 people's antibody and uses thereof

The application requires the U.S. Provisional Application 60/541 of submission on February 6th, 2004; 911, the U.S. Provisional Application of submitting on February 26th, 2,004 60/547; 584, the U.S. Provisional Application of submitting on March 18th, 2,004 60/553; The right of priority of the U.S. Provisional Application 60/599,014 that on August 6th, 948 and 2004 submitted to, the content of said application is included in this paper as a reference in full.

Background of invention

CD38 is a kind of II type membrane glycoprotein, because it has the enzymic activity of ADP ribosyl cyclase and cADP lytic enzyme, therefore belongs to extracellular enzyme family.During ontogeny, CD38 appears on the lineage committed progenitor cell of CD34+ committed stem cell and lymphocyte, erythroid cells and medullary cell.It is believed that CD38 only expresses in the lymph pedigree at T cell and the cytocerastic commitment of B.

The rise of CD38 can be used as the mark of the B cell of lymphocyte activation-especially along the differentiation of plasmocyte appearance approach.It is to cause that through its part CD31 intracellular signal takes place or intercellular communication that (altogether-) function of receptors of CD38 is inferred, and encircles in the born of the same parents of ADPr modified as second messenger in the various signal cascades simultaneously.Yet as if because it is harmless to knock out the anti-CD38 autoantibody of analogue or philtrum in the mouse, so its physiology importance is still waited to illustrate.

Except observing it expresses in hemopoietic system; The researchist finds; CD38 raises in the various clones derived from B cell tumour, T cell tumour and marrow/monocyte tumour, and said tumour comprises B-or T-cell acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL) and multiple myeloma (MM).For example, in all patient's samples of MM, great majority are all observed the strong expression of CD38.

Therefore, the over-expresses of CD38 on malignant cell is that immunotherapy provides a kind of attractive treatment target spot.Attractive especially is that the most early stage multipotential stem cell of hemopoietic system is that CD38 is negative, and the cellulotoxic effect degree of ADCC or CDC generation is well relevant with the expression level of each target spot.

The existing method of anti-CD38 treatment can be divided into two types: the interior and in vitro method of body.In vivo in the method, anti-CD38 antibody is needed the individuality of this treatment, cross the expression malignant cell and reduce to produce the antibody-mediated CD38 of anti-CD38.Minimizing can obtain through antibody-mediated ACDD and/or the CDC that the effector cell produces, or through will resist CD38 antibody as targeting moiety with the cell toxicant material for example saporin be transported to target cell and subsequently internalization obtain.In in vitro method, will contain CD38 and cross the cell mass such as the medullary cell of expressing malignant cell, from the individuality of needs treatment, shift out, and contact with anti-CD38 antibody.Target cell can use cell toxicant material such as saporin to destroy (like the description in the method in vivo), or through the anti-CD38 antibody of cell mass contact fixed is removed, from mixture, removes the target cell of over-expresses CD38 thus.The cell mass that to remove target cell afterwards feeds back to the patient.

The antibody that is specific to CD38 can be divided into different types according to different qualities.Some antibody combine with CD38 molecule (mainly being amino acid 220-300) can be in target cell initiating activity, like Ca2+ release, cytokine release, phosphorylation event and growth-stimulating, this depends on specificity (Konopleva etc., 1998 of each antibody; Ausiello etc., 2000), but do not find between binding site and their (non--) competitive characteristic of various known antibodies definite relation (Funaro etc., 1990) is arranged.

Effect about disclosed anti-CD38 antibody is understood less relatively.As if what it is now know that is that all known antibody are all discerned the epi-position (amino-acid residue 220-300) of CD38C terminal portions specifically.Up to the present, also do not know to have be specific to be arranged in the CD38N terminal portions, at the antibody of protein primary sequence away from the epi-position of avtive spot.Yet we find in clinical trial, when OKT10 is that it has relatively low avidity and effect when containing the chimeric construct body of people Fc part.In addition, OKT10 is that therefore a kind of rodent antibody is not suitable for giving the mankind.The anti-CD38scFv antibody fragment of people (WO 02/06347) has been described recently.Yet this antibody is specific to selective expression's CD38 epi-position.

Therefore, consider the very big potentiality of anti-CD38 Antybody therapy, press for killing and wounding CD38 through ADCC and/or CDC mediation and cross and express the anti-CD38 antibody of people that has high-affinity and efficient aspect the malignant cell.

As mentioned below, the present invention has satisfied these demands and other demand through complete people source and anti-efficiently CD38 antibody are provided.

Summary of the invention

An object of the present invention is to provide and effectively to mediate people and the humanized antibody that kills and wounds the CD38 overexpressing cell.

Another object of the present invention provides the antibody of administration of human safely.

A further object of the present invention provides through using one or more antibody of the present invention to treat the method that raises diseases associated and/or illness with CD38.Hereinafter has been described these purposes of the present invention and other purpose in further detail.

On the one hand; The invention provides isolated antibody or functional antibodies fragment; It contains the antigen binding domain of the epi-position that is specific to CD38; Wherein, When with human PBMC's cell as effector cell and when the ratio of effector cell and target cell is about 30:1 to 50:1, under identical or essentially identical condition, said antibody or its functional fragment can through ADCC (" ADCC ") with the chimeric OKT10 that is superior to having SEQ ID NO:23 and 24 at least 2-5 times effectiveness mediate the CD38+ target cell and kill and wound (LP-1 (DSMZ:ACC41) and RPMI-8226 (ATCC:CCL-155)).This antibody or its functional fragment can contain antigen binding domain, and this antigen binding domain comprises SEQ ID NO:5, the H-CDR3 district shown in 6,7 or 8; This antigen binding domain also can comprise SEQ ID NO:5, the H-CDR2 district shown in 6,7 or 8; And this antigen binding domain also can comprise SEQ ID NO:5, the H-CDR1 district shown in 6,7 or 8.This CD38 specific antibody of the present invention can contain antigen binding domain, and this antigen binding domain comprises SEQ ID NO:13, the L-CDR3 district shown in 14,15 or 16; This antigen binding domain also can comprise SEQ ID NO:13, the L-CDR1 district shown in 14,15 or 16; This antigen binding domain also can comprise SEQ ID NO:13, the L-CDR2 district shown in 14,15 or 16.

On the other hand; The invention provides isolated antibody or functional antibodies fragment; It contains the antigen binding domain of the epi-position that is specific to CD38; Wherein, with the said identical or essentially identical condition of epimere under, said antibody or its functional fragment can kill and wound with the Chinese hamster ovary celI of the effectiveness mediation CD38 transfection that is superior to 2 times of chimeric OKT10 (SEQ IDNO:23 and 24) through CDC at least.The antibody that satisfies these conditions can contain antigen binding domain, and this antigen binding domain comprises SEQ ID NO:5, the H-CDR3 district shown in 6 or 7; This antigen binding domain also can comprise SEQ ID NO:5, the H-CDR2 district shown in 6 or 7; And this antigen binding domain also can comprise SEQ ID NO:5, the H-CDR1 district shown in 6 or 7.This CD38 specific antibody of the present invention can contain antigen binding domain, and this antigen binding domain comprises SEQ ID NO:13, the L-CDR3 district shown in 14 or 15; This antigen binding domain also can comprise SEQID NO:13, the L-CDR1 district shown in 14 or 15; And this antigen binding domain also can comprise district SEQID NO:13, the L-CDR2 shown in 14 or 15.

Antibody of the present invention (and functional fragment) can contain the antigen binding domain of the epi-position that is specific to CD38, and wherein said epi-position contains the one or more amino-acid residues among the amino-acid residue 43-215 of CD38, shown in SEQ ID NO:22.More particularly, said antigen binding domain bonded epi-position can contain contained one or more amino-acid residues in the one or more amino acid sections (amino acid stretch) that are selected from amino acid section 44-66,82-94,142-154,148-164,158-170 and 192-206.For some antibody, this epi-position can be a line style, and for other antibody, this epi-position can be (promptly discontinuous) of conformation.Antibody or its functional fragment with one or more these characteristics can contain antigen binding domain, and this antigen binding domain comprises SEQ ID NO:5, the H-CDR3 district shown in 6,7 or 8; This antigen binding domain also can comprise SEQ ID NO:5, the H-CDR2 district shown in 6,7 or 8; And this antigen binding domain also can comprise SEQ ID NO:5, the H-CDR1 district shown in 6,7 or 8.This CD38 specific antibody of the present invention can contain antigen binding domain, and this antigen binding domain comprises SEQID NO:13,14, the L-CDR3 district shown in 15 or 16; This antigen binding domain also can comprise SEQID NO:13,14, the L-CDR1 district shown in 15 or 16; And this antigen binding domain also can comprise SEQID NO:13,14, the L-CDR2 district shown in 15 or 16.

The peptide variant of the disclosed sequence of the present invention is also contained within the scope of the present invention.Therefore, the present invention includes the anti-CD38 antibody that contains following heavy chain amino acid sequence: there is the sequence of at least 60% sequence homogeny in its CDR district and SEQ IDNO:5,6, the CDR district shown in 7 or 8; And/or there is the sequence of at least 80% sequence homology in its CDR district and SEQ ID NO:5, the CDR district shown in 6,7 or 8.Also comprise the anti-CD38 antibody that contains following light-chain amino acid sequence: there is the sequence of at least 60% sequence homogeny in its CDR district and SEQID NO:13,14, the CDR district shown in 15 or 16; And/or there is the sequence of at least 80% sequence homology in its CDR district and SEQ ID NO:13, the CDR district shown in 14,15 or 16.

For example, antibody of the present invention can be IgG (IgG for example ₁), and antibody fragment can be Fab or scFv.Therefore, antibody fragment of the present invention can be perhaps can contain the antigen binding domain with one or more characteristics of the present invention.

The invention still further relates to isolated nucleic acid sequences, each sequence is the antigen binding domain of codified people antibody or its functional fragment all, and this antigen binding domain is specific to the epi-position of CD38.The variable heavy chain of this nucleotide sequence codified antibody, and contain the sequence that is selected from down group: SEQ ID NO:1,2,3 or 4, the nucleotide sequence of perhaps under the height stringent condition, hybridizing with SEQ ID NO:1,2,3 or 4 complementary strand.The variable light chain of this nucleic acid codified isolated antibody or its functional fragment; And can contain the sequence that is selected from down group: SEQ ID NO:9,10,11 or 12, the nucleotide sequence of perhaps under the height stringent condition, hybridizing with SEQIDNO:9,10,11 or 12 complementary strand.

Nucleic acid of the present invention is fit to the reorganization preparation.Therefore, the invention still further relates to carrier and the host cell that contains nucleotide sequence of the present invention.

Compsn of the present invention can be used for treatment or preventive use.Therefore, the present invention includes the pharmaceutical composition that contains antibody of the present invention (or functional antibodies fragment) and pharmaceutically acceptable carrier or vehicle.In related fields, the invention provides treatment and the CD38 that does not hope to exist or the method for CD38 express cell diseases associated or illness.This method comprises the pharmaceutical composition of the antibody that contains of the present invention or expection of the individual effective dose that these needs are arranged.

The invention still further relates to the separation epi-position of CD38 line style or the conformation form, and their purposes in isolated antibody or its functional fragment, said antibody or antibody fragment comprise the antigen binding domain that is specific to said epi-position.In this respect; The line style epi-position can comprise amino-acid residue 192-206, and conformation (conformational) epi-position can comprise one or more amino-acid residues of the amino acid 44-66,82-94,142-154,148-164,158-170 and the 202-224 that are selected from CD38.For example; The epi-position of available CD38 is come separation antibody or its functional fragment (said every kind of antibody or antibody fragment comprise the antigen binding domain that is specific to this epi-position), comprising the epi-position contact antibody library and the step of separating said one or more antibody or its functional fragment that make said CD38.

In another embodiment, the invention provides the separation epi-position of CD38, this epi-position is made up of the aminoacid sequence of the amino acid 44-66,82-94,142-154,148-164,158-170,192-206 and the 202-224 that are selected from CD38 basically.In the present invention, one of above-mentioned aminoacid sequence of this epi-position " basically by " adds that further feature " formation ", condition are, further feature can not influence the essential characteristic and the novelty of this epi-position in itself.

In yet another embodiment, the invention provides the separation epi-position of CD38, this epi-position is made up of the aminoacid sequence of the amino acid 44-66,82-94,142-154,148-164,158-170,192-206 and the 202-224 that are selected from CD38.

The present invention also provides test kit, wherein contain (i) contain the one or more amino acid sections that are selected from 44-66,82-94,142-154,148-164,158-170,192-206 and 202-224 CD38 separate epi-position; (ii) antibody library; (iii) use this antibody library to separate one or more members' the specification sheets that specificity combines this library of this epi-position.

The accompanying drawing summary

Fig. 1 a provides the nucleotide sequence of various new antibodies variable region of heavy chain;

Fig. 1 b provides the aminoacid sequence of various new antibodies variable region of heavy chain.Represent CDR district HCDR1, HCDR2 and HCDR3 from N-to the C-end with black matrix;

Fig. 2 a provides the nucleotide sequence of various new antibodies variable region of light chain;

Fig. 2 b provides the aminoacid sequence of various new antibodies variable region of light chain.Represent CDR district LCDR1, LCDR2 and LCDR3 from N-to the C-end with black matrix;

Fig. 3 provides the aminoacid sequence based on the variable region of heavy chain of the HuCAL antibody key-gene sequence of various consensus sequences.Represent CDR district HCDR1, HCDR2 and HCDR3 from N-to the C-end with black matrix;

Fig. 4 provides the aminoacid sequence based on the variable region of light chain of the HuCAL antibody key-gene sequence of various consensus sequences.Represent CDR district LCDR1, LCDR2 and LCDR3 from N-to the C-end with black matrix;

Fig. 5 provides the aminoacid sequence (SWISS-PROT master's accession number P28907) of CD38;

Fig. 6 provides the heavy chain of chimeric OKT10 and the nucleotide sequence of light chain;

Fig. 7 provides the synoptic diagram of the epi-position of representative antibodies of the present invention;

Fig. 8 provides the dna sequence dna (bp601-2100) (SEQ IDNO:32) of _ h_IgG1_1.This carrier is based on pcDNA3.1+ carrier (Invitrogen): the aminoacid sequence of VH-padding sequence representes with black matrix, and VH-ladder sequence last read frame and the constant region gene is represented with non-black-body.Restriction site is illustrated in the sequence top.The guiding site of sequencing primer is represented with underscore; Fig. 9 provides the dna sequence dna (bp601-1400) (SEQ ID NO:33) of Ig κ light chain expression vector

_ h_Ig κ _ 1: this carrier is based on pcDNA3.1+ carrier (Invitrogen).The aminoacid sequence of V κ-padding sequence is represented with black matrix, and last reading frame of V κ-leader sequence and constant region gene are represented with non-black-body.Restriction site is illustrated in the sequence top.The guiding site of sequencing primer is represented with underscore;

Figure 10 provides the dna sequence dna (bp601-1400) (SEQ ID NO:34) of HuCAL Ig lambda light chain carrier

_ h_Ig λ _ 1: the aminoacid sequence of V λ-padding sequence is represented with black matrix, and last reading frame of V λ-terraced sequence and constant region gene are represented with non-black-body.Restriction site is illustrated in the sequence top.The guiding site of sequencing primer is represented with underscore;

Figure 11 provides the result of proliferation test: will from the PBMC (round dot with independent is represented) of 6 different healthy donors exist

antibody Mab#1 (=MOR03077); Mab#2 (=MOR03079); Mab#3 (=MOR03080); Reference antibody chOKT10; Excitability (ag.) contrast IB4; Cultivated 3 days under irrelevant

negative control IgG1 (NC) and the situation as the mouse IgG2a (Iso) of the coupling isotype contrast of IB4.Standard substance with the BrdU mark is measured proliferation activity, and mixes (RLU=relative light unit) through analyzing it based on chemiluminescent ELISA;

Figure 12 provides the result of IL-6 release test: will from the PBMC (round dot with independent is represented) of 4-8 different healthy donor exist

antibody Mab#1 (=MOR03077); Mab#2 (=MOR03079); Mab#3 (=MOR03080); Reference antibody chOKT10; Excitability (ag.) contrast IB4; Cultivated 24 hours under the situation of irrelevant

negative control (NC) and simple substratum (substratum).Through based on the amount of chemiluminescent ELISA, represent with relative light unit (RLU) from culture supernatant analysis IL-6;

It is relevant to the toxic data of CD34+/CD38+ progenitor cells that Figure 13 provides: carry in the future from the PBMC of the healthy donors of body CD34+/CD38+ progenitor cell respectively exist

antibody Mab#1 (=MOR03077); Mab#2 (=MOR03079); Mab#3 (=MOR03080); Cultivated 4 hours under the situation of positive control (PC=chOKT10) and irrelevant

negative control.Afterwards cell suspension is mixed with wetting methylcellulose gum matrix and cultivated for 2 weeks.Counting is from burst forming unit-erythrocyte (BFU-E; The B hurdle) and granulocyte/erythroid cells/scavenger cell/megalokaryocyte stem cell (CFU-GEMM; The B hurdle) and granulocyte/scavenger cell stem cell (CFU-GM; The C hurdle) colony forming unit (CFU), and according to substratum contrast (" nothing "=substratum) stdn.A representes on the hurdle CFU sum (total CFU) of all progenitor cells.Provided the MV of at least 10 different PBMC donors.Error bar is represented the standard error with MV;

Figure 14 provides the ADCC data of different clones:

A: single measurement (except the RPMI8226, being the MV of 4 measurements); E:T ratio: 30:1

B:Namba etc., 1989

C: AC is 5 μ g/ml (except the Raji, being 0.1 μ g/ml)

D: add vitamin A acid to measure stimulation [%]=[(expectation value of the killing and wounding-centre value of killing and wounding)/(value of killing and wounding in the middle of the 1-)] * 100 that the CD38 expression specificity is killed and wounded

PC: positive control (=chOKT10)

MM: multiple myeloma

CLL: chronic B cell leukemia

ALL: acute lymphoblastic leukemia

AML: acute myeloid leukemia

DSMZ: Germany mikrobe and cell culture preservation center GmbH

ATCC: US mode culture collection center

ECACC: European zooblast preservation center

MFI: average fluorescent strength;

Figure 15 provides the ADCC data of MM sample:

^a: 2-4 separate analysis;

Figure 16 provides the test-results with mean tumour volume after the MOR03080 handler myelomatosis heterograft: group 1: vector; Group 2:MOR03080 gave 1mg/kghIgG1 in 32-68 days every other day; Group 3:MOR03080 gave 5mg/kg hIgG1 in 32-68 days every other day; Group 4:MOR03080 gave 5mg/kg chIgG2a in 32-68 days every other day; Group 5:MOR03080 gave 1mg/kg hIgG1 in 14-36 days every other day; Group 6: be untreated.

Detailed Description Of The Invention

The present invention is based on the discovery new antibodies, this antibody is specific to CD38 or CD38 is had high-affinity and can have the treatment benefit to individuality.Antibody of the present invention can be people's antibody or humanized antibody, can be used for all respects of the present invention, and this will carry out more detailed description in this manual.

" people " antibody or functional human antibody fragment are defined herein as non-chimeric (for example non-" humanized ") and are not the antibody from (all or part of) inhuman species.People's antibody or functional antibodies fragment can or can be synthetic people antibody from the people." synthetic people antibody " is defined herein as the antibody with following sequence: it is partly or entirely from the computer composition sequence of analyzing based on the known person antibody sequence.The design data peptide sequence that for example, can therefrom obtain through the DB and the utilization of analyst's antibody or antibody fragment sequence is realized human antibody sequence or its segmental computer design.Segmental another example of people's antibody or functional antibodies is people's antibody or the functional antibodies segment by isolating nucleic acid encoding from the antibody sequence library in people source the library of the antibody in the natural source of people (promptly based on).

" humanized antibody " or functional humanization antibody fragment are defined herein as one of following antibody: (i) from the antibody of inhuman source (for example, having the transgenic mice of heteroimmunity system), this antibody is based on people embryo system (germline) sequence; Or (ii) chimeric antibody; Its variable region is from inhuman source and constant region is originated from the people, perhaps (iii) CDR grafted antibody, and the CDR of its variable region is from inhuman source; One or more frameworks of variable region are that the people originates, and constant region (if any) is that the people originates.

Because binding specificity is not absolute; But relative characteristic; Therefore in this manual, if certain antibody can separate with one or more certain antigen (being CD38) here with reference to antigenic region, then this antibody " specificity is incorporated into ", " being specific to " or " specific recognition " this antigen.In the most conventional form (when not mentioning the reference of regulation), " specificity combination " is meant the ability that antibody separates purpose antigen nothing to do with antigenic region, and for example, one of method of available hereinafter is confirmed.This method includes but not limited to: the test of Western trace, ELISA test, RIA test, ECL test, IRMA test and pepscan.For example, can carry out the standard ELISA test.Can mark through standard coloration reaction (for example, secondary antibodies and horseradish peroxidase, and TMB and hydrogen peroxide).Reaction in some hole is through the optical density(OD) scoring of for example 450nm.Typical background (=negative reaction) can be 0.1OD; Typical positive reaction can be 1OD.This means that the difference between the male/female can be greater than 10 times.Usually, it is single with reference to antigen to confirm that binding specificity not only uses, and is to use one group of about 3-5 irrelevant antigen such as milk powder, BSA, transferrin to wait and carry out.

Yet " binding specificity " also refers to antibody differentiation target antigen and one or more ability that is used as the closely related antigen (for example CD38 and CD157) of RP.In addition; " binding specificity " can comprise the ability of the different piece of its target antigen of antibody differentiation; For example different structure territory or the zone of CD38; Like the N-end of CD38 or the epi-position of C-stub area, or distinguish the one or more key amino acid residues of CD38 amino-acid residue or the ability of amino acid section.

In addition, " Tegeline (Ig) " is defined herein as the protein that belongs to IgG, IgM, IgE, IgA or IgD class (or its any subclass), comprises all conventional known antibody and functional fragments thereof.Therefore " functional fragment " of antibody/Tegeline is defined herein as the fragment (the for example variable region of IgG) of the antibody/Tegeline that has kept antigen binding domain." antigen binding domain " of antibody at one or more hypervariable regions of this antibody, promptly observed in CDR-1 ,-2 and/or-3 districts usually; Yet variable " framework " district also can play a significant role in antigen combines, as for CDR support being provided.Preferably; " antigen binding domain " comprises amino-acid residue 4-103 of (VL) chain that can lighten and the amino-acid residue 5-109 of variable heavy (VH) chain at least; More preferably comprise the amino-acid residue 3-107 of VL and the amino-acid residue 4-111 of VH, particularly preferably be and comprise complete VL and the VH chain (amino acid/11-113 of the amino acid/11 of VL-109 and VH; Numbering is seen WO97/08320).Being used for preferred immunoglobulins type of the present invention is IgG." functional fragment " of the present invention comprises F (ab ') ₂The structural domain of fragment, Fab fragment and scFv.F (ab ') ₂Or Fab can be designed so that C _H1And C _LIntermolecular two sulphur that exist between the structural domain interact and minimize or removed fully.

Antibody of the present invention can be from the recombinant antibodies library based on following aminoacid sequence: it is by computer design and by the synthetic nucleic acid encoding that produces.For example, can and utilize the design data peptide sequence that therefrom obtains to realize the computer design antibody sequence through analyst's sequence library.The method that design and acquisition computer produce sequence exists, for example, and Knappik etc., J.Mol.Biol. (2000) 296:57; Krebs etc., J.Immunol.Methods. (2001) 254:67; With in the USP 6,300,064 of Knappik etc. description is arranged, these documents are included in this paper as a reference in full.

Antibody of the present invention

In this manual, relate to following representative antibodies of the present invention: " antibody numbering " or " LAC " or " MOR " 3077,3079,3080 and 3100.LAC3077 representes to have corresponding to the variable region of heavy chain of SEQ ID NO:1 (DNA)/SEQ ID NO:5 (protein) with corresponding to the antibody of the variable region of light chain of SEQ ID NO:9 (DNA)/SEQ IDNO:13 (protein).LAC3079 representes to have corresponding to the variable region of heavy chain of SEQ ID NO:2 (DNA)/SEQ ID NO:6 (protein) with corresponding to the antibody of the variable region of light chain of SEQ ID NO:10 (DNA)/SEQ ID NO:14 (protein).LAC3080 representes to have corresponding to the variable region of heavy chain of SEQ ID NO:3 (DNA)/SEQ ID NO:7 (protein) with corresponding to the antibody of the variable region of light chain of SEQ ID NO:11 (DNA)/SEQ ID NO:15 (protein).LAC3100 representes to have corresponding to the variable region of heavy chain of SEQ ID NO:4 (DNA)/SEQID NO:8 (protein) with corresponding to the antibody of the variable region of light chain of SEQ ID NO:12 (DNA)/SEQ IDNO:16 (protein).

On the one hand, the invention provides the antibody with antigen binding domain, this antibodies district can specificity combines CD38 or one or more zones of CD38 is had high-affinity, and the aminoacid sequence of CD38 is shown in SEQ ID NO:22.If the avidity that records is at least 100nM (the segmental unit price avidity of Fab), claim that then antibody has " high-affinity " to antigen.Antibody of the present invention or antigen binding domain are preferably with less than about 100nM, more preferably with less than about 60nM, most preferably to combine CD38 less than the avidity of about 30nM.Again preferably with less than about 10nM, more preferably to combine the antibody of CD38 less than the avidity of about 3nM.For example, antibody of the present invention is about 10.0nM or 2.4nM (the segmental unit price avidity of Fab) to the avidity of CD38.

Table 1 has been summed up the avidity of analyzing the representative antibodies of the present invention that records through surface plasma resonance (Biacore) and FACS Scatchard:

Table 1: affinity of antibody

Antibody (Fab or IgG1)	BIACORE(Fab)K D[nM] a	FACS?Scatchard(IgG1) bK 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	Do not confirm	8.28

^a: the MV of at least 2 different avidity mensuration

^b: the RPMI8226MM clone that is used for FACS-Scatchard

Reference table 1, on fixed recombinant C D38 through surface plasma resonance (Biacore) and people RPMI8226 clone that utilize to express CD38 through flow cytometry measurement LAC3077,3079,3080 and 3100 avidity.Biacore research is directly being carried out on the fixed antigen (CD38-Fc fusion rotein).LAC3077,3079,3080 and 3100 Fab form the unit price avidity that demonstrates on the fixed CD38-Fc fusion rotein about 2.4 and 56nM between, LAC3079 demonstrates high-affinity, is Fab3100,3080 and 3077 then.

The IgG1 form is used for the affine test (FACS Scatchard) based on cell.The right hurdle of table 1 has shown the bonding strength of the LAC of this form.LAC3080 demonstrates the strongest combination, and it slightly is better than LAC3079 and 3077.

Another preferred feature of preferred antibody of the present invention is that they have specificity to the zone in the N-stub area of CD38.For example, but LAC3077 of the present invention, 3079,3080 and 3100 specificitys combine the N-stub area of CD38.

The type of the epi-position of antibodies of the present invention can be (being multistage amino acid) of line style (i.e. one section successive amino acid) or conformation.For the epi-position of confirming antibodies specific be line style or conformation, those skilled in the art can analyze the combining of overlapping peptide (for example 13 gather peptide combine with 11 amino acid eclipsed) of antibody and covering CD38 different zones.Adopt this analysis, the contriver finds, the discontinuous epi-position of the N-stub area of LAC3077,3080 and 3100 identification CD38, and the epi-position of LAC3079 can be described to be the (see figure 7) of line style.In conjunction with the knowledge that this specification sheets provided; How those of ordinary skill in the art can know that the one or more separation epi-positions with CD38 produce antibody with the antigen binding domain that is specific to said epi-position (for example, utilizing the synthetic peptide of cell expressing epi-position of epi-position or the CD38 of CD38).

Antibody of the present invention preferably with people and at least a other species generation species cross reaction, said other species can be rodents or non-human primates.Said non-human primates can be rhesus monkey, baboon and/or stump-tailed macaque.Said rodents can be mouse, rat and/or hamster.In order in multiple species, to carry out studying in the body with identical antibody, can provide with the antibody of at least a rodent cross reaction, for example be superior to the flexibility and the benefit of known anti-CD38 antibody.

Preferably, antibody of the present invention not only can combine CD38, and can mediate and kill and wound the CD38 express cell.More particularly, antibody of the present invention can pass through antibody-effector functions, through reducing its therapeutic efficiency of positive (for example pernicious) cell performance of CD38.These functions comprise ADCC (ADCC) and CDC (CDC).

Table 2 has been summed up the EC50 value of the ADCC and the CDC of representative antibodies of the present invention:

Table 2: the EC50 value of antibody

^a: the MV of at least 2 EC50 measurements

^b: one-shot measurement

^c: the MV of 2 EC50 measurements

^d: the MV of 3 EC50 measurements

^e: the MV of 4 EC50 measurements

Yet, find that CD38 not only expresses on the immunocyte of marrow (for example monocyte and granulocyte) and lymph pedigree (for example activated B and T cell, plasmocyte), also find on precursor cell separately, also to express.Because the influence (this point is important) that those cells are not killed and wounded by antibody-mediated malignant cell, therefore antibody of the present invention does not have cytotoxicity to precursor cell.

Except the catalytic activity with ring-type ADP-ribose cyclase and lytic enzyme, CD38 also demonstrates biological relevant signal transduction ability (Hoshino etc., 1997; Ausiello etc., 2000).Those functions can be through receptor-ligand binding for example or through inducing in vivo with the anti-CD38 of excitability is antibody linked, thereby causes for example calcium motion, lymphopoiesis and release cytokine.Preferably, antibody of the present invention is non-agonistic antibody.

The peptide variant

Antibody of the present invention is not limited to the specific peptide sequence that this specification sheets provides.The present invention can comprise these variant polypeptides.With reference to open and conventional obtainable technology and bibliography of the present invention; Those skilled in the art can prepare, detect and use the functional variant at this disclosed antibody, and can understand those variants with ability that mediation CD38+ target cell kills and wounds and fall within the scope of the present invention.In the literary composition, " ability that mediation CD38+ target cell is killed and wounded " is meant the functional performance that anti-CD38 antibody of the present invention is had.Therefore, the mediation CD38+ target cell ability of killing and wounding comprise through for example ADCC and/or CDC or through with the ability of killing and wounding of the toxicity construct mediation CD38+ target cell of antibody coupling of the present invention.

Variant can comprise for example, with respect to the disclosed peptide sequence of this specification sheets, having the complementary determining region (CDR) (hypermutation) of at least one change and/or the antibody in framework (FR) (variable) determining area/site.For setting forth this notion better, hereinafter antagonist structure is simply described.

Antibody is made up of two peptide chains, and every contains 1 (light chain) or 3 (heavy chain) constant regions and variable region (VL, VH), the latter is positioned at intermediary CDR by 4 FR districts and 3 and constitutes.Antigen binding site is made up of one or more CDR, and the FR district provides structural framework for CDR, therefore in antigen combines, plays an important role.Through changing one or more amino-acid residues in CDR or FR district, those skilled in the art can produce the antibody sequence of sudden change or change easily, and for example, available antigen screens these antibody sequences according to new or improved characteristic.

Table 3a (VH) and 3b (VL) have described the CDR and the FR district of some antibody of the present invention, and compare each other and compared the amino acid of specific position with corresponding consensus sequence or " key-gene " sequence (like USP 6,300,064 is said):

Data among those skilled in the art table capable of using 3a and the 3b design the peptide variant within the scope of the invention.Preferably make up variant through the amino acid that changes in one or more CDR district, variant also can have the framework region of one or more changes.According to the comparison of new antibodies and other antibody, can comprise by reformed candidate's residue, the residue 4 of LAC3080 and 3077 variable light chains or 37 and the residue 13 or 43 of variable heavy chain for example for example, this is to change because of these positions each other.Change and also can occur in framework region.For example, can change peptide FR structural domain, be that sequence is compared residue and changed with the embryo in this case.

According to the comparison of new antibodies, can comprise by reformed candidate's residue, for example with corresponding consensus sequence or " key-gene " sequence; Compare VL λ 3, the residue 27,50 or 90 of the variable light chain of LAC3080, and for example; Compare VH3, the residue 33,52 and 97 of LAC3080 variable heavy chain.Perhaps; Those skilled in the art can compare with the known array of this antibody-like of same type through the disclosed aminoacid sequence of the present invention and carry out identical analysis; For example adopt the method described in the USP 6,300,064 of (2000) such as Knappik and Knappik etc. to analyze.

In addition; Can obtain variant as starting point through following optimization with a kind of LAC: change the one or more amino-acid residues among the LAC; Amino-acid residue in preferred one or more CDR, and screen resulting variant set to obtain the having variant that improves characteristic.Particularly preferably be one or more amino-acid residues variations among the CDR-2 of CDR-1 and/or VH of CDR-3, VL of CDR-3, the VH of VL.Variation can be passed through with trinucleotide mutagenesis (TRIM) technology (Vimek s; B.; Ge, L, Plckthun; A.; Schneider, K.C., Wellnhofer; G. with Moroney S.E. (1994) Trinucleotide phosphoramidites:ideal reagents for the synthesis of mixedoligonucleotides for random mutagenesis.Nucl.Acids Res.22,5600.) set of synthetic dna molecule accomplishes.

The conservative amino acid variant

Polypeptide variants can be prepared into the whole molecular structures that keep antibody peptide sequence of the present invention.Consider each amino acid whose characteristic, those skilled in the art can know some rational replacements.For example, can carry out aminoacid replacement, i.e. " conservative property replacement " according to the similarity of polarity, electric charge, solvability, hydrophobicity, wetting ability and/or the both sexes characteristic of relevant residue.

For example, (a) nonpolar (hydrophobicity) amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine(Phe), tryptophane and methionine(Met); (b) polare Aminosaeren comprises glycocoll, Serine, Threonine, halfcystine, tyrosine, l-asparagine and Stimulina; (c) positive charge (alkalescence) amino acid comprises l-arginine, Methionin and Histidine; (d) negative charge (acidity) amino acid comprises aspartic acid and L-glutamic acid.Replace usually and can in (a)-(d) group, carry out.In addition, since glycocoll and proline(Pro) can both interrupt alpha-helix so can replace each other.Similarly; Some amino acid such as L-Ala, halfcystine, leucine, methionine(Met), L-glutamic acid, Stimulina, Histidine and Methionin are more common in alpha-helix, and Xie Ansuan, Isoleucine, phenylalanine(Phe), tyrosine, tryptophane and Threonine are more common in β-lamella.Glycocoll, Serine, aspartic acid, l-asparagine and proline(Pro) are common in the corner.Some preferably replace and can in following group, carry out: (i) S and T; (ii) P and G; (iii) A, V, L and I.In conjunction with known genetic code and reorganization and synthetic DNA technology, those skilled in the art can make up the DNA of coding conservative amino acid variant easily.In a certain embodiments, the amino acid position 3 among the SEQ ID NO:5,6,7 and/or 8 can become E from Q.

In this manual, " the sequence homogeny " between two peptide sequences is meant the per-cent of same amino acid between these two sequences." sequence similarity " is meant identical or represents the substituted amino acid whose per-cent of conservative amino acid.The sequence homogeny in preferred peptide sequence CDR of the present invention district is at least 60%, and more preferably at least 70% or 80%, more preferably at least 90%, most preferably at least 95%.The sequence similarity at least 80% in preferred antibodies CDR district, more preferably 90%, most preferably 95%.

Dna molecular of the present invention

The invention still further relates to the dna molecular of code book invention antibody.These sequences include but not limited to the dna molecular shown in Fig. 1 a and the 2a.

Dna molecular of the present invention is not limited to the disclosed sequence of this specification sheets, also comprises its variant.DNA variant of the present invention can be hybridized physical property according to it and described.Those skilled in the art can know, because DNA is double-stranded, therefore can adopt nucleic acid hybridization technique to identify its complement, its Equivalent or homologue with DNA.Can know that also hybridization can be lower than 100% complementarity generation.Yet if suitable selection condition, available hybridization technique is distinguished dna sequence dna according to the structural relation of they and particular probe.About the instruction of this condition, can be referring to Sambrook etc., 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 etc.; 1995 (Ausubel, F.M., Brent, R., Kingston; R.E., Moore, D.D., Sedman, J.G.; Smith, J.A. and Struhl, K. compiles (1995), Current Protocols in Molecular Biology.NewYork:John Wiley and Sons).

Structural similarity between two peptide sequences can be expressed as the function of " severity " of the condition of this two sequence phases mutual cross.In this manual, term " severity " condition of being meant is unfavorable for the degree of hybridizing.Stringent condition extremely is unfavorable for hybridization, has only the maximally related molecule of structure could phase mutual cross under this condition.On the contrary, nonstringent condition helps the lower molecule phase mutual cross of structural dependence degree.Therefore, the hybridization severity is directly related with the structural relation of two nucleotide sequences.Following relational expression is useful (wherein, T for association hybridization and dependency _mBe the melting temperature(Tm) of nucleic acid duplex):

a.T _m＝69.3+0.41(G+C)％

B. the T of the right 1% duplex DNA of the every increase of number of base mismatch _mTo reduce by 1 ℃

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

Wherein, μ 1 is ionic strengths of two kinds of solution with μ 2.

The hybridization severity is the function of many factors, comprising total DNA concentration, ionic strength, temperature, probe size and can interrupt hydrogen bond reagent have a situation.Promote the factor of hybridization comprise high DNA concentration, high ionic strength, low temperature, than the long probe size and there is not the reagent that can interrupt hydrogen bond.Hybridization is carried out usually in two stages: " combination " stage and " washing " stage.

At first, in the combination stage, probe combines target under the condition that helps hybridizing.This stage is usually through changing temperature control severity.Only if the use weak point (20nt) and oligonucleotide probe, the temperature of high severity is usually between 65 ℃ and 70 ℃.Typical hybridization solution contains 6X SSC, 0.5%SDS, 5XDenhardt solution and the non-specific carrier DNA of 100 μ g.Referring to Ausubel etc., 2.9 joints, supplementary issue 27 (1994).Certainly, many different but identical buffer condition is known on function.When degree of relevancy is low, can select lower temperature.Low severity combines temperature between 25 ℃ and 40 ℃.The temperature of medium severity is at least about 40 ℃ to being lower than about 65 ℃.The temperature of high severity is at least about 65 ℃.

The second, remove unnecessary probe through washing.This stage is used strict more condition usually.Therefore, " washing " stage is to confirm the most important stage of dependency through hybridizing.Washings is low salt concn normally.A kind of typical medium severity solution contains 2X SSC and 0.1%SDS.The contained amount of high severity washings is less than about 0.2X SSC (in ionic strength), and preferred strict solution contains the 0.1X SSC that has an appointment.The temperature again relevant with various severity is to the discussion of " combination ".During washing, need to change washings for several times usually.For example, typical high severity wash conditions is included in 55 ℃ of washings 2 times, and each 30 minutes, and 60 ℃ of washings three times, each 15 minutes.

Therefore, present invention resides in that high severity combines and wash conditions under with the nucleic acid molecule of the molecular hybridization shown in Fig. 1 a and the 2a, wherein this nucleic acid molecule encoding has antibody or its functional fragment of characteristic of the present invention.Preferred molecule (with mRNA) is the molecule of (preferably at least 85%, more preferably at least 90%, the most preferably at least 95%) homology that has at least 75% or 80% with one of dna molecular of the present invention or sequence homogeny.In an object lesson of variant of the present invention, the 7th nucleic acid can become G from C among the SEQ ID NO:1,2,3 and/or 4, thereby codon becomes GAA from CAA.

The function equivalence variant

Another kind of DNA variant of the present invention can be described (seeing the peptide that Fig. 1 b and 2b list) according to its coded product.These function equivalence genes are characterised in that: since the degeneracy of genetic code, they codings peptide sequence identical with the listed sequence of 2b with Fig. 1 b.SEQ ID NO:1 and 31 is examples of function equivalence variant, i.e. different but the identical polypeptide of encoding, i.e. SEQ IDNO:5 of their nucleotide sequence.

Known available several kinds of different methods make up the variant of dna molecular provided by the present invention.For example, they may be constructed such complete synthetic DNA.Effectively composition length is that 20 methods to the oligonucleotide of about 150 Nucleotide have a lot.Referring to Ausubel etc., 2.11 joints, supplementary issue 21 (1993).Available Khorana etc., J.Mol.Biol.72:209-217 (1971) at first reported method synthesize and assemble overlapping oligonucleotide; Also can be referring to Ausubel etc., the same, 8.2 joints.Synthetic DNA preferably is designed to have easily restriction site so that be cloned into suitable carriers at 5 ' and 3 ' end of gene.

As stated, the method that produces variant is to be initial with a kind of DNA of the present invention, carries out site-directed mutagenesis then.See Ausubel etc., the same, the 8th chapter, supplementary issue 37 (1997).In typical method, target DNA is cloned in the single stranded DNA phage vector.Separate single stranded DNA and with its with contain the oligonucleotide hybridization that required Nucleotide changes.Complementary strand is able to synthetic and this double stranded phage is introduced the host.In the gained filial generation some will have required sudden change, and this can confirm through dna sequencing.In addition, exist various raising progeny phages to become the method for the possibility of desired mutant.These methods are well-known to those having ordinary skill in the art, and available commercially available test kit produces this two mutants.

Recombinant DNA construction body and expression

The present invention also provides the recombinant DNA construction that contains one or more nucleotide sequences of the present invention body.Recombinant precursor of the present invention is used for and carrier, connects like plasmid or virus vector, has inserted the dna molecular of code book invention antibody in this carrier.

The said encoding sox of described technology preparation such as available Sambrook etc. (1989) and Ausubel (1989).Perhaps, available for example synthesizer is through chemical process synthetic DNA sequence.Referring to, for example, and OLIGONUCLEOTIDE SYNTHESIS (1984, Gait compiles, IRL Press, and the technology described in Oxford), the document is included this paper in as a reference in full.Recombinant precursor of the present invention contains can expressed rna and/or the expression vector of the coded protein of DNA.Said carrier also comprises regulating and controlling sequence, comprises the promotor that operationally is connected with ORF (ORF).Said carrier also comprises alternative flag sequence.In order effectively to translate the target gene encoding sequence that inserts, can also need specific start signal and bacterium secretion signal.

The present invention also provides the host cell that contains at least a DNA of the present invention.In fact this host cell can be the available cell of any expression vector.For example, it can be senior eukaryotic host cell such as mammalian cell, rudimentary eukaryotic host cell such as yeast cell, but preferred prokaryotic cell prokaryocyte such as bacterial cell.Transfection, electroporation or the phage-infect of the transfection of phosphoric acid calcium, DEAE, VISOSE mediation are introduced recombinant precursor in the host cell.

Bacterial expression

Structural dna sequence dna through the desired protein of will encoding and suitable translation initiation signal and termination signal are inserted into together and make up bacterium in the operability frame (reading phase) that has functional promotor and use expression vector.Said carrier can contain the alternative marks of one or more phenotypes and replication orgin guaranteeing to keep carrier, and in the host, increases when needed.Suitable conversion prokaryotic hosts comprises the various bacterium of intestinal bacteria (E.coli), Bacillus subtilus (Bacillus subtilis), Salmonella typhimurtum (Salmonellatyphimurium) and Rhodopseudomonas, streptomyces and Staphylococcus.

Bacteria carrier can be, for example based on phage, plasmid or phagemid.These carriers can contain from the alternative mark of commercially available plasmid and bacterium replication orgin, and these commercially available plasmids contain the element of the cloning vector pBR322 (ATCC37017) that knows usually.Transforming the appropriate host bacterial strain and making after host strain grows to suitable cell density, suppressing/induce selected promotor and cell is cultivated for some time again through suitable method (for example, changing temperature or chemically induced).Cell is usually through centrifugal collection, and is broken through physics or chemical process, and reservation gained crude extract is further purified.

In bacterial system, can advantageously select many expression vectors according to the purposes of expressed protein.For example, in the time will preparing a large amount of these proteinoid, for example just need be able to express the carrier of the fusion protein product of high-caliber easy purifying with generation antibody or screening peptide library.

Treat-ment

Treat-ment comprises the antibody of the present invention's expection of the individual treatment significant quantity that needs treatment." treatment effectively " amount is defined as the amount of said antibody in this manual; This amount is enough to reduce CD38 positive cell in the individual treatment zone-with single dose scheme or multiple doses scheme, give separately or unite with other reagent and to give; This amount can be alleviated unfavorable symptom, but on toxicology, can tolerate.Individuality can be people or non-human animal (for example, rabbit, rat, mouse, monkey or its rudimentary primate).

Antibody of the present invention can give with known drug, and said in some cases antibody itself can be modified.For example, antibody can combine with its effect of further raising with immunotoxin or ri.

Antibody of the present invention can be used as treatment or diagnostic tool at various CD38 in by the disease of expressing unfriendly or finding.The disease and the symptom that are particularly suitable for Antybody therapy of the present invention are multiple myeloma (MM) and other hemopathy, like lymphocytic leukemia (CLL), chronic granulocytic leukemia (CML), acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).Antibody of the present invention can also be used to treating inflammatory diseases, like rheumatoid arthritis (RA) or systemic lupus erythematous (SLE).

For treating above-mentioned disease, pharmaceutical composition can use one or more pharmaceutically acceptable carriers or vehicle to prepare with ordinary method used according to the present invention.Antibody of the present invention can give through any suitable mode, and this mode is variable according to the type of the disease of being treated.Possible route of administration comprises in parenteral (for example intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous), the lung and in the nose, also comprises (intralesional) administration in local immunity suppression therapy, the wound if desired.In addition, antibody of the present invention can be inculcated (pulse infusion) through pulse and is given, and for example gives with the antibody dosage that successively decreases.Preferably, dosed administration carries out through injection, and most preferably through intravenously or subcutaneous injection, this part depends on that administration is short-term or secular.The amount of administration depends on various factors, like clinical symptom, individual body weight, whether given other medicines etc.Those skilled in the art can know that route of administration is according to being changed by treatment disease or illness.

According to the present invention, the treatment significant quantity of confirming novel polypeptide depends on the characteristic, route of administration of particular patient and the characteristic of disease to be treated to a great extent.General instruction can for example found in the following document: the publication of international coordination meeting (International Conference on Harmonisation) and " Lei Mingdun pharmaceutical science " (REMINGTON ' S PHARMACEUTICAL SCIENCES) the 27th and 28 chapters; 484-528 page or leaf (the 18th edition; Alfonso R.Gennaro compiles; Easton, Pa.:Mack Pub.Co., 1990).More specifically, the factors such as toxicity and effect of confirming to depend on medicine of treatment significant quantity.Available method known in the art and that can find is in the above referred-to references confirmed toxicity.Available identical method and the method described in following examples are confirmed effect.

Diagnostic method

In some malignant tumour, CD38 expresses at the hemocyte camber; Therefore, available anti-CD38 antibody of the present invention comes video picture or manifests malignant cell possibility accumulative position among the patient.In this respect, can detect ground mark antibody through using ri, affinity labelling (like vitamin H, avidin etc.), fluorescent mark, paramagnetic atom etc.The method of carrying out this mark is well known in the art.The clinical application of antibody in diagnosing image can be with reference to Grossman, H.B., Urol.Clin.North Amer.13:465-474 (1986); Unger, E.C. etc., Invest.Radiol.20:693-700 (1985); And Khaw, B.A. etc., Science209:295-297 (1980).

The focus that detects the antibody that has this detectable label can be explained the for example position of tumor development.In one embodiment, this detection is through sampling from tissue or blood and when the antibody of detectable label exists, cultivates these samples and accomplish.In preferred embodiments, this technology waits and accomplishes through using magnetic images, fluorography with the Noninvasive mode.This diagnostic test can be used to the successful property of monitoring of diseases treatment, and wherein existing or do not have the CD38 positive cell is corresponding index.The present invention also considers anti-CD38 antibody of the present invention is used for in-vitro diagnosis.

Treatment and diagnosis composition

Can antibody of the present invention be formed in available compsn on the medicine according to currently known methods, wherein, antibody of the present invention (comprising its any functional fragment) is combined to form mixture with pharmaceutically acceptable carrier property vector.Being prepared in of suitable vector and they has description among for example " Lei Mingdun pharmaceutical science " (REMINGTON ' S PHARMACEUTICAL SCIENCES) (the 18th edition, Alfonso R.Gennaro compiles, Easton, Pa.:MackPub.Co., 1990).For processing the pharmaceutically acceptable compsn that is fit to effective administration, this compsn will contain one or more antibody of the present invention of significant quantity and an amount of carrier property vector.

Available appropriate means prepares preparation with the said active compound of sustained release.Thereby usable polymers comes complexing or absorbs anti-CD38 antibody to obtain controlled release preparation.Can control conveying through selecting the suitable macromole (for example polyester, polyamino acid, Vilaterm, pyrrolidone, ethylene vinyl acetate, methylcellulose gum, CMC 99.5 or protamine, vitriol) and the method for mixing of macromolecular concentration and sustained release.Another kind of possible method through the control preparation control action kou time is that anti-CD38 antibody is mixed polymeric materialss such as polyester, polyamino acid, hydrogel, POLYACTIC ACID or vinyl-vinyl acetate copolymer.Perhaps; Except these medicaments are mixed aggregated particles; Can these materials be wrapped in microcapsule or gluey drug delivery system or the huge emulsion; For example, microcapsule can prepare (respectively for example Walocel MT 20.000PV or gelatin microcapsule with gather (TEB 3K) microcapsule) through condensation technique or through interfacial polymerization, and gluey drug delivery system for example has liposome, albumin microsphere spheroid, microemulsion, nano particle and Nano capsule.These technology have description in " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, 1980).

Said compound can be made into through injection, for example through bolus injection or inculcate and administered parenterally continuously.Injection formulations can exist with unit dosage, for example is contained in ampoule or the multi-dose container, is added with sanitas.Said compsn can be suspension-s, solution or oiliness or aqueous emulsion form, and can contain formula agents such as suspension agent, stablizer and/or dispersion agent.Perhaps, said activeconstituents can be the powder type of before using, rebuilding with the for example aseptic apirogen water of suitable carriers.

If desired, said compsn can be present in packing or the distribution device, and said packing or distribution device can comprise one or more unit dosage that contains activeconstituents.For example, this packing can comprise metal or plastic foil, like blister package.In this packing or the distribution device working instructions can also be arranged.

Can further understand the present invention with reference to following work embodiment, therefore these embodiment just do not constitute any restriction to the present invention in order to set forth the present invention.

Embodiment

Clone

Following clone is available from European cell culture preservation center (ECACC), German microbial preservation center (DSMZ) or American type culture collection (ATCC): the hybridoma cell line (ECACC that makes CD38 mouse IgG1 monoclonal antibody OKT10; #87021903), the Jurkat cell (DSMZ, ACC282), LP-1 (DSMZ, ACC41), RPMI8226 (ATCC; CCL-155), HEK293 (ATCC; CRL-1573), CHO-K1 (ATCC, CRL-61) and Raji (ATCC, CCL-86).

Cell and culture condition

All cells is at 37 ℃ and 5%CO ₂Standard conditions under in moistening incubator, cultivate.Clone LP-1, RPMI8226, Jurkat and Raji are being added with 10%FCS (PAN biotech GmbH; #P30-3302), 50U/ml penicillium mould, 50 μ g/ml Streptomycin sulphate (Gibco; #15140-122) with 2mM Stimulina (Gibco; RPMI1640 #25030-024) (Pan biotech GmbH, #P04-16500) the middle cultivation, and Jurkat cell and Raji cell also are added with 10mM Hepes (Pan biotechGmbH; #P05-01100) with the 1mM Sodium.alpha.-ketopropionate (Pan biotech GmbH, #P04-43100).

CHO-K1 and HEK293 be grown in the DMEM that is added with 2mM Stimulina and 10%FCS (Gibco, #10938-025) in.(PAA GmbH keeps under situation P11-012) stable CD38CHO-K1 transfectant, and HEK293 also must add the 1mM Sodium.alpha.-ketopropionate there being G418.(Invitrogen #16250-078) replaces 10%FCS with utmost point low IgG FCS after the of short duration transfection HEK293.Clone OKT10 cultivate the IDMEM that is added with 2mM Stimulina and 20%FCS (Gibco, #31980-022) in.

Prepare single cell suspension from peripheral blood

The whole blood sample obtains after obtaining agreeing.With

-1077 (Sigma) according to the explanation of manufacturers from healthy donors separating periphery blood monocytic cell (PBMC).At ACK lysis buffer (0.15MNH ₄Cl, 10mM KHCO ₃, 0.1M EDTA) in cultivated 5 minutes or (Bioscience #00-4333) removes red blood cell in these cell suspensions with the commercially available prod in room temperature.Cell is used the PBS washed twice, further handles then to be used for flow cytometry or ADCC (seeing below).

Flow cytometry (" FACS ")

All dyeing are carried out in culture plate at the bottom of 96 hole circles (Nalge Nunc), wherein contain 2 * 10 in each hole ⁵Individual cell.With cell with shown in Fab or the IgG antibody of concentration at 50 μ l FACS damping fluid (PBS, 3%FCS, 0.02%NaN ₃) in cultivated 40 minutes in 4 ℃.With cell washing twice, then with R-phycoerythrin (PE) the link coupled goat-anti people or sheep anti-mouse igg (H+L) F (ab ') that are diluted in the FACS damping fluid with 1:200 ₂(Jackson Immune Research) cultivated 30 minutes in 4 ℃.Washed cell is resuspended in 0.3ml FACS damping fluid with it once more, and (BectonDickinson, San Diego pass through flow cytometry in CA) at FACSCalibur then.

For carrying out analyzing, use from 12 kinds of initial different dilution (1:2 of 12.5 μ g/ml (IgG) final concentrations based on the Scatchard of FACS ⁿ) painted.To each concentration carry out at least twice independently measure and according to the method for (1994) such as Chamow from fluorescence intensity intermediate value extrapolation KD value.

Surface plasma resonance

Adopt BIAcore3000 equipment (Biacore, Uppsala, Sweden) to confirm kinetic constant kon and koff with the serial dilution of the various Fab of the CD38-Fc fusion rotein that is attached to Covalent Immobilization.Fix in order to carry out covalency antigen, use standard EDC-NHS amine coupling chemistry.For direct coupling CD38Fc-fusion rotein, in 10mM acetate buffer (pH4.5), be coated with CM5 transmitter (senor) chip (Biacore) with about 600-700RU.For the reference flow cell, use the HSA (human serum albumin) of various amounts.Kinetic measurement is at PBS (136mMNaCl, 2.7mM KCl, 10mMNa ₂HPO0 ₄, 1.76mM KH ₂PO ₄PH7.4) flow velocity with 20 μ l/ minutes in carries out, and the Fab concentration range is 1.5-500nM.Be 1 minute the inject time of each concentration, dissociated then 2 minutes.Use 5 μ l10mM HCl regeneration.With all sensing figure (sensogram) of BIA assessment software 3.1 (Biacore) local fit.

Embodiment 1: produce antibody from the HuCAL library

For producing the treatment antibody of anti-CD38, select with MorphoSys HuCAL GOLD phage display library.HuCAL

Be a kind of based on

Notion (Knappik etc., 2000; Krebs etc., 2001) Fab library, wherein all 6 CDR are diversified, and it adopts CysDisplay ^TMTechnology is connected to phage surface (L with the Fab fragment

Hning, 2001).

A. phagemid is collected, phage amplification and purifying

HuCAL

phagemid library is in the 2 * TY substratum that contains 34 μ g/ml paraxin and 1% glucose (amplification in 2 * TY-CG).At OD600 is to infect helper phage (VCSM13) (37 ℃ of nonoscillatory 30 minutes at 0.5 o'clock; 37 ℃ with 250rpm vibration 30 minutes), then cell is got rid of down (4120g; 5min; 4 ℃), be resuspended in 2 * TY/34 μ g/ml paraxin/50 μ g/ml kantlex, and in 22 ℃ of grow overnight.Precipitate phage from supernatant with PEG, be resuspended in the PBS/20% glycerine, and in-80 ℃ of storages.Carry out the phage amplification between the two-wheeled elutriation (panning) as follows: the mid-term-logarithmic phase TG1 cell is with the phage-infect of wash-out and be applied on the LB-agar (LB-CG) that is added with 1% glucose and 34 μ g/ml paraxin.After 30 ℃ of cultivations are spent the night, scrape colony, OD600 is adjusted to 0.5 also adds helper phage as stated.

B. use HuCAL elutriation

In order to select; According to different VH key-genes HuCAL antibody-phage is divided into 3 parts (the 1st part: VH1/5 λ κ; The 2nd part: VH3 λ κ, the 3rd part: VH2/4/6 λ κ).Express at CD38 for these several parts and carry out 3 separately on the CHO-K1 cell and take turns full cell elutriation, carry out the pH-wash-out then and on the negative CHO-K1 cell of CD38, adsorb the back step to remove irrelevant antibody-phage.Come ehec infection TG1 cell with remaining antibody phage at last.Bacterial mass is resuspended in 2 * TY substratum after centrifugal, coats agar plate and spend the night 30 ℃ of cultivations.Scrape the clone who selects from flat board then, collect phage and amplification.Carrying out second as the first round selects with third round.

The Fab coding insertion fragment subclone of selected HuCAL

phage is gone into expression vector x9_Fab_FS (Rauchenberger etc., 2003) so that express solubility Fab fast.Thereby selected clone's DNA cuts the Fab coding with XbaI and EcoRI digestion and inserts fragment (ompA-VLCL and phoA-Fd), and is cloned among XbaI/EcoRI cut vector

x9_Fab_FS.The Fab that expresses in this carrier has the terminal label (FLAG of two C- ^TMh II) for detecting and purifying.

Embodiment 2: biological assay

Measure ADCC (ADCC) and CDC according to disclosed method (Naundorf etc., 2002) based on flow cytometry, as follows:

ADCC：

In order to measure ADCC, target cell (T) is adjusted to 2.0E+05 cell/ml, and (Molecular Probes, C-3099) mark is 2 minutes with 100ng/ml fluorexon AM in RPMI1640 substratum (Pan biotech GmbH) in room temperature.In the RPMMI1640 substratum, wash 3 times to remove remaining fluorexon.Simultaneously, preparation PBMC is adjusted to 1.0E+07 and mixes with the target cell of mark as (NKT) effector cell's (E) source, and making final E:T ratio according to condition determination is 50:1 or lower.Cell washing once and with cell mixture is resuspended in the RPMI1640 substratum that 200 μ l contain different dilution various antibody.Dull and stereotyped at 37 ℃ and 5%CO ₂Standard conditions under in moistening incubator, cultivated 4 hours.Analyze with iodate third ingot (PI) labeled cell and through flow cytometry (Becton-Dickinson) before the facs analysis.Each incident of measuring between the counting 50.000 and 150.000.Calculate killing activity [%] with following formula:

Wherein, ED ^A=dead cell incident (fluorexon+PI staining cell) and

EL ^A=survivaling cell incident (fluorexon staining cell)

CDC：

In order to measure CDC, (Sigma is #S-1764) with various antibody for the human serum of adding 5.0E+04CD38CHO-K1 transfectant and 1:4 dilution in microtitre orifice plate (Nunc).With all reagent and cell dilution in the RPMI1640 substratum that is added with 10%FCS (Pan biotech GmbH).With this reaction mixture at 37 ℃ and 5%CO ₂Standard conditions under in moistening incubator, cultivated 2 hours.Negative control is heat-inactivated complement or the CD38-transfectant that does not contain antibody.Cell is with the PI mark and carry out facs analysis.

Count 5000 incidents altogether, and confirm the EC50 value with the number of dead cell under the different antibodies concentration.Calculate killing activity [%] with following formula:

Wherein, ED ^C=dead cell incident (PI staining cell) and

EL ^C=survivaling cell incident (undyed)

With regard to every kind of antibody, always have 12 kinds of different antibody diluent (1:2 ⁿ) cytotoxicity values be used for ADCC in triplicate, be used for CDC in duplicate, so as with standard analysis software ( Graph Pad Software) obtains the EC-50 value.

Embodiment 3: produce stable CD38-transfectant and CD38Fc fusion rotein

In order to produce CD38 albumen, have to set up two kinds of different expression systems to be used for elutriation and screening.First method comprises and produces the CD38-Fc fusion rotein, this albumen behind of short duration transfection HEK293 cell from supernatant purifying.Second method comprises that the stable CHO-K1-clone of generation is to select antibody-phage with high CD38 surface expression through full cell elutriation.

Initial step is to produce cDNA (Invitrogen) with Jurkat cell (DSMZ ACC282), use then respectively with CD38 preceding 7 with back 9 codon complementary primers (primer MTE001 and MTE002rev; Table 4) the complete CD38 encoding sequence of amplification.The sequential analysis of CD38 insertion sequence confirms that the 49th of the disclosed aminoacid sequence of Jackson etc. (1990) be Stimulina, is different from the tyrosine of (1997) description such as Nata.In order to introduce the restriction endonuclease site and to be cloned in the different verivates of expression vector pcDNA3.1 (Stratagene); With the PCR product of purifying as template increase again its complete genome (primer MTE006 and MTE007rev; Table 4) or portion gene (primer MTE004 and MTE009rev, table 4).Under latter event, the fragment of coding extracellular domain (amino acid 45-300) is increased and it is cloned in the framework between people V κ ladder sequence and people Fc-γ 1 sequence.This carrier is to produce solvable CD38-Fc Expression of Fusion Protein carrier.The another kind of pcDNA3.1 verivate that does not contain leader sequence is used to be inserted in the CD38 full-length gene.At this moment, the terminator codon before the Fc coding region and the leader sequence of disappearance make CD38 at surface expression.With the of short duration transfection HEK293 of Fc fusion rotein carrier cell producing soluble CD38-Fc fusion rotein, and if the total length verivate then transfection CHO-K1 cell to produce stable CD38 express cell system.

Table 4:

^*Produce terminator codon (TGA) in just direction.

Embodiment 4:

clone, expression and the purifying of IgG1:

In order to express total length IgG, the variable domains fragment of heavy chain (VH) and light chain (VL) is gone into suitable

_ hIg carrier (seeing Fig. 8-10) from Fab expression vector subclone.Adopt restriction endonuclease that BlpI/MfeI (fragment is inserted in preparation) and BlpI/EcoRI (preparation carrier) are gone into

_ hIgG1 with VH structural domain fragment subclone.With endonucleases EcoRV / HpaI (λ insert) and EcoRV / BsiWI (κ insert) the VL domain fragment was subcloned into the respective carrier?

_hIgκ_1 or _h_Igλ_1 in.The of short duration transfection of employing standard calcium phosphate-DNA coprecipitation technology so that resulting IgG construct in HEK293 cell (ATCC CRL-1573), express.Pass through affinity chromatography IgG purification from the supernatant of cell culture with a-protein sepharose post.Ensuing processing comprises the IgG that carries out buffer-exchanged and sterile filtration purifying through gel-filtration.Quality control shows purity>90% (measuring), monomer I gG through reductibility SDS-PAGE>90% (measuring) through the analysis mode size exclusion chromatography.((Cambrex European Endotoxin Testing Service, Belgium) confirms the endotoxin content of this material through the assay method based on kinetics LAL.

Embodiment 5: produce and prepare chimeric OKT10 (chOKT10; SEQ ID NO:23 and 24), use the cDNA that makes from mouse OKT10 hybridoma cell line (ECACC#87021903) to pass through VH and the VL district of pcr amplification mouse for making up chOKT10.Adopt disclosed one group of primer (Dattamajumdar etc., 1996; Zhou etc., 1994).The PCR product is used for Topo-clone (Invitrogen; The pCRII-carrier) and to mono-clonal carry out sequential analysis (M13 reverse primer), analytical results shows two kinds of different κ sequence of light chain and a kind of sequence of heavy chain.According to sequence alignment (EMBL nucleotide sequence database) and document (Krebber etc., 1997), therefore the internal component that one of κ sequence belongs to tumour cell fusion partner X63Ag8.653 does not belong to OKT10 antibody.Therefore have only a new κ sequence and a VH fragment further to be cloned.These two fragments are all increased with insertion restriction endonuclease site again, and then are cloned into

IgG1-expression vector separately.The sequence of heavy chain (SEQ ID NO:23) and light chain (SEQ ID NO:24) is shown in Fig. 6.Of short duration transfection HEK293 cell and with crossing the chimeric OKT10 antibody of expression Raji clone (ATCC) bonded with CD38 in the facs analysis supernatant.

Embodiment 6: epitope mapping

1. material and method:

Antibody:

Below anti-CD38IgG be used to epitope mapping:

MOR#	LOT#	Form	Concentration [mg/ml]/volume [μ l]
				MOR03077	2CHE106_030602	The human IgG1	0.44/1500
MOR03079	2APO31	The human IgG1	0.38/500
				MOR03080	030116_4CUE16	The human IgG1	2.28/200
MOR03100	030612_6SBA6	The human IgG1	0.39/500
				Chimeric OKT10 *	030603_2CHE111	The human IgG1	0.83/500

^*Chimeric OKT10 is made up of people Fc and mouse variable region.

The CD38 sequence:

Amino acid (aa) sequence (44-300 position) is based on people CD38, and it is the open sequence of P28907 available from SWISS-PROT master's accession number.The Q (rather than T) that amino acid is the 49th is used to the peptide design.

PepSpot analyzes:

In a step-wise fashion obtain predetermined arrangement (peptide array) and antigen peptide is covalently bound on the cellulose membrane analyzing antigen peptide on the cellulose membrane.On the peptide array, directly combine test.Usually the antigen peptide array is cultivated several hours to reduce the non-specific binding of antibody with the sealing damping fluid.Cultivate with elementary (antigen peptide combination) antibody in the damping fluid in sealing, cultivate with the secondary antibodies of px (POD) mark of selective binding primary antibody then.With the antigen peptide array after secondary antibodies is cultivated directly with T (Tween)-of short duration washing of TBS damping fluid, carry out first chemoluminescence test then and obtain first overview of which antigen peptide combination primary antibody.Next combine (being attached to the non-specific antibody on the cellulose membrane) with damping fluid washing several (T-TBS damping fluid and TBS damping fluid) to remove false positive again.Carry out final chemiluminometry after these washing steps.Imaging system (Boehringer Light units, the BLU) analytical data that shows strength of signal used in single measurement as to each peptide.In order to estimate the non-specific binding of secondary antibodies (anti-human IgG), these antibody are cultivated with the peptide array as first step under the situation that does not contain primary antibody.If this primary antibody does not demonstrate combining of any and this peptide then can directly use the POD mark, can increase the sensitivity (as MOR3077 is carried out) of this system like this.In this case, carry out conventional coupling chemistry through total free aminoacids.Gather peptide (11 amino acid are overlapping) scanning antigen with 13.Can produce the array of 123 peptides like this.Directly carry out on array in conjunction with test.With the secondary antibodies of peroxidase labelling (px link coupled goat anti-human igg, the γ chain is special, affine isolated antibody; Sigma-Aldrich, A6029) detection of peptides bonded antibody MOR03077, MOR03079, MOR03080, MOR03100 and chimeric OKT10.Map with chemiluminescent substance and imaging system.In addition, with the sensitivity of the direct mark MOR03077 of POD with the raising system.

2. sum up and conclusion:

All 5 kinds of antibody show different curves in PepSpot analyzes.Fig. 7 has provided synoptic diagram, wherein can find out the different aminoacids sequence of CD38.Can know that the epi-position of finding out MOR03079 and chimeric OKT10 is a line style.The epi-position of inferring MOR03079 is between the amino acid/11 92-206 of CD38 (VSRRFAEAACDVVHV), and the epi-position of chimeric OKT10 mainly is positioned between the amino acid 284 and 298 (FLQCVKNPEDSSCTS).A kind of result in back has confirmed the public data (Hoshino etc., 1997) of parent mouse OKT10, and these data are inferred its epi-position between amino acid 280-298.Yet, confirm and the confirming of key amino acid (the main interaction sites of antigen-antibody) for accurate more epi-position, can adopt the reduced form of peptide VSRRFAEAACDVVHV and FLQCVKNPEDS SCTS and the two is carried out L-Ala and scan.

Because some peptides have covered different protein positions, so the epi-position of MOR03080 and MOR03100 is discrete beyond doubt.These peptides comprise the amino acid 82-94 of MOR03080 and amino acid 82-94,142-154,158-170,188-200 and the 280-296 of amino acid/11 58-170 and MOR03100.Yet some that can infer between these two epi-positions are overlapping, because two antibody are all discerned amino acid position 82-94 (CQSVWDAFKGAFI; Peptide #20) and 158-170 (TWCGEFNTSKINY; Peptide #58) two different loci in.

Both also can be described as the discontinuous epi-position of more piece (multisegmented) after the epi-position of MOR03077 obviously was different from.Its epi-position comprises amino acid 44-66,110-122,148-164,186-200 and 202-224.

Embodiment 7:IL-6-release/proliferation test

1. material and method:

Improve below doing according to the method for (2000) such as Ausiello and breed release test with IL-6: adopt Histopaque cellular segregation system according to supplier's (Sigma) explanation through the PBMC of density gradient centrifugation purifying, and at standard conditions (5%CO from healthy donors (obtain agreement after) ₂, 37 ℃) under in being added with the RPMI1640 substratum of 10%FCS and Stimulina (" fully RPMI1640 ") culturing cell.Following antibody is used in these two tests: anti-CD38IgG1 monoclonal antibody MOR03077, MOR03079 and MOR03080, excitability mouse IgG2a monoclonal antibody (IB4; Malavasi etc.; 1984); Irrelevant

IgG1 antibody, isotype contrast (the mouse IgG2a: anti-trinitrophenol, hapten specificity antibody of coupling; Numbering: 555571, the G155-178 clone; BectonDickinson) or substratum contrast.In order to carry out the IL-6 release test, the 1.0E+06PBMC in the complete RPMI1640 substratum cultivated 24 hours in 15ml culture test tube (Falcon) with 0.5ml when having 20 μ g/ml antibody.Harvested cell culture supernatant also discharges with the methods analyst IL-6 of Quantikine test kit according to manufacturers (R&D systems).In order to carry out proliferation test, when having 20 μ g/ml antibody, 2.0E+05PBMC was cultivated 3 days in the flat flat boards in 96 holes (Nunc).Every kind of test is carried out twice.In each hole, add BrdU after 4 days, cell was cultivated 24 hours at 37 ℃ again, then fixed cell and make the DNA sex change according to supplier's (Roche) method.In based on chemiluminescent device, measure mixing of BrdU through anti-BrdU px coupling antibody.

2. sum up and conclusion:

Proliferation test:

Except the catalytic activity with ring-type ADP-ribose cyclase and lytic enzyme, CD38 demonstrates biological relevant signal transduction ability (Hoshino etc., 1997; Ausiello etc., 2000).Those functions can be through for example receptor-ligand binding or through inducing in vivo with anti-CD38 is antibody linked.Those signal generation incidents cause for example calcium motion, lymphopoiesis and cytokine release.Yet sort signal takes place not only to depend on antigenic epitopes, and between donor and donor, also can change (Ausiello etc., 2000).It seems from the viewpoint of immunotherapy, compare agonistic antibody, more preferably non-agonistic antibody.Therefore; In proliferation test and IL-6 (important MM growth factor) release test, further characterize anti-CD38 antibody (Mab MOR03077, MOR03079, MOR03080), they and reference antibody chOKTI0 and the anti-CD38 monoclonal antibody of excitability IB4 are compared.

Like Figure 11 and shown in Figure 12, compare agonistic antibody IB4, the anti-CD38 antibody of HuCAL Mab# 1,2 and 3 and reference antibody chOKT10 and corresponding negative control show do not have or weak proliferation-inducing is only arranged, and do not have IL-6 to discharge.

Embodiment 8: the clone generates test

1. material and method:

Adopt Histopaque cellular segregation system to comprise PBMC through density gradient centrifugation from healthy individuals (after obtaining agreeing) separation, and the HuCAL different with the 10 μ g/ml anti-CD38 antibody of

IgG1 (Mab MOR03077, MOR03079 and MOR03080) and positive control (PC) chOKT10 cultivate together from body CD34+/CD38+ precursor cell according to supplier's (Sigma) explanation.Substratum and irrelevant HuCAL

IgG1 contrasts as a setting.Each ADCC test comprises cultivates 4.0E+05PBMC 4 hours in 37 ℃ in being added with the RPMI1640 substratum of 10%FCS.To generate test in order cloning, to go up inoculation 2.5E+05 from the cell of ADCC test and (37 ℃ of controlled environments at 2.50ml " fully " methylcellulose gum (CellSystems); 5%CO ₂) cultivate at least 14 days down to form colony.Colony by two independently the operator analyze and be divided into BFU-E+CFU-GEMM (burst forming unit-erythrocyte and granulocyte/erythroid cells/scavenger cell/megalokaryocyte stem cell) and CFU-GM (granulocyte/scavenger cell stem cell).

2. sum up and conclusion:

Because not only at marrow (for example monocyte, granulocyte) and lymph pedigree (for example activatory B and T cell; Plasmocyte) found the expression of CD38 on the immunocyte, also on precursor cell (CD34+/CD38+) separately, found the expression of CD38, thus those cells not influenced by antibody-mediated killing and wounding very important.Therefore, adopt the product clonogenic assay to analyze influence to the CD34+/CD38+ precursor.To cultivate with

anti-CD38 antibody (Mab#1, Mab#2 and Mab#3) or some contrasts (irrelevant

antibody, substratum and as the reference antibody chOKT10 of positive control) from the PBMC of healthy donors according to standard A DCC method, and on wetting methylcellulose gum, continue then to cultivate to produce colony.Shown in figure 13; Compare irrelevant antibody or reference antibody, the colony forming unit of all anti-CD38 antibody does not significantly reduce.

Embodiment 9: the ADCC that carries out with different clones and primary multiple myeloma cells tests

1. material and method:

The separation of MM patient's sample and ADCC: obtain the marrow aspirate from multiple myeloma patients (after obtaining agreeing).Density gradient centrifugation (Sigma) back through standard method with anti-CD38 magnetic bead (Milteny Biotec) purifying malignant cell.Carry out the ADCC test by the description of preceding text.

2. sum up and conclusion:

Some clones derived from different malignant tumours are used to ADCC to show that

anti-CD38 antibody is to comprise the cellulotoxic effect of the different sources and the clone of different CD38 expression levels to various kinds of cell.Shown in figure 14, all cells is is all killed and wounded by ADCC when constant AC (5 μ g/ml) and E:T ratio are 30:1.Some multiple myeloma samples of patient also demonstrate the cytotoxicity that produces through ADCC.All

anti-CD38 antibody can kill and wound the MM cell dose-dependently, its EC50 value 0.006 and 0.249nM between (Figure 15).

Embodiment 10: carry out the cross reactivity analysis through FACS and immunohistochemistry (IHC)

1. material and method:

Amygdaline IHC: in order to carry out IHC;

anti-CD38Mab and irrelevant negative control antibody are transformed into divalence dHLX form (Pl ü ckthun&Pack, 1997).With Leica CM3050 cryostat cut from stump-tailed macaque, rhesus monkey and people (from Graz, Austria university pathological research archives find) 5 μ m freezing microtome sections of lymphoglandula.The dry air of will cutting into slices 30 minutes to 1 hour, in ice-cold methyl alcohol fixing 10 minutes and wash with PBS.For detecting the dHLX form, mouse anti His antibody (Dianova) and Envision test kit (DAKO) are used in combination.In order to detect anti-CD38 mouse antibodies (for example with reference to mouse monoclonal OKT10), only use the Envison test kit.

Lymphocytic facs analysis: obtain the blood sample of EDTA processing and carry out density gradient centrifugation according to supplier's (Sigma) explanation with Histopaque cellular segregation system from healthy human (after obtaining agreeing), rhesus monkey and stump-tailed macaque.In order to carry out facs analysis; (HuCAL anti-CD38 and negative control Mab are as mouse IgG2a or Fab form with intermitotic cell and primary antibody; The isotype contrast of positive control murine antibody OKT10 and coupling) cultivates together, then with anti-M2Flag (Sigma; Only be used for the Fab form) and the anti-mouse conjugate (Jackson Research) of phycoerythrin (PE) mark cultivate together.On the gate lymphocyte populations, carry out facs analysis.

2. sum up and conclusion:

CD38 cross reactivity between the anti-CD38 of analysis HuCAL plants with understanding.In FACS and IHC, all anti-CD38Mab can both detect the people CD38 on the lymphocyte, have only MOR03080 and positive control OKT10 and stump-tailed macaque and rhesus monkey CD38 extra reactivity to be arranged (with table 5: the cross reaction analysis).

Table 5:

++: strong positive dyeing;-: dye-free; NC: negative control; PC: positive control (promptly with reference to cMAb)

Embodiment 11: handle the human myeloma heterograft in the mouse (using RPMI8226 clone) with MOR03080

1. set up subcutaneous mouse model:

By Aurigon Life Science GmbH (Tutzing; Germany) in female C.B-17-SCID mouse, set up the subcutaneous mouse model of the tumor cell line RPMI8226 of human myeloma deutero-; Method was following: at the-1,0 and 1 day; The anti-asialo GM1 polyclonal antibody (ASGM) that intravenously gives to remove different reactivity (xenoreactive) the NK cell in the SCID mouse (WAKO-Chemicals) so that eliminate any residual specific immune response in the C.B-17-SCID mouse.To use 5 * 10 of 50 μ l PBS preparation at the 0th day ⁶Or 1 * 10 ⁷The subcutaneous vaccination of RPMI8226 tumour cell is to the right rib (every group of 5 mouse) with ASGM (as stated) processing or untreated mouse.The tumor development situation of all 4 inoculation group all is similarly, with maybe need not anti-asialo GM1 antibody treatment or inoculate with different cell count and not find significant difference.As if tumour stagnates with size or the trend of swing is slowly grown a couple of days.The size of two tumours swings back and forth during whole research, and a tumour is arranged even from 321mm ³Peak volume completely dissolve.Every group of tumor inoculation animal that a greater number should be arranged studied in processing with this tumor model carries out.

2. handle with MOR03080:

2.1 research purpose

(Tutzing Germany) carries out antibody ( anti-CD38) that this research uses with the intraperitoneal relatively antitumor efficacy with respect to vehicle treatment (PBS) by Aurigon LifeScience GmbH.With different amounts and regimen test person antibody hMOR03080 (isotype IgG1).Tested chimeric antibody chMOR03080 (isotype IgG2a: use with embodiment 5 and make up the chimeric antibody that contains MOR03080 variable region and mouse constant region that the similar method of chimeric OKT10 (mouse VH/VL and human constant region) makes up) in addition.Select the RPMI8226 cancerous cell line also as stated female SCID mouse to be arrived in its subcutaneous vaccination as model.Judge the research terminal point according to body weight (b.w.), gross tumor volume and clinical symptom.

2.2 antibody and carrier

Antibody offers Aurigon with ready-to-use form, and its concentration is 2.13mg/ml (MOR03080hIgG1) and 1.73mg/ml (MOR03080chIgG2a), and in-80 ℃ of storages up to use.Antibody is thawed and is diluted to final concentration separately with PBS.Vector (PBS) offer Aurigon with ready-to-use form and in 4 ℃ of storages up to use.

2.3 animal specification

Kind: mouse

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

Quantity and sex: 75, female

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

Healthy state: SPF

Body weight: about 18g

Domestication: 9 days

2.4 tumor cell line

Growth tumour cell (RPMI8226 clone) also is sent to Aurigon Life ScienceGmbH, in Aurigon somatoblast and cell cycle of regrowth.Aurigon prepared the cell of injection the same day in inoculation.The substratum that is used for propagated cell is the RPMI1640 that is added with 5%FCS, 2mM L-glutaminate and PenStrep.Cell shows does not have undesirable speed of growth or behavior.

In order to inoculate, tumour cell to be suspended in PBS and with PBS final concentration to be transferred to 1 * 10 ⁷Cell/50 μ l.Thorough mixing tumour cell suspension before the injection.

2.5 process of the test

At the 0th day, with 1 * 10 ⁷The right side back of the body rib of RPMI8226 tumour cell subcutaneous vaccination to 75 a SCID mouse.After inoculation, directly select 15 animals to form first group (group 5) at random.This group was handled with 1mg/kgb.w.hIgG1-MOR03080 at 14-36 days every other day.Other 60 animals were divided into 4 groups with all at the 31st day, every group of 10 animals (about 92mm of gross tumor volume ³).Group 1-4 is made up of the average tumor size animal suitable with standard deviation.Selection demonstrates less relatively gross tumor volume, and (gross tumor volume is about 50mm ³) 5 animals form other one group (group 6) in case with pretreated group 5 compare that (except that 3, the gross tumor volume of all mouse is less than 10mm ³, an about 22mm ³, an about 44mm ³, also have an about 119mm ³).Group 1-4 used PBS (vector every other day at 32-68 days; Group 1), 1mg/kgb.w.hlgG1-MOR03080 (group 2), 5mg/kg b.w.hlgG1-MOR03080 (group 3) or 5mg/kgb.w.chIgG2a-MOR03080 (group 4) handle.Group 6 is not accepted any processing (seeing table 6).Gross tumor volume, body weight and clinical sign are measured twice weekly up to the research end.

Table 6:

Group	Number of animals	Form of medication	Material	Scheme	Therapeutic dose [mg/kg]	Administration volume [μ l/kg]
							1	10	i.p.	Vector (PBS)	At 32-68 days every other day once	-	10
2	10	i.p.	The MOR03080 human IgG1	At 32-68 days every other day once	1	10
							3	10	i.p.	The MOR03080 human IgG1	At 32-68 days every other day once	5	10
4	10	i.p.	The chimeric IgG2a of MOR03080	At 32-68 days every other day once	5	10
							5	15	i.p.	The MOR03080 human IgG1	At 14-36 days every other day once	1	10
6	5	-	-	-	-	-

2.6 result

Clinical discovery is with deadly

Do not observe the clinical discovery relevant or deadly with specific tumors or material.In group 3 (hIgG15mg/kg), 4 animals are dead when blood sample collection, and (1 at the 3rd day, and 1 at the 34th day; 2 at the 52nd day).In group 4 (muIgG2a1mg/kg), 4 animals dead (the 34th day) when blood sample collection.During studying all dead other animals all be since tumour size (excessive) by euthansia.

Weight increase

Compare and do not observe the interference to weight increase relevant with group 1 (carrier) with medicine.In group 3 (hIgG15mg/kg) and 4 (muIgG2a5mg/kg), body weight significantly receives the influence of blood specimen collection.Except this influence, the mean body weight increase of all groups all is a successive.

Tumor development (seeing Figure 16)

In group 1 (vector), find that tumor growth slowly develops with goal pace.Because this clone has clear and definite standard deviation value, therefore minimum and maximum tumour is excluded to carry out further statistical study.The tumor growth of the animal of group 1 is suitable with the tumor growth of group 6 (being untreated), is to be made up of the less animal of mean tumour volume at the 31st day although organize 6.Therefore, treatment has only minimal effect to tumor growth rate.In group 1,2 mouse since the tumour size before the 83rd day by euthansia, other have 1 before the 87th day by euthansia, so the MV of gross tumor volume no longer included representativeness after the 80th day.In group 6,1 mouse since the tumour size before the 80th day by euthansia, 2 before the 83rd day, in addition 1 before the 87th day by euthansia, so the MV of gross tumor volume no longer included representativeness after the 76th day.

In the group 2 that the hIgG1 with 1mg/kg b.w. handles, 1 animal is owing to tumor growth is excluded from further analysis to muscle tissue interior (this can improve tumor growth rate usually).Compare with control group 1 (vector), the average tumor size is significantly different since the 45th day to be finished up to research.Treatment finishes not observe the tumor growth quickening after (the 68th day).

Compare group 1 (vector), the animal of group 3 (5mg/kg b.w.hIgG1) demonstrated tumor growth and significantly reduces, and rising at the 38th day all had significance,statistical up to the 83rd day.Treatment finishes back about 2 all mean tumour volumes and begins to regrow strongly.In 10 tumours 1 disappeared at the 45th day, and did not all regrow in after treatment finishes the 19th day.

From 92mm ³The optimal representation of all treatment groups that gross tumor volume is initial appears at group 4 (5mg/kgb.w.muIgG2a), and its mean tumour volume obviously reduces, and tumour even the disappearance in 4 animals when the observation period finishes.Finish up to research since the 38th day highly significant with the difference of the mean tumour volume of organizing 1 (vector).

The early stage research of carrying out with 1mg/kg b.w.hIgG1 at 14-36 days (group 5) shows that tumor development is had early stage and long-term effect.1 animal since tumor growth in muscle tissue and from further analysis, be excluded.At the 31st day, compare with other inoculation animal, have only 5 animals measurable tumour to be arranged at inoculation position, and have only in 60 animals 2 reactionless to tumor inoculation.Tumour progression is postponed about 31 days (5 the 83rd days situation of the 52nd day situation of control group 1 and group is compared).About 50% animal does not demonstrate tumour at inoculation position when research finishes.

2.7 conclusion

Compare group 1 (contrast), do not observe the clinical discovery relevant or deadly with specific tumors or material.

Do not observe the interference to weight increase relevant with medicine.

Effect order following lower of the tumor growth of RPMI8226 tumour cell after treatment: hIgG11mg/kg; Handled (group 5) in 14-36 days every other day>muIgG2a5mg/kg; Handled (group 4) in 32-68 days every other day>hIgG15mg/kg, handled (group 3) in 32-68 days every other day>hIgG1 1mg/kg32-68 days handles (group 2) every other day.In group 2-4, mean tumour volume is increased to variable pitch once more after treatment finishes.

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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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