CN101331151A - Means and methods for treating neoplastic diseases - Google Patents

Means and methods for treating neoplastic diseases Download PDF

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Publication number
CN101331151A
CN101331151A CNA2006800471624A CN200680047162A CN101331151A CN 101331151 A CN101331151 A CN 101331151A CN A2006800471624 A CNA2006800471624 A CN A2006800471624A CN 200680047162 A CN200680047162 A CN 200680047162A CN 101331151 A CN101331151 A CN 101331151A
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cell
bispecific single
ser
chain antibody
district
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P·鲍尔勒
P·库弗
M·克灵格尔
E·里奥
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Amgen Research Munich GmbH
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Micromet GmbH
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Abstract

Means and methods are provided for preventing, treating or ameliorating indolent or aggressive B-cell non-hodgkin's lymphoma (BNHL) and B-cell leukemia, comprising administering to a patient in need thereof a bispecific single chain antibody construct; there is provided the use of said bispecific single chain antibody construct for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of indolent or aggressive B-cell non-hodgkin's lymphoma (B NHL) and B-cell leukemia, wherein said construct is administered in a specified daily dose for at least 1 week. Furthermore, the present invention relates to a kit comprising a bispecific single chain antibody construct to be used according to the present invention.

Description

The mode and the method for treatment neoplastic disease
The present invention relates to based on pharmacy mode and method to the immunity medical intervention of patient's administration of antibodies construct especially dual specific strand construct.Particularly, the present invention relates to treat the pharmacy mode and the method for inertia (indolent) or rapid spread (aggressive) B cell non-Hodgkin's (B NHL) or B cell leukemia, wherein said mode and method relate to uses bispecific single-chain antibody.The invention further relates to the purposes of bispecific single-chain antibody in the medicine of preparation treatment inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia.
Therapy based on antibody is widely used in the treatment human diseases.This type of treatment plan generally need be used repeatedly the Antybody therapy agent of heavy dose of infusion, promptly during whole treatment off and on repeatedly, be generally that intravenously ground (i.v.) and high dosage ground injection of antibodies-according to the online dictionary of Webster, " heavy dose of infusion (bolusinfusions) " expression is injected into the single dose medicine of blood vessel usually at short notice; Also referring to Www.nlm.nih.gov/medlineplusWith Www.phoenix5.org/glossaryFor example, repeatedly with dosage administering therapeutic recurrent or intractable minuent or folliculus CD20 weekly +The mono-clonal anti-CD20 antibodies Rituximab (Rituximab) of B cell non-Hodgkin's (NHL) (Mabthera (Rituxan)) surpass 4-8 week (Ghielmini M., J.Clin.Oncol.2004).Monoclonal antibody Ah the coming that heavy dose of infusion pattern also is described in the humanized anti-CD 52 that is used for chronic lymphocytic leukemia organizes monoclonal antibody (Alemtuzumab) (O ' Brian, 2003, Cancer, 98,2657-63), Herceptin (the Trastuzumab) (Trastuzumab (Herceptin) that is used for metastatic breast cancer, anti-Her2 antibody), lucky trastuzumab (the Gemtuzumab) (Mai Luota (Myelotarg) that is used for acute marrow sample leukemia (AML), anti-CD 33 antibody), Ah the coming who is used for B cell chronic lymphocytic leukemia (CLL) organizes monoclonal antibody (Campath, anti-CD 52 antibody) and be used for recurrent or intractable minuent, ibritumomab tiuxetan (the Ibritumomab) (Zevalin of the B cell NHL of folliculus or conversion, anti-CD20 antibodies) (summary is seen Cersosimo, 2003, Am.J.Health-Syst-Pharm., 60,1531-48).The monoclonal antibody Edrecolomab (Edrecolomab) of anti-17-1A is (Panorex) also as repeatedly infusion agent, per 28 days of wherein initial with high dosage (500mg), two week backs are through intravenous injection 100mg (Makower, 2003, Cancer Invest., 2,177-84).
Common phenomena is that the side effect relevant with infusion, for example release of cytokines syndrome (" CRS ") take place in antibody therapy.CRS replys the infusion of T cell bound antibody and the complication that takes place immediately.CRS is relevant with T cell/monocyte activation, and the activation with complement cascade is relevant then.These processes by can crosslinked T cell and the Fc of the antibody of monocyte and complement activation partly mediate.The pathogeny of CRS is owing to replying OKT3 to the lymphocytic stimulation of T and synthetic tumour necrosis factor (TNF) α, IL-2, IL-6 and gamma-interferon.Usually, this type of antibodies TXi Baoshouti, thereby activating T cell.Like the response class that the cytokine (for example, TNF α, interleukin-(IL-2, IL-6) and Interferon, rabbit (IFN γ)) that is discharged by the activated T cell produces and finds in severe infections and be characterized by the systemic inflammatory responses (systemic inflammatory response) of ypotension, fever and stiff type.It is very uncomfortable that the patient feels, as have a high fever-really, CRS is actually one type non-infectious fever.Described other side effect relevant with CRS is that fatigue, vomiting, heartbeat are overrun, hypertension, headache and backache.
When using multiple monoclonal antibody, also observe CRS.For example, utilize the anti-tumor activity of anti-CD20 antibodies Rituximab to treat B cell chronic lymphocytic leukemia (B-CLL).The dosage of finishing by time increment on every Wendesdays increase gradually promptly increase dosage to Rituximab as single agents bring into play remarkable clinical activity be essential (Lin, 2003, Seminar Oncol.30,483-92).Yet this application program triggers the release of cytokine TNF-α and IL-6, and wherein the blood plasma level of TNF-α and IL-6 reached peak value respectively in 90 minutes behind initial infusion.The rising of this cytokine be accompanied by fever, shiver with cold, ypotension and feel sick (Winkler, 1999, Blood, 94,2217-24).The toxicity of infusion can reduce by suitable pre-pharmaceutical preparation (pre-pharmaceutical agent) and the application program of strengthening (Lin, 2003, Seminar Oncol.30,483-492).
For example also observe CRS during bi-specific antibody when using other antibody formation.The bi-specific antibody MDX-2H12 (anti-Fc γ acceptor I * anti-Her-2/neu) of single infusion associating GCSF (granulocyte colony-stimulating factor) highest level that caused TNF-α and IL-6 in 2 and 4 hours behind infusion MDX-2H12 respectively in the patient with breast cancer.The peak level of TNF-α and IL-6 not relevant with the dosage of the bi-specific antibody of being used (Repp, 2003, Br.J.Cancer, 89,2234-43).
As described in the WO 99/54440, CRS is using the bispecific single-chain antibody of anti-CD19 * anti-CD30 and (is finding in the inside clinical study of bscCD19 * CD3) carry out to repeat infusion in the cell-derived property of B chronic lymphocytic leukemia (B-CLL) patient.Shown in Figure 19 and 20 of WO 99/54440, discovery is replied each infusion and is discharged TNF, IL-6 and IL-8, wherein said infusion is 20 minutes infusion of administered twice (being respectively 3 μ g and 10 μ g bispecific single-chain antibodies), follows and uses back release cytokine at every turn.After using 10 μ g bispecific single-chain antibodies, observe maximum release of cytokines.
People such as Ten Berge, 1996, Transplant.Proc.28,3217-20 have described two hours the research of monoclonal antibody OKT3 that relates to the anti-CD3 of continuous infusion.The purpose of this research is to determine whether the known thromboembolic complication complication relevant with using OKT3 depends on the mode that this molecule is used.Usually, using OKT3 by continuous infusion tolerates preferably than the heavy dose of infusion of single.
Continuous infusion also is described in the prior art of antibody related agents.For example, continuous infusion GD3 Sphingolipids,sialo monoclonal antibody specific R24 is mainly 25 and 50mg/m 2Dosage cause the relevant toxicity of R24 (Alpaugh, 1998, Med.Oncol., 15,191-8).The special people IgM monoclonal antibody infusion of twice 24 hours property GD3 of continuous infusion Sphingolipids,sialo 48 hours can be during infusion and is induced little side effect (Irie afterwards in suffering from the patient of metastatic melanoma, 2004, Cancer Immunol.Immunother.53,110-7).At this paper, the rank that need high therapeutic antibodies rate of application, promptly restrains antibody is finished clinical effectiveness.
In addition, the bi-specific antibody of the anti-CD16 * anti-CD30 that will produce as mouse IgG1 from hybridoma be applied to suffer from patient's the different infusion scheme of Hodgkin's disease (Hartmann, 2001, Clin.Cancer Res., 7,1873-1881).This antibody is by activating the NK cell in conjunction with CD16 (Fc-γ receptor II I).Infusion is to surpass 24 hours continuous infusion in continuous 4 days or to carry out with every other day 1 hour infusion.The absolute dosages of the bi-specific antibody of each infusion is 4 * 25mg in 5% human albumin solution.No matter which kind of application program is not observed consistent the variation, although the latter is completely different between individuality in the periphery cytometry behind the infusion bi-specific antibody.No matter the Antybody therapy scheme is continuously or intermittently, the patient is developed and fever.Can not alleviate the generation or the seriousness of the side effect that the patient suffers continuously and non-intermittently with heavy dose of infusion of therapeutic antibody.Discussed and for example reached effector cell's quantity of target and/or the increase of active state may also be essential for the anticancer effectiveness that increases the bi-specific antibody therapy by the while administer cytokines.Although in research mentioned above, use the therapeutic antibodies of total amount 100mg, be defined as the limited utilization ratio of bi-specific antibody but bi-specific antibody becomes the major obstacle of the therapy of success and widespread use.The author proposes, and can come to improve clinical efficacy by following approach very bigly, and described approach is: the dosage that 1) increases bi-specific antibody; With 2) increase treatment cycle quantity.
Therefore, need to reduce undesirable side effect.
Therefore, the objective of the invention is to provide the pharmacy mode and the method for the patient tolerability of tool increase for medical therapy based on antibody.Particularly, purpose is that the antibody that this type of mode and method allow to be used keeps maximum bioactivity, minimizes this not the wishing and disadvantageous side reaction of causing of using simultaneously.
The present invention relates to the purposes of bispecific single-chain antibody construct in the pharmaceutical composition for preparing prevention, treats or improve cancer or non-entity or solid tumor, wherein said bispecific single-chain antibody construct is used for using long period of time enduringly.
Therefore, first aspect of the present invention provides the purposes of bispecific single-chain antibody construct in the pharmaceutical composition for preparing prevention, treats or improve inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, wherein said bispecific single-chain antibody construct comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：2)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：4)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19) (SEQ ID NO.:6), perhaps
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)(SEQ ID NO.：8)，
And wherein said bispecific single-chain antibody construct used for 1 week with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface at least, and wherein said per daily dose was used above at least 6 hours.
In another embodiment, the present invention relates to prevent, treat or improve the method for inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, described method comprises to its individuality of needs uses the pharmaceutical composition that comprises the bispecific single-chain antibody construct, described bispecific single-chain antibody construct comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：2)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：4)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19) (SEQ ID NO.:6), perhaps
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)(SEQ ID NO.：8)，
Wherein said bispecific single-chain antibody construct used for 1 week with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface at least, and wherein said per daily dose was used above at least 6 hours.
In another embodiment, the present invention relates to medicine box, it comprises pharmaceutical composition and the specification sheets that is used for preventing, treating or improve the people inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, the bispecific single-chain antibody construct that wherein said pharmaceutical composition comprises comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：2)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：4)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19) (SEQ ID NO.:6), perhaps
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)(SEQ ID NO.：8)，
And described specification sheets has been described the application program of bispecific single-chain antibody construct, comprise with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface using at least 1 week, and wherein said per daily dose is used above at least 6 hours.
Another embodiment of the present invention relates to the medicine box that is used for prevention in the people, treats or improve inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, comprising using the bispecific single-chain antibody construct that comprises people CD3 and people CD19 specific combination structural domain, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：2)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：4)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19) (SEQ ID NO.:6), perhaps
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)(SEQ ID NO.：8)，
Wherein said bispecific single-chain antibody construct used for 1 week at least with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface, and wherein said per daily dose uses and surpass at least 6 hours, and wherein said medicine box contains following component:
(a) the independent per daily dose of at least 7 part of 140 μ g-320 μ g bispecific single-chain antibody construct; With
(b) with the device (means) of component to promote the mode of treatment plan conformability is arranged.
Therefore, the average body surface area of calculating according to the present invention in the context of method, medicine box or purposes of patient is 1,7-2,2m 2Scope.
Advantageously, medicine box of the present invention further comprises the residue reagent or the material of optional (a) reaction buffer, storage solution and/or described purposes needs.In addition, the component bag of medicine box of the present invention be packaged in independently the pipe or bottle in or packaged in combination in container or many container unit.Standard scheme well known by persons skilled in the art is preferably followed in the preparation of medicine box.
For assess anti-CD19 described herein * anti-CD3 bispecific single-chain antibody (security and the tolerance of bscCD19 * CD3), to two patients that suffer from recurrent non-Hodgkin lymphoma (B NHL) by long-continued infusion administered compound.
Be diagnosed as follicular lymphoma first place patient in 2000.Though this patient has accepted number of chemical therapy and immunotherapy in the past, disease has still worsened.According to purposes provided herein and method with after anti-CD19 as herein described * anti-CD3 bispecific single-chain antibody construct is treated, disease is not only stopped first, and has realized significantly dwindling of lymphoma focus.Described in following non-limiting example, when this patient with 15 μ g/m 2When the described herein anti-CD3 of/24 hours dosage levels * anti-CD19 bispecific single-chain antibody construct was accepted 4 all continuous infusions, treatment was well tolerated, and does not promptly observe pronounced side effects.Treatment causes strong T cell activation and propagation.The T cell of tool cytotoxicity phenotype mainly is because CD8 +T cell proliferation.The activation of T cell and propagation are induced by the B lymphoma cell that anti-CD3 described herein * anti-CD19 bispecific single-chain antibody is modified in tumour.The B cell count of measuring during the treatment shows, described construct can fully be eliminated round-robin B (lymphoma) cell to the cytotoxicity of lymphoma cell owing to it.(restaging) more by stages after treatment, discovery clearly reduces according to the lymphoma piece of the reaction assay of NHL: by reaction (the Partial Response of part in the corresponding tumor response test, PR) computational x-ray tomography art (CT), (SumProduct Diameters SPD) is diagnosed as decline 58.0% to six gross product diameters with reference to the lymphoma focus.
The 2nd patient diagnosis was for suffering from small lymphocytic lymphoma (SLL or B-CLL) in 1999.In the history in 7 years of disease, the patient has accepted 7 kinds of different chemotherapy schemes and immunotherapy and radiotherapy, but do not show any principal reaction, and provide successful therapy first with anti-CD19 * anti-CD3 bispecific single-chain antibody construct treatment this patient according to pharmacy mode of the present invention and method, wherein can realize being higher than 50% with reference to dwindling of lymphoma focus.When described second place patient with 15 μ g/m 2Treatment is during two weeks continuously for/24 hours described herein anti-CD19 * anti-CD3 bispecific single-chain antibody construct, and round-robin B (lymphoma) cell is effectively exhausted.In addition, observe total lymphoma cell in the marrow by whole eliminations of anti-CD19 as herein described * anti-CD3 bispecific single-chain antibody construct.This finds consistent with the complete myelomatosis reaction that is difficult to realize by for example conventional chemical therapy.Treatment more by stages, find that lymphoma knurl piece clearly reduces: by the computational x-ray tomography art (CT) of the reaction (PR) of part in the corresponding tumor response test, six sizes with reference to the lymphoma focus are diagnosed as and descend 57.2%.
According to term of the present invention " bispecific single-chain antibody " or " single chain bispecific antibody " or relational language represent by will at least two antibody variable regions be connected the antibody construct that produces in the single polypeptide chain that lacks the constant and/or Fc part of complete immunoglobulin (Ig)." joint " used herein connects mutually homospecific V structural domain, and " spacer " used herein connects not homospecific V structural domain.For example, bispecific single-chain antibody can be tool two antibody variable regions construct in two VH districts for example altogether, wherein said each variable region is other antigen of combination branch specifically, and connect so that these two antibody variable regions and insertion spacer wherein exist with single continuous polypeptide chain by short (being less than 10 amino acid usually) synthetic property polypeptide spacer each other.The example of another bispecific single-chain antibody can be the single polypeptide chain with three antibody variable regions.Herein, a two antibody variable regions for example VH and a VL can be formed scFv, wherein these two antibody variable regions are connected to each other by synthetic property peptide linker, usually described peptide linker are carried out gene design so that minimize immunogenicity, keep resistance to the proteolyzing maximum simultaneously.This scFv can be specifically in conjunction with specific antigen, and be connected in can further combined with the different antigenic antibody variable region of scFv institute conjugated antigen, for example VH district.In addition, the example of another bispecific single-chain antibody is the single polypeptide chain of four antibody variable regions of tool.Herein, preceding two antibody variable regions for example VH district and VL district can form one can be in conjunction with an antigenic scFv, and second VH district and VL district can form second energy in conjunction with another antigenic scFv.In single continuous polypeptide chain, a species specific independent antibody variable region can advantageously separate by synthetic property peptide linker as indicated above, and each scFv can be advantageously by small peptide spacer as indicated above separately.WO99/54440, WO 2004/106381, Mack, J.Immunol. (1997), 158,3965-70; Mack, PNAS, (1995), 92,7021-5; Kufer, Cancer Immunol.Immunother., (1997), 45,193-7;
Figure A20068004716200131
Blood, (2000), 95,6,2098-103; Br ü hl, J.Immunol., (2001), and 166, shown the limiting examples of bispecific single-chain antibody and production method thereof among the 2420-2426.
As used herein, " people CD3 " is illustrated in the human T-cell and goes up the antigen of expressing as the part of polymolecular TXi Baoshouti complex body, and CD3 is made up of five kinds of different chains: CD3-ε, CD3-γ, CD3-δ, CD3-η and CD3 ζ.As indicated above, CD3 for example causes the T cell to be similar to conjugated antigen by the cluster of anti-cd 3 antibodies on the T cell but does not rely on the activation of T cell subsets clone-specific.Therefore, as used herein, term " with the antigenic bispecific single-chain antibody of one specific specificity specific combination people CD3 " relates to and can be combined in the people CD3 complex body of expressing on the T cell and can induce target cell elimination/cracked CD3 specific construct, and another the non-CD3 bound fraction bonded antigen with bispecific single-chain antibody is carried/shown to wherein said this type of target cell.As known in the art, combining of CD3 complex body and CD3 specific junction mixture (for example bispecific single-chain antibody of using according to pharmacy mode of the present invention and method) causes the T cell activation, sees, for example WO 99/54440 or WO 2004/106381.Therefore, the structure physical efficiency that is fit to pharmacy mode of the present invention and method advantageously in vivo and/or external elimination/cracking target cell.Corresponding target cell comprises for example cell of CD19 of expressing tumor antigen, and wherein said tumour antigen is by second specific specificity (being the non-CD3 bound fraction of the bispecific single-chain antibody) identification of described construct.Preferably, described second specific specificity is the specificity of describing in WO 99/54440 or WO 2004/106381 to people CD19.According to this embodiment, each antigen-specific of bispecific single-chain antibody partly comprises antibody VH district and antibody VL district.The favourable variant of this bispecific single-chain antibody from the N-terminal to the C-terminal is:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：2)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)(SEQ ID NO.：4)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19) (SEQ ID NO.:6), perhaps
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)(SEQ ID NO.：8)。
More specifically, in implication of the present invention, with term " specifically in conjunction with " or relational language for example " specificity " be interpreted as mainly by following two parameter characterizations: qualitative parameter is (in conjunction with epi-position, or antibody binding site) and quantitative factor (binding affinity, or its bonded position of this antibodies has many strong).Which epi-position of antibodies can be advantageously by for example FACS method, ELISA, a peptide-epitope mapping or mass spectrometric determination.The intensity of antibodies defined epitope can advantageously be measured by for example known Biacore and/or ELISA method.Uniting these technology makes and can will calculate signal: noise likens the representative measure into binding specificity to.At described signal: noise than in, signal is represented the intensity of antibodies purpose epi-position, other is different from the intensity of the irrelevant epi-position of purpose epi-position and noise is represented antibodies.With the signal noise ratio of each purpose epi-position by for example at least 50 but preferably approximately 80 as assessment antibody with special mode binding purposes epi-position, be the index of " specific junction mixture ", wherein said signal noise ratio is for example measured by Biacore, ELISA or FACS.Term " be bonded to/with ... interact " also relate to two conformational epitope, structure epi-position or discontinuous epitopes that the zone is formed by people's target molecule or its part.In the context of the invention, conformational epitope is by two or more isolating discontinuous aminoacid sequence definition in primary sequence, the described discontinuous aminoacid sequence (Sela that on molecular surface, gathers together when polypeptide is folded into natural protein wherein, (1969) Science166,1365 and Laver, (1990) Cell 61,553-6).The non-linear epi-position that the residue by the polypeptide chain compartment is assembled into represented in term " discontinuous epitope " in the context of the invention.When polypeptide chain was folded into three-dimensional structure, these residues gathered together on molecular surface and constitute conformation/structure epi-position.
According to the present invention, " variable region " of using with Ig deutero-AI in the literary composition comprises the fragment and the derivative of polypeptide, and wherein said polypeptide comprises a CDR derived from antibody, antibody sheet placate or derivatives thereof at least.The present invention has considered following situation, and promptly described at least one CDR is preferably CDR3, more preferably is the CDR3 of heavy chain of antibody (CDR-H3).But other antibody deutero-CDR also is included in the term " variable region " especially.
Term " antibody fragment or derivatives thereof " relates to single-chain antibody or its fragment, synthetic property antibody, antibody fragment for example Fab, F (ab2) ', Fv or scFv fragment, single domain antibody etc. or arbitrary these derivative of chemically modified.The antibody or its corresponding immunoglobulin chain that adopt according to the present invention can further be modified with routine techniques known in the art outside motif, for example by separately or unite and use aminoacid deletion known in the art, insertion, replacement, interpolation and/or reorganization and/or other modification arbitrarily (for example posttranslational modification and chemically modified, for example glycosylation and phosphorylation).The method of introducing this type of modification in the dna sequence dna of coding immunoglobulin (Ig) chain amino acid sequence is known for those skilled in the art, sees, for example people such as Sambrook; Molecular Cloning:ALaboratory Manual; Cold Spring Harbor Laboratory Press, second edition 1989 and third edition calendar year 2001.
Term used herein " peptide " or " polypeptide " have been described the molecule that a class comprises peptide group and polypeptide group.The peptide group is by being no more than 30 amino acid whose molecular compositions, and the polypeptide group is by more than 30 amino acid whose molecular compositions.Term " antibody fragment or derivatives thereof " relates in particular to peptide or the polypeptide construct that comprises at least one CDR, preferred CDR-H3.The fragment of described antibody molecule or derivative are defined to be the part of above-mentioned antibody and/or peptide or the polypeptide of modifying by chemical/biological chemistry or molecular biology method.Correlation method is known in the art, and especially is described in laboratory manual and (sees people such as Sambrook, in above-mentioned quoted passage; People such as Gerhardt; Methods for General andMolecular Bacteriology; ASM Press, 1994; Lefkovits; ImmunologyMethods Manual:The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions:A MolecularCloning Manual; Cold Spring Harbor Laboratory Press, 2002).
The method of in giving amino acid sequence of polypeptide, identifying motif for it be known to those skilled in the art that and in some laboratory manuals (for example, people such as Sambrook, in above-mentioned quoted passage; M ü lhardt; Der Experimentator:Molekularbiologie/Genomics; SpektrumAkademischer Verlag is in 2001) be described.In addition, described method has been carried out illustration in the attached embodiment in back.
Term administering used herein " expression is to the above-mentioned bispecific single-chain antibody of individual administering therapeutic significant quantity.The dosage that " treatment significant quantity " expression tells on to its object of using.Precise dosage depends on therapeutic purpose, and determines with known technology by those skilled in the art.As known in the art with ground mentioned above, according to systemic delivery and local delivery, age, body weight, overall health, sex, diet, time of application, drug interaction and disease serious property adjustment can be essential, and is determined by the normal experiment method by those skilled in the art.
Attending doctor and clinical factor decision dosage.Know ground as medical field, any patient's dosage depends on many factors, comprising patient's size, body surface area, age, the specific compound of using, sex, time of application and route of administration, overall health and other medicine of using simultaneously.
Know ground as medical field, any patient's dosage depends on many factors, comprising patient's size, body surface area, age, the specific compound of using, sex, time of application and route of administration, overall health and other medicine of using simultaneously.General dosage can for example be set in the scope of embodiment of the present invention and appended embodiment, yet, especially consider above-mentioned factor, the dosage that is below or above this exemplary range is fine.
As described herein, previous research is preferred uses heavy dose of infusion to come administering therapeutic antibody.As used herein, term " heavy dose of infusion " refers to interrupted infusion before 6 hours, and " continuous infusion " refers to and can carry out at least 6 hours and not interrupted infusion enduringly." continuous infusion " refers to the infusion of using enduringly.Therefore, in the context of the invention, term " persistent " and " successive " are as synonym.Within meaning of the present invention, term administering was above at least 6 hours " the expression under conditions, promptly during the required whole cycle of pharmacy mode of the present invention and method with the bispecific single-chain antibody that continues, the constant mode is used for pharmacy mode and method according to the present invention to the patient body continuous administration.Avoid interrupting the importing of bispecific single-chain antibody, promptly except needs replenish to be supplied with the bispecific single-chain antibody used or medical science mediation etc., during required whole the using of pharmacy mode of the present invention and method,, describedly change the state that the state-transition used from this antibody forward patient body is no longer used to patient body for this antibody into because following transformation appears or occur indistinctively in other reason.With regard to this type of essential additional of short duration termination of importing that causes institute's administration of antibodies, still this type of is used " continual " or " persistent " that be interpreted as in pharmacy mode of the present invention and the method meaning.In most applications, to such an extent as to be very little this type of expeced time of replenishing that the time length be generally weak point like this do not compare the whole application program of pharmacy mode and method according to the present invention to the time that patient body imports antibody.At least 1 week or 2,3,4,5,6,7,8,9,10,11 or 12 weeks or even longer uninterruptedly or enduringly take place in the application program of especially preferred bispecific single-chain antibody.In most of embodiment preferred, continuous 4-8 week of bi-specific antibody promptly 4,5,6,7 or 8 uses above 24 hours allly.For example, it can be the patient of treatment by stages after 4 all continuous administration, diagnosable reaction (as giving a definition) to or part minimum to the bispecific single-chain antibody treatment.In this case, the prolongation continuous administration is finished even better treatment result, for example reaction (complete response) completely.Mode with pharmacy mode and method according to the present invention continues to use incessantly bispecific single-chain antibody to allow following favourable T cell activation, comes advantageously to remove all diseased cells from health thereby make it bring into play enough long effect.Because it is very low that the ratio of the bispecific single-chain antibody that will uninterruptedly use remains on, thus can continue the application of treatment agent longer time, and not to the risk of patient's harmful side effect.
Find, by with every day every square metre of body surface area 10 μ g to 80 μ g dosage use bispecific single-chain antibody and can obtain pharmacy mode of the present invention and the useful and unexpected effect of method.It is constant that per daily dose keeps during using.Yet, following method of application is also within the scope of this embodiment, wherein before pharmaceutical methods described herein, use the more bispecific single-chain antibody of low dosage (" predose ") initial several days of infusion cycle, and use more high dosage (" maintenance dose ") in the residue infusion phase.For example, can use the per daily dose of every square metre of body surface area 15 μ g then at Remaining Stages at the bispecific single-chain antibody of using every square metre of body surface area 5 μ g in first day of infusion phase.Perhaps, use the per daily dose of every square metre of body surface area 45 μ g then at Remaining Stages at the bispecific single-chain antibody of using every square metre of body surface area 15 μ g in first day of infusion phase.Also consider following manner, promptly use the bispecific single-chain antibody of the long-pending 5 μ g of every square metre of body surface first day of infusion phase, use the bispecific single-chain antibody of every square metre of body surface area 15 μ g then in second of the infusion phase day, use (keeping) dosage every day of every square metre of body surface area 45 μ g then at Remaining Stages.Therefore, at first day that treats or at first and second days, bispecific single-chain antibody construct as herein described can be used with (every day) predose that is lower than the long-pending 10 μ g-80 μ g of every square metre of body surface and slowly make patient tissue adapt to treatment.After this, can use the actual sustain dosage of the long-pending 10 μ g-80 μ g of every square metre of body surface.Therefore, the average body surface area of calculating in the context of the method according to this invention, medicine box or purposes of patient is 1,7-2,2m 2Scope.Find, maintain the bare minimum for some time that reaches the expection result of treatment, can be best implemented in and above be described as extremely beneficial T cell activation increase by the bispecific single-chain antibody ratio that will uninterruptedly use (being continuous infusion).Except hereinafter enumerate with simulation natural T cell activation relevant advantage, the feasible bispecific single-chain antibody absolute magnitude of using of bispecific single-chain antibody of using lower ratio is lower.This causes the lower expense of individual patient, makes that the treatment of according to the present invention pharmacy mode and method is that its patient burden of wider needs must rise.
In implication of the present invention, term " B cell non-Hodgkin's " or " the cell-derived non-Hodgkin lymphoma of B " comprise inertia and rapid spread B cell non-Hodgkin's (B NHL).As used herein, the cell-derived neoplastic disease of term " inertia or rapid spread B cell non-Hodgkin's (B NHL) " expression Malignant B.Inertia B NHL is low malignant lymphoma.Rapid spread BNHL is high malignant lymphoma.B cell non-Hodgkin's (B NHL) can advantageously be follicular lymphoma, lymph-plasma cell sample lymphoma, marginarium cell lymphoma, lymphoma mantle cell (MCL), diffuse large B cell lymphoma (DLBCL), Burkitt lymphoma, small lymphocytic lymphoma (SLL/CLL) and the cell-derived subgroup of other B arbitrarily.Term used herein " B cell leukemia " can be advantageously be B cell leukemia (for example chronic lymphocytic leukemia or acute lymphoblastic leukemia) arbitrarily.Further with reference to for example seeing Http:https:// www.cancer.orgPreferably, inertia non-Hodgkin B cell lymphoma can as described in the following embodiment with the bispecific single-chain antibody treatment of anti-people CD3 and people CD19.
Term used herein " prevents from " to understand as follows: with chemotherapy or radiotherapy lymphoma focus, fully alleviate the lymphoma focus of human patients after, situation is not to be that all lymphoma cells can both be removed from health.Yet these remaining tumour cells may produce the lymphoma of sending out again.Pharmacy mode of the present invention and method can be used for killing these remaining tumour cells, thereby prevent lymphoma (originating from the hiding property lymphoma cell that remains in after the initial therapy in the health) recurrence.By this way, this pharmacy mode and method help prevent the patient to recur B NHL or B cell leukemia.
In the clinical study in vivo, further assessed the safety spectrum of anti-CD19 * anti-CD3 bispecific single-chain antibody.As a result of, after the patient who suffers from intractable B cell tumour is with the bispecific single-chain antibody of the heavy dose of infusion same dose of multiple, finding undesirable first-dose response.Shown in hereinafter, CRS intensity is the highest after using first dosage, to the reaction of the anti-CD19 of infusion * anti-CD3 bispecific single-chain antibody subsequently decrescence.Behind 6 infusions, compare with baseline values, almost do not observe inducing of pair cell factor level.In addition, when increasing the dosage of the bispecific single-chain antibody of using to the patient who suffers from recurrence or intractable B cell chronic lymphocytic leukemia, after using bispecific single-chain antibody repeatedly, observe the CRS strength degradation.Be not bound by theory, described phenomenon most probable results from the adaptation (" T cell silence ") of T cell to repetitious stimulation.In addition, when monitoring T cell quantity in subject patient, it is very big to find that the T cell quantity rises and falls based on each infusion, shows the T cell activation of short-term, explosive type.The biologic activity of anti-CD19 * anti-CD3 bispecific single-chain antibody can obtain proof in these researchs, cause T cell activation, release of cytokines and B cell counting to descend, but do not determine best biological dose (OBD).In addition, observe the side effect relevant with CRS.
Find surprisingly on the contrary and in the context of the invention, for example by using the bispecific single-chain antibody at least 1 week with the per daily dose of the long-pending 10 μ g-80 μ g of every square metre of body surface, can finish the biology effect of expection by bispecific single-chain antibody as herein described, be secular T cell activation with expression and to the toxicity of tumour cell (reduce simultaneously, minimize or abrogate fully the undesirable negative interaction relevant with heavy dose of administering therapeutic antibody), wherein said per daily dose was used above at least 6 hours.As described in accompanying non-limiting example, surpass the i.e. 15 μ g/m of 24 hours at least two weeks of ground continuous administration bispecific single-chain antibodies in a small amount to the patient who suffers from small lymphocyte lymphoma (SLL/CLL) or follicular lymphoma 2Bispecific single-chain antibody be enough to secular T cell activation in the body and propagation, and cause suitable antitumor reaction: two patients have the reaction (PR) of part, promptly compare six and shrink with reference to lymphoma and to be higher than 50%.In addition, in these patients, confirm to have consumed lymphoma cell from marrow, this by ordinary method for example chemotherapy be unapproachable.Importantly, the bispecific single-chain antibody of this low dosage causes using the more physiological t cell responses of bispecific single-chain antibody than heavy dose in the patient.In addition, method provided herein and purposes do not cause usually relevant with the T cell activation, pronounced side effects, for example CRS.These low dosage still can be induced useful antitumor reaction in the patient who suffers from inertia or rapid spread B cell non-Hodgkin's (BNHL) or B cell leukemia.
Formerly use in the application program of repeated heavy dose of infusion, even use minimum initial dose in the I clinical trial phase in vivo, observe also that white blood cell count(WBC) reduces suddenly in the peripheral blood, wherein said white corpuscle comprises CD3 +, CD4 +And CD8 +T cell (seeing embodiment).An explanation of this reduction is an activated T cell and other activatory white corpuscle adheres to suddenly and migrate into tissue to small part.Use this explosive type T cell activation that bispecific single-chain antibody causes by heavy dose and cause local cells factor spectrum endothelial function unusual and disorderly, this is considered to the major cause of observed side effect in CRS usually.On the contrary, as described in following embodiment, can activate (and conversion) T cell colony more lenitively according to pharmacy mode of the present invention and method, the cytokine quantity that shows release is violent the minimizing.Therefore, supply according to pharmacy mode of the present invention and method and just avoided for example disorderly suddenly phenomenon of local cells factor network.So not appearing in the newspapers according to the patient of the inventive method and purposes treatment has pronounced side effects.In addition, pharmacy mode and method are used bispecific single-chain antibody and are not caused that the viewed violent T cell of any as heavy dose of infusion bispecific single-chain antibody rises and falls according to the present invention.
As shown in embodiment, slightly reduce (most likely because the steroid of using altogether) though can observe the T cell quantity when initial in the infusion phase, cause more according to treatment of the present invention that the activated T cell quantity increases in the blood.Particularly, Cytotoxic CD8 +The T cell count is owing to increasing.This is to showing the adverse effect cell: (E: T) neoplastic disease of ratio is especially to be fit to target cell, and wherein said neoplastic disease is the interior E of body for example: the T ratio reaches 1: 100-1: 1000 B cell lymphoma focus.Previous experiment in vitro shows that this type of disadvantageous E: the T ratio causes B cell killing more slowly.Under this kind condition, must suppose that identical T cell has the B cell killing potentiality that multiple causes tumor mass significantly to reduce.In addition, suppose that enlarging the T cell bank that is positioned at (for example in B cell lymphoma) in the B cell tumour improves E: the T ratio also is essential to finishing treatment result.Because according to pharmacy mode of the present invention and method provide in the blood flow constant at least or even the activated T cell quantity that increases, so improved for a long time to the tumour cell cytotoxicity potential of the tumprigenicity B cell in the B cell lymphoma for example.
In addition, cause CD8 according to pharmacy mode of the present invention and method +T cell such as T cell activation mark HLA-DR (activating mark for a long time) compare the long-term activation of institute's illustration with CD69 or CD25 (short-term activation mark).Ground as noted above, continuing to use low anti-CD19 * anti-CD3 bispecific single-chain antibody of measuring is enough to increase activatory CD8 in long-time +The T cell quantity is described in for example appended embodiment and Fig. 6-8.This with use in heavy dose that observed explosive type T cell activation forms sharp contrast in anti-CD19 * anti-CD3 bispecific single-chain antibody; Referring to for example Fig. 1.Therefore, that pharmacy mode and method according to the present invention and the bispecific single-chain antibody that prolongs existence allow is mild, secular T cell activation and propagation, and this especially is fit to treatment and is characterized by unfavorable E: the neoplastic disease of T ratio, for example B cell lymphoma.In addition, observe after restarting continuous infusion (for example after medical intervention), faster and stronger cytotoxicity CD8 takes place +T cell activation and propagation.Therefore, preferably several take turns infusion circulation after during the no infusion.For example, during the bispecific single-chain antibody infusion of this paper definition around the patient can accept continuously, the no infusion around then, this also within the scope of the present invention.After this, this order can repeat once usually, twice, three times or even more times.
In addition, though can observe secular T cell activation in the patient of pharmacy mode according to the present invention and method treatment, related side effects significantly reduces or even eliminates.Therefore, can minimize or prevent the side effect of CRS dependency by the bispecific single-chain antibody of using pharmacy mode and method according to the present invention.
At last, be stressed that, though in purposes described herein, method and medicine box, only used the bispecific single-chain antibody of low amount, thus Antybody therapy causes in the detectable antitumor reaction it is very effective in vivo at elimination tumprigenicity B cell.This descends by the B cell counting in Fig. 9 and 11 and by the size of the lymphoma shown in Figure 10 and 13 and has carried out illustration.Particularly, the reaction that the patient of two pharmacy mode and method treatments according to the present invention has part promptly is higher than 50% contraction than 6 with reference to the lymphoma focus.In addition, in these patients, confirm to have eliminated lymphoma cell effectively from marrow; Referring to Figure 12.Therefore, when using pharmacy mode of the present invention and method, side effect is reduced or eliminates, but does not follow any Cytotoxic decline.
On the contrary, the antibody therapy of heavy dose of mode of administration causes in a large number, the T cell activation of short-term and subsequently the activated T cell to the reorientating/move of endothelium and tissue, and in blood flow only remaining effective T cell in a small amount.As known in the art, endothelium can be by a large amount of T cell activations, the T cell adhesion is in endotheliocyte and migrate into tissue and cause and be badly damaged subsequently.Under pathological conditions, can observe this type of unexpected T cell activation, for example in the serious septicemia that causes by bacteriotoxin, (see Li (2004) for example, Br.J.Pharmacol.141 (4): 709-16; Matzen (2004), VirusRes.104 (2): 145-55; Jacob HS. (1980) Arch.Pathol.Lab.Med.104 (12): 617-20; Salyer (1990), Am.J.Pathol.136 (4): 831-41; Okajima (2004) Curr.Vasc.Pharmacol.2 (2): 125-33; Ferrero (2004) Methods Mol.Med.98:127-36).In addition, the T cell activation of this explosive type is relevant with the side effect that cytokine causes.Formerly use in the dose study of repeated heavy dose of infusion, even use minimum initial dose in the I clinical trial phase in vivo, observe also that white blood cell count(WBC) reduces suddenly in the peripheral blood, wherein said white corpuscle comprises CD3 +, CD4 +And CD8 +The T cell.An explanation of this reduction is an activated T cell and other activatory white corpuscle adheres to suddenly and migrate into tissue to small part.Suppose the unexpected activation of these a large amount of activated T cell colonys (the human T-cell stock of random time about 70% is in the peripheral blood) and migration cause organizing in the disorder of T Cell Homeostasis and local cytokine spectrum.
Therefore, and compare than previous I phase research, the remarkable decline that significant prolongation ground is exposed to bispecific single-chain antibody and dosage that each time unit gives has increased drug tolerance potentially and has maximized the anti-tumor activity of medicine.
Formerly investigate in the clinical study of the security of the anti-CD19 * anti-cd 3 antibodies of dual specific strand of repeated heavy dose of infusion and tolerance, behind each infusion, observe undesirable T cell count fluctuation, show that the T cell depends on each infusion explosive type activatable.It should be noted that using described bispecific single-chain antibody identical and increase dosage all observes this effect.
The pharmacy mode shows that with the supply that method is carried out the T cell activation pattern that obtains with heavy dose of infusion greater amount antibody is in different in nature T cell activation patterns according to the present invention.Particularly, have been found that, the bispecific single-chain antibody of using pharmacy mode and method according to the present invention cause that the initial stage slowly increases in the body, it is constant to keep then or even the cell immune response that increases, described immune response is with for example observed response class is seemingly in the virus infection at natural infection.In natural infection, when meeting this antigen in the Lymphoid tissue of the cyclicity T of pathogenic antigen-specific cell in drainage, it can activate.After this type of activation in natural infection, the T cell subsets of pathogenic antigen-specific is bred.Because CD8 +The propagation of T cell, this increase with to invasion pathogenic agent or to being increased by the cytotoxicity potential of the cell of this pathogenic infection.Therefore, pharmacy mode of the present invention and method stimulate the immune response to natural infection such as virus infection, because activated T cell count slow and that increase gradually takes place.
In a word, the invention provides following major advantage:
-simulate the t cell responses of physiology more, there is not violent T cell fluctuation, constant or even the increase of the effector cell in the blood flow (cytotoxicity) T cell quantity
-release of cytokines reduce/does not have owing to hang down the first-dose response of amount bispecific single-chain antibody, tool effective antitumour (CTL) active (two patients have the reaction of part, and wherein a people eliminates lymphoma cell effectively from marrow)
The adverse side effect of-reduction
-minimize the abnormal T cell activation that causes by repeatedly T cell activation and inactivation cycle
The long-term activation and the propagation of-cytotoxic T cell
The prolongation of-medicine exists and helps to overcome the unfavorable E than the slower B cell killing of original expectation of causing that is present in usually in the lymphoma focus: the T ratio
The prolongation of-medicine exists permission by one and identical T cell generation B cell killing repeatedly, and therefore guarantees that tumor mass significantly reduces
-propagation partial T cell bank and improve E: T ratio and treatment result
-external activity exists significant difference (to be reflected in EC between different peripheral blood lymphocytes (PBMC) donor 50The difference of value).The exposure duration that medicine is prolonged in the patient of significant proportion, improved anti-CD19 at cytotoxic activity.
Therefore, compare with previous I phase research, the remarkable decline that significant prolongation ground is exposed to medicine and dosage that each time unit gives has increased tolerance potentially and has maximized the anti-tumor activity of medicine, produces better to compose safely.
Can every day surpass 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or according to using of the present invention program even above 24 hours.Preferably, using according to the present invention program to surpassing 10 hours, being more preferably and surpassing 12 hours or even be more preferably above 24 hours.
In the preferred embodiment of method of the present invention, purposes or medicine box, the per daily dose of described bispecific single-chain antibody construct surpasses at least 8 hours for using, is more preferably at least 10 hours.
In method of the present invention, purposes or medicine box even the embodiment that is more preferably, per daily dose is for using above at least 12 hours, 14 hours, 16 hours, 18 hours, 20 hours or 22 hours.Most preferably, per daily dose is that whole day is used, promptly above 24 hours.
In another preferred embodiment of method of the present invention, purposes or medicine box, the described V of the structural domain that described CD3 is special HAnd V LThe district is derived from the special antibody of CD3, and the special antibody of wherein said CD3 is selected from: OKT-3, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87,12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2 and F101.01.Each these antibody is all described in detail in prior art.
More specifically, the described V of the special structural domain of described CD3 HThe district comprises at least one CDR3 district, and described CDR3 district comprises or is aminoacid sequence shown in the SEQ ID NO.11, the described V of the structural domain that described CD3 is special HThe district comprises at least one CDR2 district, and described CDR2 district comprises or is aminoacid sequence shown in the SEQ ID NO.10, and/or the described V of the special structural domain of described CD3 HThe district preferably comprises at least one CDR1 district, and described CDR1 district comprises or is aminoacid sequence shown in the SEQ IDNO.9.
Construct of the present invention also comprises V LThe district.The described V of the structural domain that described CD3 is special LThe district comprises at least one CDR3 district, and described CDR3 district comprises or is aminoacid sequence shown in the SEQ ID NO.14, the described V of the structural domain that described CD3 is special LThe district comprises at least one CDR2 district, and described CDR2 district comprises or is aminoacid sequence shown in the SEQ ID NO.13, and/or the described V of the special structural domain of described CD3 LThe district comprises at least one CDR1 district, and described CDR1 district comprises or is aminoacid sequence shown in the SEQ ID NO.12.
Be interpreted as, in most of preferred embodiments, the CDR (CDR1, CDR2, CDR3) of definition as mentioned be included in the independent dual specific construct and carry out using according to of the present invention.
In another preferred embodiment of method of the present invention, purposes or medicine box, the V of the structural domain that described CD3 is special HThe district comprises or is SEQ ID NO 17, the V in described CD19 ad hoc structure territory HThe district comprises or is SEQ ID NO 15, the V of the structural domain that described CD3 is special LThe district comprises or is SEQ ID NO 18, and/or the V in described CD19 ad hoc structure territory LThe district comprises or is SEQ ID NO 16.
Method of the present invention, purposes or medicine box also consider to utilize the bispecific single-chain antibody construct that comprises the aminoacid sequence that is selected from following sequence:
(a) aminoacid sequence of in SEQ ID NO 2,4,6 or 8, describing;
(b) by the aminoacid sequence of nucleic acid sequence encoding shown in SEQ ID NO 1,3,5 or 7;
(c) had the aminoacid sequence of the nucleic acid sequence encoding of at least 70%, 80%, 90%, 95% or 99% identity by the nucleotide sequence with (b), wherein said aminoacid sequence can (specifically) be bonded to CD3 and CD19; And
(d) by with the aminoacid sequence of the nucleic acid sequence encoding of the nucleotide sequence genetic code degeneracy of (b), wherein said aminoacid sequence can (specifically) be bonded to CD3 and CD19.
Whether any specific nucleic acid molecule or polypeptide can advantageously be measured with known computer program at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the Nucleotide or the aminoacid sequence of this paper definition.The preferred method that measure best overall coupling between search sequence (sequence of this paper definition) and the target sequence, is also referred to as whole sequence alignment can be used based on the FASTDB computer program of people's algorithms such as Brutlag (Comp.App.Biosci.6:237-245 (1990)) and measure.In sequence alignment, inquiry and target sequence all are dna sequence dna.The RNA sequence compares by U is converted to T.
In the preferred embodiment of method of the present invention, purposes or medicine box, described variable domains connects by the extra contacts and/or the spacer sequence of definition as mentioned, and as shown in WO 2004/106381.
In the preferred embodiment of method of the present invention, purposes or medicine box, use day continued at least 2 weeks, at least 3 weeks, at least 4 all or at least 8 weeks.Therefore, use and also consider to carry out, at least 6 hours every days, preferred at least 8 hours every days, be more preferably at least 10 hours every days, 24 hours every days most preferably, carried out at least 1,2,3,4,5,6,7 or 8 weeks or even longer with persistent basis.Also consider several do not have after taking turns infusion circulation infusion during.For example, the bispecific single-chain antibody of this paper definition of continuous infusion around the patient can accept, do not have around accepting then infusion during.After this, this order can repeat once, twice, three times or even more times, be lower than the detectable level of for example calculating by ordinary method of x-ray tomography art until the lymphoma focus.Advantageously, prolong and to use, promptly kill all or all substantially lymphoma cells until reaching reaction completely.In addition, (for example at no infusion after the phase or after medical intervention) observes cytotoxicity CD8 after restarting continuous infusion +Faster and stronger activation and propagation take place in the T cell.Therefore, preferably several do not have after taking turns infusion circulation infusion during.
In another preferred embodiment of method of the present invention, purposes or medicine box, pharmaceutical composition is united one or more other medicines preparations and is used.
The present invention also is used for the scheme of combination therapy (co-therapy) method and conjoint therapy.In some embodiments, bispecific single-chain antibody and one or more other therapies of this paper definition are co-administered.In some embodiments, bispecific single-chain antibody and one or more other therapies of this paper definition are applied to the patient simultaneously.Preferably, this type of therapy is used for the treatment of as defined herein BNHL or B cell leukemia.Term " side by side " not limit drug or therapeutical agent was used in the definite identical time, but the bispecific single-chain antibody of expression this paper definition and other medicament are used so that the bispecific single-chain antibody of this paper definition can provide than they independent effects of using increase together with other reagent to the experimenter in order and at certain time intervals.For example, every kind of medicine or therapeutical agent can in turn be used with any order in the identical time or in different time points; Yet if do not use in the identical time, they should be used in the enough approaching time so that the treatment or the effect of drugs of expection are provided so.
Every kind of (associating) therapeutical agent can be used with suitable arbitrarily form or by any suitable approach individually.In other embodiments, the bispecific single-chain antibody of this paper definition before operation, simultaneously or use afterwards.Preferably, the size of performing the operation and removing partial lymphoma fully or reducing big tumour.Operation also can be used as preventive measure and carries out or be used for alleviating pain.
Dose quantity provided herein and frequency of administration are included in the term " treatment effectively ".Dosage and frequency are general further according to every patient's specific factor and different, and wherein said factor depends on the particular treatment used or severity and type, route of administration and patient's age, body weight, reaction and the passing medical history of pharmaceutical preparation, B NHL or B cell leukemia.Suitable scheme can be by those skilled in the art by considering this type of factor and deferring to the dosage of recommending in the report and Physicians ' Desk Reference (the 59th edition, 2005) in the document for example and select.
In some embodiments, the therapy of using bispecific single-chain antibody described herein with use for example chemotherapy, radiotherapy, hormonotherapy and/or the biotherapy/immunotherapy associating of one or more therapies.Medicine or therapeutical agent include, but are not limited to protein molecule, comprising but be not limited to peptide, polypeptide, protein, the protein that comprises posttranslational modification, antibody etc.; Perhaps small molecules (being less than 1000 dalton), inorganic or organic compound; Perhaps nucleic acid molecule, comprising but be not limited to two strands or single stranded DNA or two strands or single stranded RNA and 3 strands of spiral nucleic acid molecule.Medicine or therapeutical agent can be derived from any known biology (including but not limited to animal, plant, bacterium, fungi and protobiont or virus) or synthetic property molecular library.
Described conjoint therapy is included in and uninterruptedly uses before the bispecific single-chain antibody, during and/use one or more therapeutical agents or pharmaceutical preparation afterwards jointly.Described therapeutical agent or the pharmaceutical preparation used jointly with bispecific antibody are preferably the chemotherapeutics that is used for standard non-Hodgkin lymphoma (NHL) therapy, as platinum, anthracene nucleus class, alkylating agent, metabolic antagonist, topoisomerase enzyme inhibitor, microbiotic, mitotic inhibitor and antitublin etc.Yet conjoint therapy also is intended to reduce inflammation.Especially preferably use anti-inflammatory agent, for example glucocorticosteroid jointly with any-mode listed above herein.As described in the appended embodiment of this paper, before the anti-CD19 of continuous administration * anti-CD3 bispecific single-chain antibody, used the cytokine that the 100mg steroid suppresses the continuous administration initial period and generated to the patient.Alternatively, steroid also can be used at the anti-CD19 of continuous administration * initial period of the 1st day to the 4th day of anti-CD3 bispecific single-chain antibody.The dosage of using steroid as 1 hour before at the 1st day continuous infusion for 500mg, at the 2nd day (behind the continuous infusion 24 hours) for 250mg, be 125mg at the 3rd day (behind the continuous infusion 48 hours), and be 125mg at the 4th day (behind the continuous infusion 72 hours).Yet, also consider before the anti-CD19 of continuous administration * anti-CD3 bispecific single-chain antibody, during or do not use steroid afterwards.Certainly, chemotherapeutics and anti-inflammatory drug/medicament also can be used (co-administration) altogether.In the incident that takes place to use altogether during the continuous administration bispecific single-chain antibody, this type of is used altogether and can occur in the whole time length of using described antibody or only be its one or more parts.For example, this type of uses can be before using bispecific single-chain antibody initial and can finish behind beginning continuous administration bispecific single-chain antibody altogether, and this is all in the scope in embodiment of the present invention.In such cases, it is favourable omitting any further using altogether at the Remaining Stages of uninterruptedly using bispecific single-chain antibody, perhaps alternatively during uninterruptedly using bispecific single-chain antibody and/or time point after a while afterwards again altogether administration of pharmaceutical preparations also be favourable.Conjoint therapy is designed so that to use altogether arbitrarily occurs over just all that uninterruptedly to use before the bispecific single-chain antibody and/or occur in afterwards and not during uninterruptedly using also be favourable.So, must " conjoint therapy scheme " in the embodiment of the present invention meaning be interpreted as and on the sequential meaning, comprise the continuous administration bispecific single-chain antibody.This means that in some cases the conjoint therapy scheme can carry out than uninterruptedly using the longer time of bispecific single-chain antibody, because may be before the continuous administration bispecific single-chain antibody and/or use the part of medicament afterwards jointly as conjoint therapy.On the contrary, the scheme of conjoint therapy can be carried out the time shorter than continuous administration bispecific single-chain antibody, because use jointly in time period during conjoint therapy occurs over just the continuous administration bispecific single-chain antibody.Preferably, described steroid is followed according to demonstration or scheme and dosage mentioned above among the embodiment and is used.
If in the whole time period of using incessantly, all use jointly with continuous infusion according to bispecific single-chain antibody of the present invention, so just can be with a kind of solution of the medicament of two kinds of medicaments-be used for conjoint therapy and bispecific single-chain antibody-be combined in.The medicament that is used for conjoint therapy is directly added to the bispecific single-chain antibody solution/preparation that is applied to the patient.Further, also can be with the medicament that is used for conjoint therapy and bispecific single-chain antibody as independent solution parallel using of identical time.
In some cases, conjoint therapy is favourable or essential, and this depends on that the patient of acceptance pharmacy mode and method treatment according to the present invention exists, suspects have or expect other disease that has.This type of is used jointly can be before uninterruptedly the using of pharmacy mode and method according to the present invention, during or/and afterwards with one or more heavy dose of application.Alternatively, as according to as shown in the inventive method, medicine box or the purposes, this using jointly also can be uninterrupted character, and can simultaneously and even can carry out in use in carrier identical with the bispecific single-chain antibody of uninterruptedly using in this case.
The bispecific single-chain antibody of this paper definition and other therapeutical agent can act synergistically.As used herein, term " collaborative " than any two or more independent therapies (for example refers to, one or more medicines or therapeutical agent) the more effective conjoint therapy (for example, the bispecific single-chain antibody of associating this paper definition and (a) other medicines or the therapeutical agent listed of this paper) of additive effect.Conjoint therapy (for example, the bispecific single-chain antibody of this paper definition and (a) other medicines listed of this paper or the associating of therapeutical agent) synergistic effect allow to use more one or more therapies of low dosage (for example, one or more medicines or therapeutical agent) and/or more low frequency use described therapy to the B NHL that suffers from this paper definition or the patient of B cell leukemia.The therapy of utilizing low dosage more (for example, medicine or therapeutical agent) and/or more use to low frequency the ability minimizing of described therapy and use the relevant toxicity of described therapy to the experimenter, and do not reduce described therapy in prevention or treatment disease, the B NHL of for example this paper definition or the effectiveness in the B cell leukemia.In addition, synergistic effect can be created in prevention, manages, treats and/or improve the therapy (for example, medicine or therapeutical agent) that tool in the B NHL of this paper definition or the B cell leukemia improves curative effect.At last, the synergistic effect of conjoint therapy (for example, medicine or therapeutical agent) can be avoided or reduce and the side effect of using relevant unfavorable of any therapy or not expecting.
In another embodiment, conjoint therapy is intended to reduce inflammation.Especially preferably use anti-inflammatory agent, for example glucocorticosteroid jointly with any-mode listed above herein.
The conjoint therapy of this embodiment of the present invention also advantageously provides the activation signals to immune effector cell, cell proliferation or cytositimulation.
Uninterruptedly using bispecific single-chain antibody can be intravenous, parenteral, subcutaneous, endermic, endoperitoneal, intramuscular or lung.In most of the cases, intravenous mode of administration is to select to be used for uninterruptedly using the pattern of bispecific single-chain antibody, and is that selection is used for using jointly the pattern as the pharmaceutical preparation of conjoint therapy part instructions about how to take medicine in this case.So, especially preferred intravenous using.In this case, can advantageously select suitable metering outfit as many treatments infusion pump model 6060 by the Baxter preparation.No matter select what metering outfit, it all should design and be built into and minimize or prevent better that therapeutical agent is applied in the interruption in cartridge case exchange and/or battery altering or the process of charging.This can for example realize by the equipment of selecting to have the bispecific single-chain antibody secondary containers of separating with the exchange cartridge case, so selects so that even also can continue to be infused into the patient from secondary containers when removing empty or almost empty cartridge case and changing new cartridge case.
The pattern that intravenously is used and in the situation of the part drug regimen of using conjoint therapy jointly, relate to and be implanted into pump to patient's body and measure this type of and use.Those of ordinary skills know this type of volume pump, the model 6060 by the Baxter preparation for example listed above.
As limiting examples, it can be, and is uninterrupted, be that successive is used by carrying or implant the pony pump system that metering therapeutical agent in patient's body enters influx in patient's body and realized.This type of pump system is generally known in the art, and depend on usually exchange periodically, contain the cartridge case that remains to be injected therapeutical agent.When exchange cartridge case in this pump system, flow into the intravital therapeutical agent of patient incessantly temporary transient interruption can take place thereupon.In this case, still in the meaning scope of pharmacy mode of the present invention and method, think and change the application stages before the cartridge case and change common " uninterruptedly using " of forming a kind of this type of therapeutical agent of application stages after the cartridge case.Identical system will use very long using, wherein need to change cartridge case more than once or need change driving pump wherein battery, this causes therapeutical agent temporarily to interrupt flowing into patient body.
Because long-term wound infects especially easily, so will take appropriate measure to minimize the risk of infection of using the puncture site that enters patient body.The intramuscular administration that also is applied to by similar delivery system mentioned above.
Continuous administration can be for coming through skin by the paster that is attached on the skin, and replace at interval with certain hour.Those skilled in the art will know that and be fit to this purpose drug delivery patch chip system.It should be noted that, especially be fit to continuous administration through dermal administration, because changing first paster that uses up can realize easily by simultaneously it being replaced with second new paster, for example be attached on the skin surface of first paster that uses up of next-door neighbour, and carry out immediately before putting up sheet removing first.Flow disruption problem or battery dies problem can not occur.
In a further preferred embodiment, continuous administration realizes that by the lung approach for example by being worn over the conduit in one or two nostril of nose, described conduit is connected with pressurized tank, and its content is by accurate measurement.
Yet described in this paper and appended embodiment, most preferred mode of administration is used for the intravenously in preset time/time period.
Bispecific single-chain antibody used herein advantageously is applied to human patients with the form of pharmaceutical composition.Though can only use bispecific single-chain antibody used herein, preferably in pharmaceutically acceptable carrier, use.The suitable pharmaceutical carrier example is well known, and comprises phosphate buffered saline(PBS), water, liposome, polytype wetting agent, sterile solution etc.The composition that contains examples of such carriers can carry out preparation by well-known ordinary method.These pharmaceutical compositions can be applied to the experimenter with proper dosage.Dosage is by attending doctor and clinical factor decision.As medical field as everyone knows, any patient's dosage depends on many factors, comprising patient's size, body surface area, age, the specific compound of being used, sex, time of application and approach, general health and other medicine of using simultaneously.The preparation of parenteral administration comprises aseptic moisture or anhydrous solution or suspension.The example of anhydrous solvent is a for example ethyl oleate of propylene glycol, polyoxyethylene glycol and injectable alicyclic organic.Aqueous carrier comprises water, aqueous solution or suspension, comprising salt and buffering medium.The parenteral carrier comprises sodium chloride solution, woods lattice dextrose, dextrose and sodium-chlor, perhaps lactic acid Ringer's solution.Intravenous carrier comprises fluid and nutritious supplementary, electrolyte replenisher electrolyte replenisher of woods lattice dextrose (for example based on) etc.Also can there be sanitas and other additive, for example biocide, antioxidant, sequestrant and rare gas element etc.In addition, composition can comprise proteinaceous carrier, as for example serum albumin or immunoglobulin (Ig), wherein preferred people source.Considered that also composition also comprises further biologic activity reagent except comprising proteinaceous bispecific single-chain antibody, this depends on the desired use of pharmaceutical composition.This type of medicament can be as the medicament of the medicament of cytostatic agent, prevention antihyperuricemic, suppress immunoreactive medicament (glucocorticosteroid for example, FK506), act on the medicine of the recycle system and/or such as the medicament or the cytokine known in the art of T cell co-stimulatory molecules.
Preferably, bispecific single-chain antibody preparation in damping fluid, stablizer and tensio-active agent that this paper is defined.Damping fluid can be phosphoric acid salt, Citrate trianion, succinate or acetate buffer.Stablizer can be amino acid and/or sugar.Tensio-active agent can be stain remover, PEG etc.More preferably, the bispecific single-chain antibody preparation of this paper definition is in Citrate trianion, Methionin, trehalose and tween 80.Preferred isoosmotic salt and tween 80 are as the thinner of pharmaceutical composition of the present invention.
Preferably, in purposes of the present invention, method or medicine box, pharmaceutical composition is used to human patients.
The success of bispecific single-chain antibody therapy can be monitored by the standard method to various diseases of having set up:
For the treatment of B cell leukemia, can use for example white blood cell count(WBC), differentiation thing (differentials), fluorescent activation cell sorting art (FACS), bone marrow aspiration and special clinical chemistry parameters and other standard method of having set up of multiple leukemia.To the treatment of B cell lymphoma, can use for example computer assisted x-ray tomography art, X ray, nucleus magnetic resonance x-ray tomography art (for example, to be used for assessing (Cheson (1999), J.Clin.Oncol. based on the reaction of National Cancer Institute's standard; 1244), clinical chemistry parameters (for example serum lactic dehydrogenase) and other standard method of having set up of the scanning of positron radiation x-ray tomography art, white blood cell count(WBC), differentiation thing, fluorescent activation cell sorting art, bone marrow aspiration, lymphoglandula vivisection/histology and multiple lymphoma-specific 17 (4):.
Cytotoxicity can be by methods known in the art and is detected with the method described in the appended embodiment hereinafter.
Fig. 1: (dosage of bscCD19 * CD3) increases research-to periphery CD3 gradually to use anti-CD19 * anti-CD3 bispecific single-chain antibody to patient's intravenously of suffering from recurrent B cell non-Hodgkin's (B NHL) +, CD4 +And CD8 +The influence of lymphocyte count.Be diagnosed as the patient 0202 who suffers from recurrent B cell non-Hodgkin's (B NHL) and in semiweekly treatment plan, accepted 6 times above the bscCD19 * CD3 that used by intravenously in 4 hours.The dosage of bscCD19 * CD3 increases gradually by following dosage: 1,2,4,7,10 and 13 μ g/m 2Body surface area.CD3 +T lymphocyte (grey rhombus), CD4 +T lymphocyte (black square) and CD8 +The number of T lymphocyte (black triangle) is measured by flow cytometry before 4 hours infusions of the 0th, 2,7,9,14 and 16 day and when finishing respectively, and is wherein said that various T lymphocyte numbers are represented with the percentage ratio that accounts for total lymphocyte number.Indicate with arrow and grey band during 4 hours the infusion.
Fig. 2: patient's intravenously of suffering from intractable B cell malignancies is used anti-CD19 * anti-CD3 bispecific single-chain antibody, and (dosage of bscCD19 * CD3) increases research-to the influence of TNF α serum-concentration gradually.Diagnosed the patient 1003 that suffers from lymphoma mantle cell (MCL) accept the 0th, 2,4,14,16 and 18 day of research 6 times, surpass bscCD19 * CD3 infusion of 4 hours, 1,5 μ g (initial dose) and 3 μ g (in the maintenance dose of search time subsequently) at the 0th day.Before during 4 hours infusions and measure the serum-concentration of TNF α afterwards.Indicate with arrow and grey band during 4 hours infusions.
Fig. 3: the dosage of using bscCD19 * CD3 to patient's 1003 intravenouslys described in Fig. 2 legend increases research-to the influence of IFN γ serum-concentration gradually.
Fig. 4: the dosage of using bscCD19 * CD3 to patient's 1003 intravenouslys shown in Fig. 2 legend increases research-to the influence of IL6 serum-concentration gradually.
Fig. 5: shown in Fig. 2 legend, increase research-gradually to the influence of IL10 serum-concentration to the dosage of the patient 1003 intravenous bscCD19 of using * CD3.
Fig. 6: CD3 +/ CD8 +And CD3 +/ CD4 +The T cell counting is analyzed.Behind beginning infusion bscCD19 * CD3, CD8 +And CD4 +The T cell disappears in a large number from peripheral blood, and this is interpreted as: bscCD19 * CD3 mediation periphery blood T cell and B cell are crosslinked, cause the T cell activation, thereby trigger the distribution phenomenon.Yet, behind the treatment half cycle, CD8 +And CD4 +The T cell reappears in the blood, and quantity further increases respectively until the 7th day and the 21st day.Compare CD8 with initial value +And CD4 +The T cell is shown as has increased by 3.5 to 4 times in blood.The T cell quantity in treatment began in the 4th week reduce before, CD8 +And CD4 +The quantity of T cell keeps high in the 2nd and 3 weeks of treatment.At infusion bscCD19 * CD34 after week, when diagnosing out suitable tumour to dwindle the standard of the reaction of satisfying part, CD8 +And CD4 +The T cell counting still is higher than the preceding numerical value of corresponding treatment.
Fig. 7: the CD8+T cell subsets is analyzed.The effector cell remembers subgroup TEM and is responsible for by bscCD19 * CD3 inductive CD8 +Unique subgroup of T cell proliferation.CD8 +The T cell is responsible for the most cytotoxicity of all T cells, and TEM cell and the responsible together CD8 of TEMRA subgroup +The most cells toxicity of T cell.Except the TEM subgroup, at other CD8 +Fail to observe the noticeable change in the cell counting between the T cell subsets, can not breed based on the single activation signals that bscCD19 * CD3 provides as inmature T cell, perhaps the TEMRA subgroup can not be bred fully.Therefore, the CD8 that can breed +The selective proliferative of TEM cell can be clearly owing to replying the CD8 that modified by bscCD19 * CD3 +Cell fission and propagation that the TEM cell contacts with the B lymphoma cell.Since cyclicity B lymphoma cell the 3rd day, promptly before the T cell proliferation of observing, from peripheral blood, exhaust, so can get rid of the remarkable contribution of B lymphoma cell that round-robin bscCD19 in the blood * CD3 modifies T cell proliferation.
Fig. 8: CD8 +T cell activation labeled analysis.Highly activating CD8 +After the T cell, CD8 +T cell proliferation, wherein CD8 +The height activation of T cell is represented by the activation mark HLA-DR that continues to raise.Other activation mark is CD69 and the only of short duration rise of demonstration behind initial infusion bscCD19 * CD3 of CD25 for example, the activation of the B lymphoma cell that this can modify by cyclicity bscCD19 in the peripheral blood * CD3 is explained, as long as cyclicity B lymphoma cell has just been exhausted during first 3 days of treatment.On the contrary, surpass the lasting rise HLA-DR in 3 weeks and reflected CD8 in the tumour +The T cell is by contacting the activation that causes with the intrinsic B lymphoma cell of tumour, and wherein said B lymphoma cell is subjected to the modification of bscCD19 * CD3.The activation of T cell produces the proliferative T lymphocyte reaction in the tumour in the knurl, and then causes T cell proliferation, and this also appears in the circulation blood then.These T cells that activate and migrate into subsequently blood in tumour still show long-term activation mark HLA-DR, but short-term activation mark CD69 and CD25 have reduced.
Fig. 9: the B cell counting of patient #105003.The patient begins with 140 B of about every milliliter of blood (lymphoma) cell before treating.Behind initial infusion, cyclicity B (lymphoma) cell descends rapidly in first 3 days of treatment, and finally disappears from peripheral blood fully at first all treatments end.Therefore, because bscCD19 * CD3 is to the cytotoxicity of described cell, so it can remove cyclicity B (lymphoma) cell fully.
Figure 10: (A): (left side before treating for 4 weeks with bscCD19 * CD3; On September 26th, 05) and afterwards (the right; On October 31st, 05) measure the size of lymphoma focus #2 (as shown in Figure 10 (B) table) by calculating auxiliary x-ray tomography art (CT).(B): other selects and carries out the lymphoma focus decline in size that effect judges and quantize to lymphoma shown in (A) and 5, as described in the table as shown in (B).The percentage ratio decline that total lymphoma size descends and is expressed as these 6 selected lymphoma focus maximum cross section diameter product summations (being gross product diameter or SPD).Therefore, lymphoma is shunk 58.0% and be diagnosed as the standard that reaches reaction (PR) partly.
Figure 11: the B cell counting of patient #102003.The patient begins with 920 B of about every milliliter of blood (lymphoma) cell before treating.Behind initial infusion bscCD19 * CD3, cyclicity B (lymphoma) cell in first 3 days of treatment, show with cell redistribute consistent rapid decline, then in the limited recovery of 600 every milliliter cell place short-terms.In further therapeutic process, the reaction (PR) of part is exhausted, diagnosed to B (lymphoma) cell fully after treating for two weeks from circulation.These data have affirmed that as its part clinical effectiveness, bscCD19 * CD3 can fully remove cyclicity B (lymphoma) cell.
Figure 12: the myeloid tissue pathology of patient #102003 before with bscCD19 * CD3 treatment and after two weeks.
Left side figure is before with bscCD19 * CD3 treatment: marrow shows the marrow migration of small lymphocytic lymphoma cell 40-50%.Right figure is with bscCD19 * CD3 treatment two all backs: no longer can find the evidence of small lymphocytic lymphoma cell migration, significantly increase and observe the T cell.
Figure 13: (A): (left figure before treating with bscCD19 * CD3; On May 2nd, 05) (right figure and after 2 weeks; On May 31st, 05) measures the size (shown in the table of table among Figure 13 (B)) of 4 mediastinal lymphoma focuses of patient #102003 by computer assisted x-ray tomography art (CT).(B): other selects lymphoma focus (the retroperitoneal and membranous) decline of effectively judging in size to quantize to lymphoma shown in (A) and 2, as described in the table as shown in (B).The percentage ratio decline that total lymphoma size descends and is expressed as these 6 selected lymphoma focus maximum cross section diameter product summations (being gross product diameter or SPD).Therefore, lymphoma is shunk 57.2% and be diagnosed as the standard that reaches reaction (PR) partly.
Figure 14: CD8 +T cell proliferation is relevant with different clinical responses.2 patient CD8 have been shown +The cell processes of TEM subgroup, wherein said patient promptly reaches the patient #109002 (reaction completely) and the #105003 (reaction of part) of different clinical responses.Also described respectively first and second 4 weeks to the contribution of overall clinical reaction and first 4 week area under TEM subgroup curve (AUC).Contribution to patient's clinical response maximum occurred in first 4 week of treatment, and well and CD8 +The propagation of TEM subgroup is relevant.The propagation of these height cytotoxic T cells is the precondition of clinical effectiveness seemingly, and this is based on following observation, and the absolute cell count that reaches when promptly breeding helps best clinical response with the relative increase both who compares the level before handling.
Figure 15: in patient #109002 by the detected ilium of CT scan (Iliacal) lymphoma (using circles mark): the size before the treatment of anti-CD19 * anti-CD3 bispecific single-chain antibody is: 3.2 * 2.2cm 2Behind the anti-CD19 of 4 all continuous infusions * anti-CD3 bispecific single-chain antibody, same lymphoma focus narrows down to 1,7 * 1.2cm 2Size.Behind the anti-CD19 of 8 all continuous infusions * anti-CD3 bispecific single-chain antibody, lymphoma disappears, and promptly size is normal to normal lymphoglandula size (0.8 * 0.8cm 2).
Figure 16: the spleen size normalizing that before the anti-CD19 of patient #109002 continuous infusion * anti-CD3 bispecific single-chain antibody makes treatment, sharply increases.
Now, the present invention will by with reference to following only for exemplary and be not interpreted as the restriction scope of the invention embodiment be described.
Embodiment 1:
1. make up and comprise CD19 * CD3 and the CD3 * CD19 bispecific single-chain antibody that various structural domains are reset
Usually, comprise specificity in conjunction with the antigenic structural domain of people CD3 and specificity in conjunction with each bispecific single-chain antibody molecule of the antigenic structural domain of people CD19 as name as described in the table 1 hereinafter.
Table 1: the shape that comprises anti-CD3 specificity and the specific bispecific single-chain antibody molecule of anti-CD19 Formula
The construct numbering SEQ ID No:(Nucleotide/protein) The form of protein construct (N → C)
1 1/2 VL(CD19)-VH(CD19)-VH(CD3)-VL(CD3)
2 3/4 VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3)
3 5/6 VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19)
4 7/8 VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19)
PCR method (Orlandi according to standard, Proc.Natl.Acad.Sci.USA 86 (1989), 3833-7) variable light chain (VL) structural domain of clone's HD37 hybridoma and variable heavy chain (VH) structural domain (Pezzutto, J.Immunol.138 (1997), 2793-9).Carrying out cDNA with oligomerization dT primer and Taq polysaccharase synthesizes.Based on D ü bel, J.Immunol.Methods 175 (1994), the described primer of 89-95 use with the primer 5 ' L1 (SEQ ID NO:19) and the 3 ' K (SEQ IDNO:20) of VL structural domain flank and be used for the 5 ' H1 (SEQ ID NO:21) of heavy chain and 3 ' G (SEQ ID NO:22) primer by the anti-CD19V structural domain of pcr amplification.Anti-CD3 scFv cDNA fragment is so kind as to give (Traunecker, EMBO be (1991) 3655-9 J.10) by Traunecker.
Construct 1 generation as mentioned below shown in the table 1.With corresponding VL and VH regional cloning to dividing in other plasmid vector, as with Oligonucleolide primers 5 ' VLB5RRV (SEQ IDNO:23)/3 ' VLGS15 (SEQ ID NO:24) and 5 ' VHGS15 (SEQ ID NO:25)/3 ' VHBspE1 (SEQ ID NO:26) being carried out the template of VL and VH specific PCR respectively, to obtain anti-CD19scFv fragment.Introduce overlapping complementary sequence in the PCR product, its combination is to form the 15-amino acid (Gly that merges subsequently among the PCR 4Ser 1) 3The encoding sequence of-joint.With primer 5 ' VLB5RRV (SEQ ID NO:23)/3 ' VHBspE1 (SEQ ID NO:26) is carried out amplification step, the fusion product (perhaps anti-CD19 scFv fragment) that obtains with Restriction Enzyme EcoRV and BspE1 cutting thereby and be cloned in the bluescript KS carrier (Statagene), it contains the encoding sequence (Kufer, Cancer Immunol.Immunother.45 (1997) 193) of (the EcoR1/Sal clone's) the anti-CD3 bispecific single-chain antibody of anti-17-1A/ (being actually the form that does not contain the Flag label).Therefore, anti-17-1A specificity is substituted by anti-CD19-scFV fragment, keeps to connect segmental 5 the amino acid whose Gly of the anti-CD3 scFv of C-terminal 4The Ser joint.Subsequently, coding had VL CD19-VH CD19-VH CD3-VL CD3(Mack, Proc.Natl. Acad.Sci.USA 92 (1995), 7021-5) to the EcoR1/Sal1 site of described expression vector pEF-DHFR for the dna fragmentation subclone of the anti-CD3 bispecific single-chain antibody of anti-CD19/ that structural domain is arranged.By electroporation with the plasmid DNA transfection that obtains in the Chinese hamster ovary celI of DHFR defective.As indicated abovely select, (Mack, Proc.Natl.Acad.Sci.USA 92 (1995), 7021-5) for gene amplification and protein production.Be shown in SEQ ID NO:1 corresponding to the dna sequence dna of the described construct 1 of table 1 as mentioned.The protein translation of this dna sequence dna (having leader sequence) is shown in SEQ ID NO:2.
Structure as described below is described other construct of table 1 above.Use dna sequence dna (its protein translation is shown in SEQ ID NO:2) as pcr template when stating the anti-CD19 single chain bispecific antibody of the various anti-CD3/ shown in the table 1 in design corresponding to SEQ ID NO:1 (construct 1).
Arrange in site A1 and A2, (to be defined) VH-VL that produces CD19 with dividing other combination of primers 5 ' VHCD19BsrGI (SEQ ID NO:36) and 3 ' VHCD19GS15 (SEQ ID NO:37) or 5 ' VLCD19GS15 (SEQ ID NO:38) and 3 ' VLCD19BspEI (SEQ ID NO:39) to carry out PCR as Figure 1A among the WO2004/106381 and 1B.In these PCR working cyclees, in the PCR product, introduce overlapping complementary sequence to form the encoding sequence that merges 15 amino acid joints among the PCR subsequently.(merging PCR) merges the VL and the VH structural domain of amplification in second PCR reaction, wherein only uses outside primer (i.e. 5 ' VHCD19BsrGI (SEQ ID NO:36) and 3 ' VLCD19BspEI (SEQ ID NO:39)) and needs this two kinds of amplicons.
Use utilizes the similar approach of other combination of primers to make up other structural domain and arranges.Design one group of suitable primer to carry out clone's step, finally obtain various VL-VH structural domains and arrange based on multiplex PCR.Used combination of primers is listed in the table below:
Table 2: make up the site A1 of the construct 1-4 shown in the table 1 and the gram of the employed PCR-based of A2 Grand overview of steps
Figure A20068004716200381
The following primer that design comprises described Restriction Enzyme recognition site carries out clone's step of PCR-based to change the VH-VL structural domain arrangement of C end position, i.e. defined site B1 of the Figure 1A of WO2004/106381 and 1B and B2.
Table 3: make up the site B1 of the construct 1-4 shown in the table 1 and the gram of the employed PCR-based of B2 Grand overview of steps
Figure A20068004716200391
The corresponding PCR product cloning that with flank is two BspEI sites is in the plasmid that is called BS-CTI with BspEI and the digestion of XmaI Restriction Enzyme.Insert in the Bluescript KS carrier (GenBank accession number X52327) so that other cleavage site and coding G to be provided with Restriction Enzyme cutting site XbaI and SalI at the polylinker that will be called CTI (SEQ ID NO:45) before 4The sequence of S joint, 6 successive histidine residues and terminator codon.In this clone's step, thereby the fusion of the XmaI site of the BspEI site of VH structural domain and plasmid destroys this two sites.Correct direction according to the method sequence verification variable domains of standard.
All molecular biology methods mentioned above are according to Sambrook, Molecular Cloning (laboratory manual, the 3rd edition, cold spring port experiment press (Cold Spring Harbor LaboratoryPress), New York, the described standard method in cold spring port (2001) is carried out.
With DNA (SEQ ID NO:1,3,5, the 7) transfection of the bispecific single-chain antibody of coding schedule 1 in the Chinese hamster ovary celI of DHFR defective with (Mack as described in people such as Mack, Proc NatlAcad Sci USA 92 (1995) 7021-25) carries out eukaryotic protein and expresses in the Chinese hamster ovary celI of DHFR defective.The final concentration that is increased to 20nM by the concentration with methotrexate (MTX) is induced the gene amplification of construct.Enlarge institute's cells transfected then and produce 1 and go up clear liquid.
2. express and the bispecific single-chain antibody of anti-CD3 of purifying and CD9
(CHO) expresses bispecific single-chain antibody in Chinese hamster ovary cell.Handle (" Molecular Cloning ", people such as Sambrook, 1989) according to the calcium phosphate of cell and carry out the expression vector transfection.In rolling bottle, use the CHO DMEM substratum (HiQ of improvement HiClone) culturing cell was gathered in the crops in 7 days then.By the centrifugal cell that removes, will contain expressed proteinic supernatant liquor and be stored in-20 ℃.
With
Figure A20068004716200402
FPLC System (Pharmacia) and Unicorn
Figure A20068004716200403
Software carries out chromatography.All chemical reagent be research grade and available from Sigma (Deisenhofen) or Merck (Darmstadt).The method that provides according to manufacturers is with being loaded with ZnCl 2Fractogel Post (Merck) carries out fixed metal chelate affinity chromatography (" IMAC ").With buffer A 2 (20mM sodium phosphate buffer, pH 7.5,0.4M NaCl) balance columns and with the flow velocity of 3 ml/min cell culture supernatant (500ml) is added in the post (10ml).Wash post to remove unconjugated sample with buffer A 2.With the protein of buffer B 2 (20mM sodium phosphate buffer pH 7.5,0.4MNaCl, 0.5M imidazoles) elution of bound of two step gradients, as described below:
20% buffer B 2 of step 1:6 column volume;
100% buffer B 2 of step 2:6 column volume.
The elute protein fraction of combining step 2 is used to be further purified.
On with PBS (Gibco) equilibrated Sephadex S200 HiPrep post (Pharmacia), carry out gel permeation chromatography.SDS-PAGE and western blotting that the protein example of wash-out (flow velocity 1 ml/min) carries out standard detect.Calibrate post with molecular weight determination (molecular weight standard test kit, Sigma MW GF-200) before the purifying.With the protein determination dyestuff (MicroBCA, Pierce) and IgG (Biorad) determine protein concn as standard protein.
In the two-step purifying method of IMAC and gel-filtration, separate bispecific single-chain antibody.Determine that by gel-filtration in PBS main product has the molecular weight of about 52kDa under natural condition.This molecular weight is corresponding to bispecific single-chain antibody.All constructs are according to this method purifying.
The bispecific single-chain antibody protein of purifying is analyzed by carry out SDS PAGE under reductive condition with prefabricated 4-12%Bis Tris gel (Invitrogen).Carry out specimen preparation and application according to the method that manufacturers provides.With MultiMark protein standard (Invitrogen) determining molecular weight.Gel dyes with colloid coomassie (Invitrogen method).Measure by SDS-PAGE, isolating proteinic purity is greater than 95%.
The method Optitran that provides according to manufacturers
Figure A20068004716200411
BA-S83 film and Invitrogen BlotModule carry out western blotting.Used antibody is that (Penta His, Qiagen) antibody and alkaline phosphatase (AP) the anti-mouse Ig of labelled goat (Sigma) use BCIP/NBT (Sigma) as substrate to anti-His Tag.Bispecific single-chain antibody can detect specifically by western blotting.Detect single band at 52kD place corresponding to the bispecific molecule of purifying.
3.CD19 the flow cytometry binding analysis of * CD3 specific polypeptide
Carry out facs analysis to detect construct in conjunction with the CD19 function relevant with the CD3 ability.Positive NALM-6 cell (human B cell precursor leukemia) of CD19 and the positive Jurkat cell (HTL) of CD3 have been used for this purpose.200, the pure cell conditioned medium liquid of Chinese hamster ovary celI culture (second section is described as mentioned) of the bi-specific antibody that different VH that 000 NALM-6 cell and 200,000 Jurkat cells express to have CD19 and CD3 with 50 μ l respectively and VL structural domain are arranged was hatched on ice 30 minutes.The combination of twice of washed cell and following detection construct in PBS.The cell of Chu Liing contacts with unlabelled mouse Penta His antibody (being diluted among the 50 μ l PBS that contain 2%FCS at 1: 20) as mentioned above, the C end of wherein said unlabelled mouse Penta His antibody by construct histidine-tagged with the construct specificity combination that combines cell.Carry out washing step afterwards to remove unconjugated mouse Penta His antibody.The Fc γ specific antibody (Duanova) that is conjugated with phycoerythrin that was diluted in the 50 μ l PBS that contain 2%FCS with 1: 100 detects the anti-His antibody of bonded.Fresh culture substitutes culture supernatants as negative control.
(Becton Dickinson Heidelberg) goes up analysis of cells at the FACS-Calibur instrument by flow cytometry.As described FACS dyeing and the fluorescent strength determining of carrying out of Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).The binding ability that several structural domains are arranged clearly can detect shown in WO2004/106381.In facs analysis, have different all constructs arranged of CD19 and the specific VH of CD3 and VL structural domain and all show and the combining of CD3 with comparing with the negative controls of substratum and 1,2 detection antibody.In the construct that table 1 is mentioned, observe strong in conjunction with the active fluorescence intensity variation＞5 * 10 that causes 1
4.CD19 and the biological activity of CD3 bispecific single-chain antibody
Use the determination of cytotoxic activity method that discharges based on fluorochrome to measure the cytotoxic activity of the bi-specific antibody of VH and the rearrangement of VL structural domain.The positive NALM-6 cell of CD19 is as target cell (1.5 * 10 7Individual), its in 37 ℃ in cell culture medium with 10 μ M fluorexon AM (MolecularProbes, Leiden, Netherland, numbering C-1430) mark 30 minutes.After the washed twice, pair cell is counted and is mixed with CD4 positive T cell clone's CB15 cell (doctor Fickenscher of German University of Erlangen/Nuernberg is so kind as to give) in cell culture medium.Every milliliter is mixed 2 * 10 6Individual CB15 cell and 2 * 10 5Individual NALM-6 cell (E: T=1: use 50 these suspensions of μ l 10) and in each hole of 96 hole circle base plates.Antibody is diluted to desired concn and 50 these solution of μ l are added in the cell suspension in RPMI/10%FCS.At 37 ℃/5%CO 2In hatch the reaction 2 hours of standard.After the cell-cytotoxic reaction, hatch the dyestuff that discharges in the substratum can be in the fluorescence reader (Tecan, Crailsheim, Germany) quantitatively and with the fluorescent signal of control reaction (not containing bi-specific antibody) and the fluorescent signal (in 1% saponin(e, hatching 10 minutes) of whole lysing cell compare.On the basis of these readings, calculate than cytotoxicity according to following formula: [fluorescent value (sample)-fluorescent value (contrast)]: [fluorescent value (all cracking)-fluorescent value (contrast)] * 100.
Prism software (GraphPad Software Inc., San Diego, USA) definite S shape dose response curve generally have＞0.97 R 2Numerical value.EC by routine analyzer calculating 50Value is used for the comparison biological activity.
Shown in WO2004/106381, all constructs are at the NALM-6 cell showed cell toxicity of expressing CD19.In the construct shown in the table 1 above, detected the strong biological activity of EC50 value＜500pg/ml.
Embodiment 2:
Clinical application bscCD19 * CD3 in the B cell lymphoma patient
In the application of approval, the patient (women, nineteen thirty-seven gives birth to) who suffers from the cell-derived chronic lymphocytic leukemia of B (B-CLL) is as describing the treatment with bispecific single-chain antibody bscCD19 * CD3 (SEQ ID NO.2) in detail among the WO 99/54440.Facs analysis shows that patient's 95% peripheral blood cells is the CD19 positive cell, and 77% cell expressing CD20 antigen.Peripheral blood of patients cell and bscCD19 * CD3 are hatched the remarkable exhaustion that has shown the positive B cell of CD19.For preventing any acute cytokine reaction and tumour cracking and freezing, use the 2mg clemastine (Tavegil of the IV dosage of prevention to the patient ) and 200mg Cimitidine Type A/AB (Tagamet
Figure A20068004716200432
) and 300mg Zyloric and 20mg omeprazole (Antra
Figure A20068004716200433
).
The first administration of patient is 3 μ g bscCD19 * CD3 that 20 minutes infusions are dissolved in the isotonic phosphate buffer liquid that contains 5% human serum albumin (HSA).The patient does not show any untoward reaction in infusion process.About 1 hour patient feels cold and perspired then in about 5 minutes behind the infusion, and the blood pressure appropriateness reduces about 10mmHg and the body temperature appropriateness increases (+0.5 ℃) a few hours.In addition, her headache symptom slightly worsens.The patient is with other 2mg Tavegil
Figure A20068004716200434
With 200mg Tagamet
Figure A20068004716200435
250mg Ultracortene-H (Solu-Decortin
Figure A20068004716200436
) and 50mg Pethidine (Dolantin
Figure A20068004716200437
) treatment.All symptoms are all obtaining release on the same day, and do not have sequela.
Using second dose after one day under above-mentioned the same terms, is 10 μ g bscCD19 * CD3.About 1 hour patient obviously feels cold, has a fever (39.2 ℃), slightly breathes and hypotension behind the infusion.The patient uses 2mg clemastine, 200mg Cimitidine Type A/AB, 300mg Solu-Decortin and 15mg Piritramide (Dipidolor ) treatment.For stablizing her cardiovascular function, the patient accepts the Dopamine HCL infusion and carries out volume and replace.Symptom significantly reduces after this treatment.
In ensuing 3 days, the patient continues as low-grade fever body degree (about 37.2 ℃), 1 day slight slight edema of pleural effusion and lower end of appearance after second dose of administration.
The ultrasonic examination of spleen, 5 lymphonodi abdominaleses and nodi lymphatici axillares is carried out in second dose of 1 day and 4 days of using after bscCD19 * CD3.Lymphoglandula before 1 day lymphoglandula after the 10 μ g dosed administrations and spleen and the treatment is compared with spleen and is dwindled about 20%.This observes in ultrasonic evaluation for the second time and has obtained checking.The weight of spleen has reduced 350g (from treating the 1280g after preceding 1630g is reduced to treatment).
In several days after the therapeutic process neutralization, the quantity of white corpuscle (comprising most of Malignant B cells) reduces.C reactive protein (CRP) is the acute phase reactive protein of reflection T cell activation and pro-inflammatory cytokine effect.It significantly increases after using 10 μ g bscCD19 * CD3, afterwards continuous decrease in ensuing 3 days observe.
Measure reflection administered compound acute immunoreactive serum cytokines level afterwards a plurality of intervals before administered compound and after the administered compound.Explanation according to manufacturers is measured the serum cytokines level by the quantitative ELISA assay method.Use that tumor necrosis factor TNF-alpha significantly increases in dose-dependent mode in first hour after bscCD19 * CD3.Interleukin 6 (IL-6) and interleukin 8 (IL-8) also show remarkable and dose-dependent increase.Their highest level was observed after using bscCD19 * CD3 in 2-4 hour.
In a word, bscCD19-CD3 can use for safely the patient who suffers from intractable B-CLL.The observed release of cytokines though disadvantageous side effect is likely, the tolerance of the bscCD19 * CD3 of 3 μ g and 10 μ g dosage are acceptables and can control well by the treatment of preventative mensuration and symptom.Importantly, bscCD19 * CD3 causes dwindling of the spleen that becomes big before the patient and lymphoglandula shown in ultrasonic examination.Since the increase of spleen and lymphoglandula is dwindled so and has been reflected owing to use the destruction that bscCD19 * CD3 causes the Malignant B cell because the Malignant B cellular infiltration causes.
Embodiment 3:
The dosage escalation I phase is studied-patient 0202
The purpose that this dosage escalation I phase is studied is to study the dose-dependently that immunocompetence changes in B cell non-Hodgkin's (NHL) the patient tumors target tissue that recurs.For this purpose, use anti-CD19 * anti-CD3 bi-specific antibody (SEQ ID NO.2 6 times for each patient's intravenously; Also see WO 99/54440), the 0th, 2, surpass 4 hours in the treatment plan of 7,9,14 and 16 days weekly or twice and use.In individual, increase the dosage of anti-CD19 * anti-cd 3 antibodies gradually: 1,2,4,7,10 and 13 micrograms/square metre body surface area with following dosage.
Collect before the application and the whole blood sample in the process to estimate increase bscCD19 * CD3 dosage to the quantity of circulating lymphocyte subgroup and the influence of distribution.Measure the quantity of lymphocyte subgroup by the lymphocytic sum of differential blood count assay and by facs analysis, and as described in embodiment 2, be expressed as total lymphocyte percentage ratio.Especially, measured CD3 in the peripheral blood lymphocytes by flow cytometer (FACS) +, CD4 +And CD8 +The T cell quantity.
As the illustrative patient (being patient 0202) who finishes the pharmacological agent phase of Fig. 1,, still observe CD3 in during 4 hours infusion even use the bi-specific antibody of minimum initial dose +, CD4 +And CD8 +Suddenly the reduction of T cell quantity (showing) with total lymphocyte percentage ratio.The numerical value of T cell quantity before 24-38 hour returns to about administration.In other patient that this research comprises, observe the Cytometric similar curve process of T.A kind of T of being interpreted as cell of this reduction and other activated lymphocytes are sticked suddenly and are moved in the tissue at least in part.A kind of hypothesis is that this kind activation suddenly and the migration of a large amount of activated T cell colonys (about 80% of human T-cell storehouse all rests in the peripheral blood at any time) causes the destruction that the local cells factor is formed in imbalance of T Cell Homeostasis and the tissue.Positively, cause disadvantageous side effect for patient's drug administration according to mode shown in Figure 1.This result shows progressively activating and move more near the physiologic immunity reaction and can avoiding lacking of proper care suddenly and causing the phenomenon of adverse side effect thus as local cells factor network of T cell.
Embodiment 4:
I clinical trial phase-patient 1003
In another clinical trial, in intractable inertia or rapid spread B cell non-Hodgkin's (B NHL) and B cell leukemia patient, detect the anti-CD19 * anti-cd 3 antibodies of repeated intravenous infusion dual specific strand (SEQ ID NO.2; Also see among the WO 99/54440) security, maximum tolerated dose and best biological dose.Gave 6 anti-CD19 * anti-cd 3 antibodies of each patient's infusion above two hours or above four hours at the 0th, 2,4,14,16 and 18 day that studies.Totally 15 patients are with 3.0 μ g/m 2Dosage treatment.
Different time points before drug administration or in the application gets that blood sample is biochemical to follow the tracks of, hematology and amynologic parameter.Measure the T cell quantity as mentioned above.In addition, use enzyme-linked immunosorbent assay (ELISA) and fluidic cell bead microarray art (CBA) to measure the serum level of patient's cytokine.
When the T cell quantity among the subject patient of monitoring, observe the big ups and downs of T cell quantity each time after the infusion, show the short-term in the research before also having observed, the T cell activation of burst sample; See embodiment 3.In addition, example shown in Fig. 2-5 is diagnosed as patient's (being patient 1003) of lymphoma mantle cell (MCL), behind the infusion bi-specific antibody, in the patient of treatment, find the violent release of cytokines of TNF α, interferon-gamma, interleukin-6 and interleukin 10.Release of cytokines intensity after using initial dose is the highest, and the reaction of the anti-CD19 of infusion * anti-CD3 bi-specific antibody subsequently reduces (first-dose response).Almost do not observe inducing of cytokine levels after 6 infusions.Research shown in embodiment 3, unexpected T cell activation and release of cytokines cause the disadvantageous side effect to the patient.Great majority in these incidents have the seriousness of gentleness or appropriateness, and all are temporary transient.The most often observe blood parameters and the laboratory abnormalities of solidifying parameter, temporary transient when same, and be gentle to appropriate mostly on character and clinical significance.The result who obtains in this research shows that more progressive T cell activation is to avoid necessary as the disorderly suddenly phenomenon of observed local cells factor network in the above-mentioned clinical trial.
Embodiment 5:
1. non-with the treatment of anti-CD19 * anti-CD3 bispecific single-chain antibody (SEQ ID NO.2) continuous infusion Hodgkin lymphoma (B NHL) patient's research
Estimate anti-CD19 * anti-CD3 bispecific single-chain antibody (bscCD19 * CD3 by long-term infusion administered compound; Aminoacid sequence shown in the SEQ ID NO.2) security and tolerance.B cell non-Hodgkin's (B NHL) patient of two example recurrences is shown in part 2 and the 3rd part (patient #105003 and patient #102003) hereinafter.At first day of treatment, two patients accepted the 5 μ g/m of " initial exposure dosage " with continuous intravenous infusion (i.v.) 2/ 24 hours anti-CD19 * anti-CD3 bi-specific antibody.Afterwards dosage is increased to gradually 15 μ g/m 2/ 24 hours " maintenance dose ", described dosage are used with continuous intravenous infusion in during the infusion in 2 weeks of remainder (patient #102003) or 4 weeks (patient #105003).The grade that the per daily dose of the medicine of studying contains 5%HAS at 500ml is oozed in the NaCl solution and is used.Each patient's acceptor concentration is 1 hour release of cytokines with the inhibition infusion starting stage of combination therapy (i.v.) of the glucocorticosteroid (methyl meticortelone) of 1 * 100mg before the beginning infusion bi-specific antibody.
The 4th part of present embodiment 5 provides with " initial exposure dosage " (0.5-5 μ g/m 2/ 24 hours) and 15 μ g/m 2Other summary data of the patient of " maintenance dose " treatment of/24 hours.
2. be diagnosed as the patient #105003 of follicular lymphoma (B cell non-Hodgkin's)
This male patient of 44 years old was diagnosed as follicular lymphoma in 2000.The patient accepted repeatedly chemotherapy (CHOP in the past; Bendamustin) and immunotherapy (Rituximab; Anti-CD-20 monoclonal antibody).Disease was once more serious after participating in the inoculation test in 2 years.Autumn in 2005, the patient experience bone marrow infiltration causes anaemia; Lung soaks into and causes the breathing function damage.In addition, this patient's quality of life is owing to the deterioration of B-symptom (splenomegaly, Sleep hyperhidrosis increase and 3 all weight loss 3kg) obviously reduces.It is essential that this clinical condition becomes new therapy.
Hereinafter, the patient describes 4 weeks of continuous infusion in detail as mentioned with 15 μ g/m 2/ 24 hours dosage level is accepted anti-CD19 * anti-CD3 bispecific single-chain antibody (SEQ ID NO.2).Treatment is not promptly observed remarkable disadvantageous side effect since on October 3rd, 2005 and well tolerance.
2.1 T cell activation and propagation that anti-CD19 * anti-CD3 bispecific single-chain antibody causes
With after following 4 kinds of different antibodies or the surface marker compound staining by four look flow cytometry according to the monocyte of research method in the patient #105003 of different time points peripheral blood sample:
The 1st group: CD2 * CD3 * CD4 * CD8 (T cell)
The 2nd group: CD3 * CD16 * CD19 * CD56 (lymphocyte count: B cell, T cell and NK cell)
The 3rd group: CD8 * CD25 * CD69 * HLA-DR (CD8 +The T cell activation)
The 4th group: CD8 * CD28 * CD45RA * CCR7 (CD8 +The T cell subsets)
Calculate the mean absolute lymphocyte subgroup by the 2nd batch total and (promptly be defined as CD19 respectively +/ CD3 -, CD3 +And CD3 -/ CD56 +The B cell of cell, T cell and NK cell) number, be expressed as in (B cell, T cell or NK cell)/(%B cell+%T cell+%NK cell) multiply by the total lymphocyte number of each blood sample that routine test measures.Calculate absolute CD4 by the 1st batch total +And CD8 +The T cell count is represented %CD3 respectively +CD4 +CD8 -Cell/%CD3 +Cell or %CD3 +CD4 -CD8 +Cell/%CD3 +Cell multiply by the absolute T cell count of being calculated by the 2nd batch total.CD8 +The absolute CD8 that calculates from the 2nd batch total is multiply by in the absolute cell counting of T cell subsets by the following percentage that obtains from the 4th group +The T cell counting is calculated:
%CD8 +CD45RA +CD28 +Cell/%CD8 +Cell is (corresponding to inmature CD8 +The T cell),
%CD8 +CD45RA -CCR7 +Cell/%CD8 +Cell is (corresponding to maincenter memory CD8 +T cell=T CM),
%CD8 +CD45RA -CCR7 -Cell/%CD8 +Cell is (corresponding to effect memory CD8 +T cell=T EM) and
%CD8 +CD45RA +CD28 -Cell/%CD8 +Cell is (corresponding to the positive effect memory of CTL (the cytotoxic T cell)=CD45RA CD8 of end differentiation eventually +T cell=T EMRA).
T cell activation among this patient is determined as expresses all CD8 +The %CD8 of the cell surface of T cell activation mark CD25, CD69 or HLA-DR (flow cytometry the 3rd group) +The T cell.
At first, analyzed CD4 +And CD8 +The T cell counting.As shown in Figure 6, CD8 +And CD4 +The T cell disappears from peripheral blood after the anti-CD19 of beginning infusion * anti-CD3 bispecific single-chain antibody to a great extent, and it is interpreted as the T cell by the anti-CD19 * periphery blood T cell of anti-CD3 bispecific single-chain antibody mediation and the distribution of the crosslinked initiation of B cell.Yet, after the treatment half cycle, CD8 +And CD4 +The T cell reappears in blood and further accelerates respectively up to the 7th day and the 21st day.Compare CD8 with their initial value +And CD4 +The T cell shows 3.5-4 amplification doubly in blood.CD8 +And CD4 +The T cell counting during the 2nd week of treatment and the 3rd week and the 4th week treatment T cell quantity remain high before beginning to reduce.When the tumour when diagnosis essence as mentioned below is dwindled the reaction normal that satisfies part, the anti-CD19 of infusion * anti-CD3 bispecific single-chain antibody CD8 after 4 weeks +And CD4 +The T cell counting still is higher than the preceding numerical value of corresponding treatment.
Next, analyzed CD8 in further detail +The T cell subsets.As shown in Figure 7, CD8 +T cell subsets analysis revealed, effect memory subgroup TEM almost ad hoc is responsible for by anti-CD19 * anti-CD3 bispecific single-chain antibody inductive CD8 +The propagation of T cell.CD8 +The T cell is responsible for most cytotoxic activity in all T cells, and TEM-cell and the responsible together CD8 of TEMRA subgroup +Most cells cytotoxic activity in the T cell.Except the TEM-subgroup, at other CD8 +Fail to observe the noticeable change in the cell counting between the T cell subsets, can not breed based on the single activation signals that anti-CD19 * anti-CD3 bispecific single-chain antibody provides as inmature T cell, perhaps the TEMRA-subgroup can not be bred fully.Therefore, the CD8 that can breed +The selective proliferative of TEM cell can be subjected to the CD8 that anti-CD19 * anti-CD3 bispecific single-chain antibody is modified owing to replying clearly in the tumour +Cell fission and propagation that the TEM cell contacts with the B lymphoma cell; Also see Figure 14.Since cyclicity B lymphoma cell the 3rd day, promptly from peripheral blood, exhaust before observing T cell proliferation, so can get rid of the remarkable contribution of B lymphoma cell that the anti-CD19 of cyclicity in the blood * anti-CD3 bispecific single-chain antibody is modified T cell proliferation.
At last, analyzed CD8 +The activation of T cell is because the marker of T cell activation as shown in Figure 8.Highly activating CD8 +CD8 after the T cell +T cell proliferation, wherein CD8 +The height activation of T cell is represented by the activation mark HLA-DR that continues to raise.Other activation mark is CD69 and the only of short duration rise of demonstration behind initial infusion construct of the present invention of CD25 for example, this can explain by the activation of the anti-CD19 of cyclicity in the peripheral blood * B lymphoma cell that anti-CD3 bispecific single-chain antibody is modified, because cyclicity B lymphoma cell has just been exhausted during first 3 days of treatment.On the contrary, surpass the lasting HLA-DR of rise of 3 weeks and reflected CD8 in the tumour +The T cell is by contacting the activation that causes with the intrinsic B lymphoma cell of tumour, and wherein said B lymphoma cell is subjected to the modification of anti-CD19 * anti-CD3 bispecific single-chain antibody.The activation of T cell causes the proliferative T lymphocyte reaction in the tumour in the knurl, and then causes T cell proliferation, and this also appears in the circulation blood then.These activate and move the T cell that enters blood and still show long-term activation mark HLA-DR in tumour, but short-term activation mark CD69 and CD25 have reduced.
As conclusion, use the treatment of anti-CD19 * anti-CD3 bispecific single-chain antibody to cause secular T cell activation and propagation.The T cell of tool cytotoxicity phenotype is mainly owing to CD8 +The propagation of T cell.T cell activation and propagation are induced by the B lymphoma cell that anti-CD19 * anti-CD3 bispecific single-chain antibody is modified in tumour.The T cell activation reduces and reduces and can explain by the anti-CD19 * effectiveness of anti-CD3 bispecific single-chain antibody in this patient in T cell count between the period 4 between the period 3 of infusion, this causes tumour shrinking 58.0% (seeing below) with being diagnosed as after anti-CD19 * anti-CD3 bispecific single-chain antibody treated for 4 weeks, also therefore reducing B lymphoma cell sum, and this can induce T cell activation and propagation by anti-CD19 * anti-CD3 bispecific single-chain antibody mediation.
2.2 consume peripheral blood B (lymphoma) cell by anti-CD19 * anti-CD3 bispecific single-chain antibody
Use at the monocyte that reacts the peripheral blood sample of the research patient #105003 that obtains after the antibody compound staining of following four kinds of cell surface markers according to the method for standard by four look flow cytometry in different time points up to optimal clinical: CD3 (T cell sign thing) * CD16 (NK cell/scavenger cell mark) * CD19 (B cell sign thing) * CD56 (NK cell sign thing).
Be defined as CD19 respectively +/ CD3 -, CD3 +And CD3 -/ CD56 +The lymphocyte subgroup of cell is each blood sample of Routine Test Lab mensuration is multiply by in the absolute cell counting of B cell, T cell and NK cell by % (B, T or NK cell)/(%B+%T+%NK cell) total lymphocyte counting calculating.Therefore, the absolute quantity of B (lymphoma) cell is calculated as CD19 +/ CD3 -The sum of cell.
As shown in Figure 9, the patient begins with 140 B of about every milliliter of blood (lymphoma) cell before treating.Behind the anti-CD19 of beginning infusion * anti-CD3 bispecific single-chain antibody, cyclicity B (lymphoma) cell descends rapidly in first 3 days of treatment, and finally disappears from peripheral blood fully at first all treatments end.In addition, B (lymphoma) cell remains in the peripheral blood and does not exist, until the reaction of diagnosing part after 4 weeks of treatment; See below.These data show, because anti-CD19 * anti-CD3 bispecific single-chain antibody is to the cytotoxic activity of described cell, so it can remove cyclicity B (lymphoma) cell fully.
2.3 reduce lymphadenomatous size by anti-CD19 * anti-CD3 bispecific single-chain antibody
Use the stdn reaction normal of non-Hodgkin lymphoma (NHL) reaction evaluating to estimate the effect that anti-CD19 * anti-CD3 bispecific single-chain antibody is treated.Select 6 representative lymphoma focuses according to these standard needs.The product summation of lymphoma focus maximum cross section diameter is defined as baseline SPD (Sum Product Diameters).Estimate in the whole therapeutic process and the rule basis (regular base) in research later stage with the variation of this SPD then.For example, Bu Fen reaction be defined as SPD reduce by 50% or more than.As shown in figure 10, (restaging) more by stages after 4 weeks of treatment find obviously to reduce according to NHL reaction evaluating lymphoma piece mentioned above: diagnose 6 to reduce with reference to 58% SPD in the lymphoma focus, the reaction (PR) of the part in the tumor response of estimating corresponding to X ray computer tomography art (CT).With 8 week of anti-CD19 * anti-CD3 bispecific single-chain antibody treatment the back verified the reactions of part by X ray computer tomography art, shown that 66% tumour is dwindled (SPD).
3. be diagnosed as the patient #102003 of small lymphocytic lymphoma (B-CLL)
60 years old male patient of this example is diagnosed as small lymphocytic lymphoma in April, 1999.Relevant discovery in patient's medical history has sleep apnea comprehensively card, acute renal failure, zoster, intermittent bronchitis and intermittent hepatopathy.The patient has repeatedly chemotherapy (Chlorambucil, fludarabine, Knospe, endoxan), immunotherapy (Rituximab; Anti-CD-20 monoclonal antibody) and the history of abdomen part radiotherapy.
In the medical history in 7 years, the patient accepts 7 kinds of different chemotherapy regimens and Rituximab as monotherapy or conjoint therapy and the radiotherapy that do not show any principal reaction.
Using anti-CD19 * anti-CD3 bispecific single-chain antibody is this patient's the 13rd kind of treatment plan.The patient with the continuous infusion that above describes in detail with 15 μ g/m 2/ 24 hours dosage is accepted anti-CD19 * anti-CD3 bispecific single-chain antibody (SEQ ID NO.2) 2 weeks.Treatment was since on May 9th, 2005.The treatment back took place completely more by stages in two weeks, and its result shows hereinafter.
3.1 peripheral blood B (lymphoma) cell that anti-CD19 * anti-CD3 bispecific single-chain antibody causes consumption Exhaust
The described definite B cell count of 2.2 parts as mentioned.As shown in figure 11, in every milliliter of blood of patient 920 B (lymphoma cell) cell is arranged before the treatment.After the anti-CD19 of beginning infusion * anti-CD3 bispecific single-chain antibody, circulation B (lymphoma) cell descends rapidly, is the limited temporary transient stage that returns to every milliliter of 600 cells in initial 3 days afterwards, and this is consistent with the cell redistribution.In further therapeutic process, B (lymphoma) cell is exhausted the reaction that is diagnosed as part after two weeks of treatment fully from circulation.These digital proofs are as the clinical efficacy of its part, and anti-CD19 * anti-CD3 bispecific single-chain antibody can be eliminated round-robin B (lymphoma) cell fully.
3.2 eliminating the lymphoma of marrow, anti-CD19 * CD3 bispecific single-chain antibody soaks into
In addition, before with anti-CD19 * anti-CD3 bispecific single-chain antibody treatment and after treating, carry out myelosis experiment of science.Shown in the left figure of Figure 12, can be observed the bone marrow infiltration of small lymphocytic lymphoma 40-50% before the treatment.Proved the diagnosis of small lymphocytic lymphoma/CLL.
Shown in the right figure of Figure 12, with not finding after the treatment of anti-CD19 * anti-CD3 bispecific single-chain antibody that tangible small lymphocytic lymphoma soaks into, and observe the remarkable increase of T cell.These data show that anti-CD19 * anti-CD3 bispecific single-chain antibody eliminated lymphocyte from marrow, and wherein said lymphocyte is under detectability.This finds that with myeloid reaction is consistent fully this for example is to be difficult to realize under conventional chemotherapy.
3.3 the lymphoma that anti-CD19 * anti-CD3 bispecific single-chain antibody causes size reduces
In addition, the lymphoma piece of by stages observing again after the treatment of 2 weeks obviously reduces: as shown in figure 13, X ray computer tomography art (CT) diagnoses 6 sizes with reference to the lymphoma focus to reduce by 57.2%, the reaction (PR) of the part in estimating corresponding to tumor response.
In the medical history in 7 years, the patient accepts 7 kinds of different chemotherapy regimens and immunotherapy and radiotherapy, do not show any main reaction, the therapy of a success is provided, has dwindled more than 50% with reference to the lymphoma focus with anti-CD19 * anti-CD3 bispecific single-chain antibody treatment patient for the first time because realized.
4. clinical data is summed up
According to the 3+3 dosage escalation regimens of classics with 0.5 μ g/m 2The patient of the inertia NHL of/24 hours predose treatment recurrence.Begin to use the release of cytokines symptom that steroid is mentioned with alleviation.By CTC-AE (common adverse events standard) evaluate safety and tolerance.The common adverse events standard of NCI (American National ICR) is for can be used for the descriptive term of adverse events (AE) classification.For each AE standard provide classification (seriousness) scope and only after data are checked the security that the council (data review committee) sums up from 14 days the first predose with DLT (dose-limiting toxicity (doselimiting toxicity)) just permissible dose improve.By the periphery immunocyte being carried out quantitatively and description monitoring biological activity with specific ELISA research cells in vivo factor level and by facs analysis.Anti-CD19 * contrast CT (X ray computer tomography art) scanning after anti-4 weeks of CD3 bispecific single-chain antibody treatment.If according to standardized Cheson standard (by the investigation of center radiology department) patient is stable at least, so the patient is carried out extra 4 all anti-CD19 * anti-CD3 bispecific single-chain antibody cycle again.The Cheson standard can be set up by National Cancer Institute and Inpharm industry the guidance of estimating the NHL clinical response is provided.According to this standard, the clinical and radiation sign completely dissolve when detecting of all diseases and disease related symptom, all lymphoglandula recover normal size, and spleen has obtained reaction completely when recovering not have lymphoma in size and the marrow.Concerning the reaction of part, the size of the obvious lymphoglandula of 6 maximums is essential to reduce by 50%, and the size of other lymphoglandula, liver and spleen does not increase, and spleen tubercle, liver tubercle must dwindle at least 50% and must not have new disease location.
So far, 19 patients that comprised the median of the chemotherapy/immunotherapy before having 4.From DL1 (0.5 μ g/m 2/ 24 hours) (DL=dosage level) to DL3 (5 μ g/m 2/ 24 hours) dosage enlarge during, observe dose-limiting toxicity, and AE (side effect) is generally medium.At DL4 (first day 5 μ g/m 2/ 24 hours, maintenance dose 15 μ g/m 2/ 24 hours), treated 7 patients, 2 acceptance wherein are less than 14 days treatment (1DLT interrupts 1 time when each investigator determines).
But in 14 assess patient (＞2 weeks of treatment, and before the anti-CD19 of infusion * anti-CD3 bispecific single-chain antibody, B cell in the peripheral blood is detectable) in, there are 9 after finishing CD19 * CD3 bispecific single-chain antibody infusion, to observe cyclicity B (lymphoma) cell and exhaust fully.At DL4, the frequency that dose-dependently increases reaches 100%.At DL4,3 among 7 patients have relevant marrow (BM) and soak into (＞10%), and wherein 1 patient shows improvement, and 2 patients show that lymphoma cell is from the BM completely dissolve.In 14 assess patient (＞2 weeks of treatment, and scan all relevant ranges) in, best whole tumor response (overall tumourresponse) is 1 CR (reaction completely in patient #109002, described patient is the 61 years old male patient who is diagnosed as 1 grade of follicular lymphoma, IIIa phase), 2 PR (reaction of part), 1 MR (little reaction (minor response)), 7 SD (stable disease) and 3 PD (disease of deterioration).Especially, patient #109002 has shown further impressive result, promptly eliminates fully (CR); See Figure 14-16.This elimination completely in described patient treated first day 5 μ g/m by 4 weeks 2/ 24 hours predose and 15 μ g/m 2/ 24 hours follow-up dosage is realized.After this 4 week, the reaction of patient #109002 display part, and the patient accepts further 15 μ g/m of other 4 weeks immediately 2/ 24 hours dosage, and then cause reacting completely.
Be not subjected to hereinafter to limit, this area is as give a definition " reaction " completely, " reaction of part ", " disease of deterioration " and " little reaction ":
If detect less than focus by CT imaging and other test, so just be commonly referred to reaction completely.If CT imaging and marrow test show there is not disease indication, it has just defined reaction completely more rigorously so.
The reaction of part is described wherein to detect and can be measured descend at least reaction of 50% therapy of tumor load.
Increase or observe the lymphoglandula of new expansion when patient experience symptom (fever, night perspire etc.) and when the lymphoglandula size, so just think that it suffers from the disease of deterioration.
When the patient does not experience symptom and do not increase or observe and degenerating when lymphoglandula, so just think that it suffers from disease stable or that degenerate.Sometimes this illness or the observed clinically passivity of this disease are described as alleviating.
" little reaction " roughly represents contraction in a small amount.Little reaction is not the term of real standard, but it is used more and more.In general, little reaction be higher than total tumor long-pending 25% but be lower than 50%, the latter be reaction (PR) partly.
Sequence table
＜110〉Micromet AG
＜120〉mode and the method for treatment neoplastic disease
<130>L1410 PCT S3
<160>45
＜170〉PatentIn version 3 .3
<210>1
<211>1587
<212>DNA
＜213〉artificial
<220>
＜223〉dual specific scFv CD 19 * CD3
<400>1
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ccactccgat atccagctga cccagtctcc agcttctttg gctgtgtctc tagggcagag 120
ggccaccatc tcctgcaagg ccagccaaag tgttgattat gatggtgata gttatttgaa 180
ctggtaccaa cagattccag gacagccacc caaactcctc atctatgatg catccaatct 240
agtttctggg atcccaccca ggtttagtgg cagtgggtct gggacagact tcaccctcaa 300
catccatcct gtggagaagg tggatgctgc aacctatcac tgtcagcaaa gtactgagga 360
tccgtggacg ttcggtggag ggaccaagct cgagatcaaa ggtggtggtg gttctggcgg 420
cggcggctcc ggtggtggtg gttctcaggt gcagctgcag cagtctgggg ctgagctggt 480
gaggcctggg tcctcagtga agatttcctg caaggcttct ggctatgcat tcagtagcta 540
ctggatgaac tgggtgaagc agaggcctgg acagggtctt gagtggattg gacagatttg 600
gcctggagat ggtgatacta actacaatgg aaagttcaag ggtaaagcca ctctgactgc 660
agacgaatcc tccagcacag cctacatgca actcagcagc ctagcatctg aggactctgc 720
ggtctatttc tgtgcaagac gggagactac gacggtaggc cgttattact atgctatgga 780
ctactggggc caagggacca cggtcaccgt ctcctccgga ggtggtggat ccgatatcaa 840
actgcagcag tcaggggctg aactggcaag acctggggcc tcagtgaaga tgtcctgcaa 900
gacttctggc tacaccttta ctaggtacac gatgcactgg gtaaaacaga ggcctggaca 960
gggtctggaa tggattggat acattaatcc tagccgtggt tatactaatt acaatcagaa 1020
gttcaaggac aaggccacat tgactacaga caaatcctcc agcacagcct acatgcaact 1080
gagcagcctg acatctgagg actctgcagt ctattactgt gcaagatatt atgatgatca 1140
ttactgcctt gactactggg gccaaggcac cactctcaca gtctcctcag tcgaaggtgg 1200
aagtggaggt tctggtggaa gtggaggttc aggtggagtc gacgacattc agctgaccca 1260
gtctccagca atcatgtctg catctccagg ggagaaggtc accatgacct gcagagccag 1320
ttcaagtgta agttacatga actggtacca gcagaagtca ggcacctccc ccaaaagatg 1380
gatttatgac acatccaaag tggcttctgg agtcccttat cgcttcagtg gcagtgggtc 1440
tgggacctca tactctctca caatcagcag catggaggct gaagatgctg ccacttatta 1500
ctgccaacag tggagtagta acccgctcac gttcggtgct gggaccaagc tggagctgaa 1560
acatcatcac catcatcatt agtcgac 1587
<210>2
<211>523
<212>PRT
＜213〉artificial
<220>
＜223〉dual specific scFv CD 19 * CD3
<400>2
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val
20 25 30
Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val
35 40 45
Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly
50 55 60
Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly
65 70 75 80
Ile Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
85 90 95
Asn Ile His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
100 105 110
Gln Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
115 120 125
Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
145 150 155 160
Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser
165 170 175
Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
180 185 190
Ile Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys
195 200 205
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala
210 215 220
Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe
225 230 235 240
Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
260 265 270
Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro
275 280 285
Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr
290 295 300
Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
305 310 315 320
Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln
325 330 335
Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
340 345 350
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
355 360 365
Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly
370 375 380
Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly
385 390 395 400
Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr
405 410 415
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met
420 425 430
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln
435 440 445
Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val
450 455 460
Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser
465 470 475 480
Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr
485 490 495
Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr
500 505 510
Lys Leu Glu Leu Lys His His His His His His
515 520
<210>3
<211>1533
<212>DNA
＜213〉artificial
<220>
＜223〉CD 19 VH/VL * CD3 VH/VL (BsrG I to Sal I)
<400>3
tgtacactcc caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc 60
agtgaagatt tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt 120
gaagcagagg cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga 180
tactaactac aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag 240
cacagcctac atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc 300
aagacgggag actacgacgg taggccgtta ttactatgct atggactact ggggccaagg 360
gaccacggtc accgtctcct ccggtggtgg tggttctggc ggcggcggct ccggtggtgg 420
tggttctgat atccagctga cccagtctcc agcttctttg gctgtgtctc tagggcagag 480
ggccaccatc tcctgcaagg ccagccaaag tgttgattat gatggtgata gttatttgaa 540
ctggtaccaa cagattccag gacagccacc caaactcctc atctatgatg catccaatct 600
agtttctggg atcccaccca ggtttagtgg cagtgggtct gggacagact tcaccctcaa 660
catccatcct gtggagaagg tggatgctgc aacctatcac tgtcagcaaa gtactgagga 720
tccgtggacg ttcggtggag ggaccaagct cgagatcaaa tccggaggtg gtggatccga 780
tatcaaactg cagcagtcag gggctgaact ggcaagacct ggggcctcag tgaagatgtc 840
ctgcaagact tctggctaca cctttactag gtacacgatg cactgggtaa aacagaggcc 900
tggacagggt ctggaatgga ttggatacat taatcctagc cgtggttata ctaattacaa 960
tcagaagttc aaggacaagg ccacattgac tacagacaaa tcctccagca cagcctacat 1020
gcaactgagc agcctgacat ctgaggactc tgcagtctat tactgtgcaa gatattatga 1080
tgatcattac tgccttgact actggggcca aggcaccact ctcacagtct cctcagtcga 1140
aggtggaagt ggaggttctg gtggaagtgg aggttcaggt ggagtcgacg acattcagct 1200
gacccagtct ccagcaatca tgtctgcatc tccaggggag aaggtcacca tgacctgcag 1260
agccagttca agtgtaagtt acatgaactg gtaccagcag aagtcaggca cctcccccaa 1320
aagatggatt tatgacacat ccaaagtggc ttctggagtc ccttatcgct tcagtggcag 1380
tgggtctggg acctcatact ctctcacaat cagcagcatg gaggctgaag atgctgccac 1440
ttattactgc caacagtgga gtagtaaccc gctcacgttc ggtgctggga ccaagctgga 1500
gctgaaacat catcaccatc atcattagtc gac 1533
<210>4
<211>505
<212>PRT
＜213〉artificial
<220>
＜223〉CD 19 VH/VL * CD3 VH/VL (sophisticated protein w/o leader sequence)
<400>4
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr
130 135 140
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
145 150 155 160
Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu
165 170 175
Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
180 185 190
Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly Ser
195 200 205
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Lys Val
210 215 220
Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr
225 230 235 240
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser
245 250 255
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
260 265 270
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
275 280 285
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
290 295 300
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
305 310 315 320
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
325 330 335
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
340 345 350
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
355 360 365
Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
370 375 380
Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser
385 390 395 400
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
405 410 415
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser
420 425 430
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser
435 440 445
Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
450 455 460
Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
465 470 475 480
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
485 490 495
Glu Leu Lys His His His His His His
500 505
<210>5
<211>1531
<212>DNA
＜213〉artificial
<220>
＜223〉CD3VH/VL * CD19VH/VL (BsrGI to SalI)
<400>5
tgtacactcc gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc 60
agtgaagatg tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt 120
aaaacagagg cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta 180
tactaattac aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag 240
cacagcctac atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc 300
aagatattat gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt 360
ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca 420
gctgacccag tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg 480
cagagccagt tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc 540
caaaagatgg atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg 600
cagtgggtct gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc 660
cacttattac tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct 720
ggagctgaaa tccggaggtg gtggatccca ggtgcagctg cagcagtctg gggctgagct 780
ggtgaggcct gggtcctcag tgaagatttc ctgcaaggct tctggctatg cattcagtag 840
ctactggatg aactgggtga agcagaggcc tggacagggt cttgagtgga ttggacagat 900
ttggcctgga gatggtgata ctaactacaa tggaaagttc aagggtaaag ccactctgac 960
tgcagacgaa tcctccagca cagcctacat gcaactcagc agcctagcat ctgaggactc 1020
tgcggtctat ttctgtgcaa gacgggagac tacgacggta ggccgttatt actatgctat 1080
ggactactgg ggccaaggga ccacggtcac cgtctcctcc ggtggtggtg gttctggcgg 1140
cggcggctcc ggtggtggtg gttctgatat ccagctgacc cagtctccag cttctttggc 1200
tgtgtctcta gggcagaggg ccaccatctc ctgcaaggcc agccaaagtg ttgattatga 1260
tggtgatagt tatttgaact ggtaccaaca gattccagga cagccaccca aactcctcat 1320
ctatgatgca tccaatctag tttctgggat cccacccagg tttagtggca gtgggtctgg 1380
gacagacttc accctcaaca tccatcctgt ggagaaggtg gatgctgcaa cctatcactg 1440
tcagcaaagt actgaggatc cgtggacgtt cggtggaggg accaagctcg agatcaaatc 1500
cgggcatcat caccatcatc attgagtcga c 1531
<210>6
<211>504
<212>PRT
＜213〉artificial
<220>
<223>CD3VH/VL×CD19VH/VL
<400>6
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile
130 135 140
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
145 150 155 160
Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser
165 170 175
Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro
180 185 190
Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
195 200 205
Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
210 215 220
Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
225 230 235 240
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
245 250 255
Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
260 265 270
Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly
275 280 285
Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr
290 295 300
Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu
305 310 315 320
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp
325 330 335
Ser Ala Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg
340 345 350
Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
355 360 365
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380
Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu
385 390 395 400
Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr
405 410 415
Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro
420 425 430
Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro
435 440 445
Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
450 455 460
His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser
465 470 475 480
Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
485 490 495
Ser Gly His His His His His His
500
<210>7
<211>1528
<212>DNA
＜213〉artificial
<220>
＜223〉CD3VH/VL * CD19VL/VH (BsrGI to SalI)
<400>7
tgtacactcc gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc 60
agtgaagatg tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt 120
aaaacagagg cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta 180
tactaattac aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag 240
cacagcctac atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc 300
aagatattat gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt 360
ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca 420
gctgacccag tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg 480
cagagccagt tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc 540
caaaagatgg atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg 600
cagtgggtct gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc 660
cacttattac tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct 720
ggagctgaaa tccggaggtg gtggatccga tatccagctg acccagtctc cagcttcttt 780
ggctgtgtct ctagggcaga gggccaccat ctcctgcaag gccagccaaa gtgttgatta 840
tgatggtgat agttatttga actggtacca acagattcca ggacagccac ccaaactcct 900
catctatgat gcatccaatc tagtttctgg gatcccaccc aggtttagtg gcagtgggtc 960
tgggacagac ttcaccctca acatccatcc tgtggagaag gtggatgctg caacctatca 1020
ctgtcagcaa agtactgagg atccgtggac gttcggtgga gggaccaagc tcgagatcaa 1080
aggtggtggt ggttctggcg gcggcggctc cggtggtggt ggttctcagg tgcagctgca 1140
gcagtctggg gctgagctgg tgaggcctgg gtcctcagtg aagatttcct gcaaggcttc 1200
tggctatgca ttcagtagct actggatgaa ctgggtgaag cagaggcctg gacagggtct 1260
tgagtggatt ggacagattt ggcctggaga tggtgatact aactacaatg gaaagttcaa 1320
gggtaaagcc actctgactg cagacgaatc ctccagcaca gcctacatgc aactcagcag 1380
cctagcatct gaggactctg cggtctattt ctgtgcaaga cgggagacta cgacggtagg 1440
ccgttattac tatgctatgg actactgggg ccaagggacc acggtcaccg tctcctccgg 1500
gcatcatcac catcatcatt gagtcgac 1528
<210>8
<211>503
<212>PRT
＜213〉artificial
<220>
＜223〉CD3VH/VL * CD19VL/VH (sophisticated protein w/o leader sequence)
<400>8
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile
130 135 140
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
145 150 155 160
Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser
165 170 175
Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro
180 185 190
Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
195 200 205
Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
210 215 220
Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
225 230 235 240
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser
245 250 255
Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser
260 265 270
Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln
275 280 285
Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu
290 295 300
Val Ser Gly Ile Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
305 310 315 320
Phe Thr Leu Asn Ile His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr
325 330 335
His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr
340 345 350
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
355 360 365
Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
370 375 380
Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
385 390 395 400
Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly
405 410 415
Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr
420 425 430
Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser
435 440 445
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala
450 455 460
Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr
465 470 475 480
Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
485 490 495
Gly His His His His His His
500
<210>9
<211>10
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-H1
<400>9
Gly Tyr Thr Phe Thr Arg Tyr Thr Met His
1 5 10
<210>10
<211>17
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-H2
<400>10
Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210>11
<211>10
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-H3
<400>11
Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr
1 5 10
<210>12
<211>10
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-L1
<400>12
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn
1 5 10
<210>13
<211>7
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-L2
<400>13
Asp Thr Ser Lys Val Ala Ser
1 5
<210>14
<211>9
<212>PRT
＜213〉artificial
<220>
<223>CD3CDR-L3
<400>14
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210>15
<211>124
<212>PRT
＜213〉artificial
<220>
＜223〉anti-CD19VH
<400>15
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>16
<211>111
<212>PRT
＜213〉artificial
<220>
＜223〉anti-CD19VL
<400>16
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210>17
<211>119
<212>PRT
＜213〉artificial
<220>
＜223〉anti-CD3VH
<400>17
Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210>18
<211>106
<212>PRT
＜213〉artificial
<220>
＜223〉anti-CD3VL
<400>18
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210>19
<211>36
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' L1
<400>19
gaagcacgcg tagatatckt gmtsacccaa wctcca 36
<210>20
<211>30
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' K
<400>20
gaagatggat ccagcggccg cagcatcagc 30
<210>21
<211>33
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' H1
<400>21
cagccggcca tggcgcaggt scagctgcag sag 33
<210>22
<211>39
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' G
<400>22
accaggggcc agtggataga caagcttggg tgtcgtttt 39
<210>23
<211>38
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLB5RRV
<400>23
aggtgtacac tcctgatatc cagctgaccc agtctcca 38
<210>24
<211>48
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VLGS15
<400>24
ggagccgccg ccgccagaac caccaccttt gatctcgagc ttggtccc 48
<210>25
<211>53
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VHGS15
<400>25
ggcggcggcg gctccggtgg tggtggttct caggtsmarc tgcagsagtc wgg 53
<210>26
<211>39
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHBspE1
<400>26
aatccggagg agacggtgac cgtggtccct tggccccag 39
<210>27
<211>38
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLL2KBspEIGS
<400>27
cttccggagg tggtggatcc gacattcagc tgacccag 38
<210>28
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHL2KBspEI (C end)
<400>28
cctccggagg agactgtgag agtgg 25
<210>29
<211>38
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VHCD19BspEIGS
<400>29
cttccggagg tggtggatcc caggtgcagc tgcagcag 38
<210>30
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VLCD19BspEI (C end)
<400>30
cctccggatt tgatctcgag cttgg 25
<210>31
<211>35
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLCD19BspEIGS
<400>31
cttccggagg tggtggatcc gatatccagc tgacc 35
<210>32
<211>31
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VHL2KBsrGI
<400>32
aggtgtacac tccgatatca aactgcagca g 31
<210>33
<211>50
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHL2KGS15
<400>33
ggagccgccg ccgccagaac caccaccacc tgaggagact gtgagagtgg 50
<210>34
<211>53
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLL2KGS15
<400>34
ggcggcggcg gctccggtgg tggtggttct gacattcagc tgacccagtc tcc 53
<210>35
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VLL2KBspEI
<400>35
aatccggatt tcagctccag cttgg 25
<210>36
<211>31
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VHCD19BsrGI
<400>36
aggtgtacac tcccaggtgc agctgcagca g 31
<210>37
<211>50
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHCD19GS15
<400>37
ggagccgccg ccgccagaac caccaccacc ggaggagacg gtgaccgtgg 50
<210>38
<211>53
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLCD19GS15
<400>38
ggcggcggcg gctccggtgg tggtggttct gatatccagc tgacccagtc tcc 53
<210>39
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VLCD19BspEI
<400>39
aatccggatt tgatctcgag cttgg 25
<210>40
<211>35
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VLL2KBsrGI
<400>40
aggtgtacac tccgacattc agctgaccca gtctc 35
<210>41
<211>50
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VLL2KGS15
<400>41
ggagccgccg ccgccagaac caccaccacc tttcagctcc agcttggtcc 50
<210>42
<211>53
<212>DNA
＜213〉artificial
<220>
＜223〉primer 5 ' VHL2KGS15
<400>42
ggcggcggcg gctccggtgg tggtggttct gatatcaaac tgcagcagtc agg 53
<210>43
<211>30
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHL2KBspEI
<400>43
aatccggatg aggagactgt gagagtggtg 30
<210>44
<211>25
<212>DNA
＜213〉artificial
<220>
＜223〉primer 3 ' VHCD19BspEI (C end)
<400>44
cctccggagg agacggtgac cgtgg 25
<210>45
<211>73
<212>DNA
＜213〉artificial
<220>
＜223〉primer CTI polylinker
<400>45
tctagaattc ttcgaatccg gaggtggtgg atccgatatc cccgggcatc atcaccatca 60
tcattgagtc gac 73

Claims (21)

1. the bispecific single-chain antibody construct is used for the purposes that preparation prevents, treats or improve the pharmaceutical composition of inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, described bispecific single-chain antibody construct comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19), or
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)，
And wherein said bispecific single-chain antibody construct used at least 1 week with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface, and wherein said per daily dose was used above at least 6 hours.
2. prevent, treat or improve the method for inertia or rapid spread B cell non-Hodgkin's (BNHL) or B cell leukemia, described method comprises to its patient of needs uses the pharmaceutical composition that comprises the bispecific single-chain antibody construct, described bispecific single-chain antibody construct comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19), or
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)，
Wherein said bispecific single-chain antibody construct used at least 1 week with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface, and wherein said per daily dose was used above at least 6 hours.
3. medicine box, it is included in the philtrum prevention, treats or improve the pharmaceutical composition and the specification sheets of inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, described pharmaceutical composition comprises the bispecific single-chain antibody construct of pharmaceutical active, described bispecific single-chain antibody construct comprises people CD3 and the special binding domains of people CD19, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19), or
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)，
And described specification sheets has been described the application program of bispecific single-chain antibody construct, comprise with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface using at least 1 week, and wherein said per daily dose is used above at least 6 hours.
4. be used in the people, preventing, treating or improve the medicine box of inertia or rapid spread B cell non-Hodgkin's (B NHL) or B cell leukemia, use the treatment plan that comprises the bispecific single-chain antibody construct of the special binding domains of people CD3 and people CD19 according to comprising at least one week, wherein corresponding variable heavy chain district (V H) and corresponding variable light chain district (V L) hold the C end to arrange from N with following order:
V L(CD19)-V H(CD19)-V H(CD3)-V L(CD3)，
V H(CD19)-V L(CD19)-V H(CD3)-V L(CD3)，
V H(CD3)-V L(CD3)-V H(CD19)-V L(CD19), or
V H(CD3)-V L(CD3)-V L(CD19)-V H(CD19)，
Wherein said bispecific single-chain antibody construct used at least 1 week with the per daily dose of long-pending 10 μ g to the 80 μ g of every square metre of body surface, and wherein said per daily dose uses and surpass at least 6 hours, and wherein said medicine box contains following component:
(a) the independent per daily dose of the bispecific single-chain antibody construct of at least 7 part of 140 described pharmaceutical active of μ g to 320 μ g; With
(b) with the device of component to promote the mode of treatment plan conformability is arranged.
According to the purposes of claim 1, according to the method for claim 2 or according to the medicine box of claim 3 or 4, wherein said per daily dose was used above at least 10 hours.
According to the purposes of claim 1, according to the method for claim 2 or according to the medicine box of claim 3 or 4, wherein said per daily dose was used above at least 12 hours.
According to the purposes of claim 1, according to the method for claim 2 or according to the medicine box of claim 3 or 4, wherein said per daily dose was used above at least 24 hours.
According to any described purposes in the claim 1,5,6 or 7, according to any described method in the claim 2,5,6 or 7 or according to any described medicine box among the claim 3-7, the described V of the structural domain that wherein said CD3 is special HDistrict and V LThe district is derived from the CD3 specific antibody that is selected from OKT-3, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87,12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2 and F101.01.
9. according to any described purposes, method or a medicine box among the claim 1-8, the described V of the structural domain that wherein said CD3 is special HThe district comprises the CDR3 district that at least one comprises aminoacid sequence SEQ ID NO.11.
10. according to any described purposes, method or a medicine box among the claim 1-9, the described V of the structural domain that wherein said CD3 is special HThe district comprises the CDR2 district that at least one comprises aminoacid sequence SEQ IDNO.10.
11. according to any described purposes, method or a medicine box among the claim 1-10, the described V of the structural domain that wherein said CD3 is special HThe district comprises the CDR1 district that at least one comprises aminoacid sequence SEQ IDNO.9.
12. according to any described purposes, method or a medicine box among the claim 1-11, the described V of the structural domain that wherein said CD3 is special LThe district comprises the CDR3 district that at least one comprises aminoacid sequence SEQ IDNO.14.
13. according to any described purposes, method or a medicine box among the claim 1-12, the described V of the structural domain that wherein said CD3 is special LThe district comprises the CDR2 district that at least one comprises aminoacid sequence SEQ IDNO.13.
14. according to any described purposes, method or a medicine box among the claim 1-13, the described V of the structural domain that wherein said CD3 is special LThe district comprises the CDR1 district that at least one comprises aminoacid sequence SEQ IDNO.12.
15. according to any described purposes, method or a medicine box among the claim 1-14, the described V of the structural domain that wherein said CD3 is special HThe district comprises SEQ ID NO.17, the described V of the structural domain that described CD19 is special HThe district comprises SEQ ID NO.15, the described V of the structural domain that described CD3 is special LThe district comprises SEQ ID NO.18, and/or the described V of the special structural domain of described CD19 LThe district comprises SEQ ID NO.16.
16. according to any described purposes, method or a medicine box among the claim 1-15, wherein said bispecific single-chain antibody construct comprises and is selected from following aminoacid sequence:
(a) aminoacid sequence of in SEQ ID NO 2,4,6 or 8, describing;
(b) by the aminoacid sequence of nucleic acid sequence encoding shown in SEQ ID NO 1,3,5 or 7;
(c) had the aminoacid sequence of the nucleic acid sequence encoding of at least 70%, 80%, 90%, 95% or 99% identity by the nucleotide sequence with (b), wherein said aminoacid sequence can be bonded to CD3 and CD19 specifically; And
(d) by with the aminoacid sequence of the nucleic acid sequence encoding of the nucleotide sequence genetic code degeneracy of (b), wherein said aminoacid sequence can be bonded to CD3 and CD19 specifically.
17. according to any described purposes, method or a medicine box among the claim 1-16, wherein said variable region connects by extra joint sequence.
18. according to any described purposes, method or a medicine box among the claim 1-17, use wherein said day continued at least 2 weeks, at least 3 all or at least 4 weeks.
19. according to any described purposes, method or a medicine box among the claim 1-18, other medicament of wherein said pharmaceutical composition and one or more is co-administered.
20. according to any described purposes, method or a medicine box among the claim 1-19, wherein said pharmaceutical composition is used to human patients.
21. according to any described purposes, method or a medicine box among the claim 1-20, wherein said bispecific single-chain antibody construct was used with the per daily dose that is lower than long-pending 10 μ g to the 80 μ g of every square metre of body surface at first day.
CNA2006800471624A 2005-12-16 2006-11-29 Means and methods for treating neoplastic diseases Pending CN101331151A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP05027606 2005-12-16
EP05027606.2 2005-12-16
EP06004144.9 2006-03-01

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ZA (1) ZA200806095B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
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CN102711822A (en) * 2009-10-27 2012-10-03 米克罗麦特股份公司 Dosage regimen for administering a CD19xCD3 bispecific antibody
CN103533943A (en) * 2010-11-10 2014-01-22 安进研发（慕尼黑）股份有限公司 Prevention of adverse effects caused by cd3 specific binding domains
CN105017422A (en) * 2014-04-30 2015-11-04 山东百因制药技术有限公司 Anti-CD3/anti-CD19 dual-specific antibody and application thereof
CN105251003A (en) * 2010-10-27 2016-01-20 安进研发（慕尼黑）股份有限公司 Means and methods for treating dlbcl
CN107184977A (en) * 2008-11-07 2017-09-22 安进研发（慕尼黑）股份有限公司 The treatment method of ALL
US10071158B2 (en) 2005-04-26 2018-09-11 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10071158B2 (en) 2005-04-26 2018-09-11 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids
US10576149B2 (en) 2005-04-26 2020-03-03 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids
CN107184977A (en) * 2008-11-07 2017-09-22 安进研发（慕尼黑）股份有限公司 The treatment method of ALL
US10662243B2 (en) 2009-10-27 2020-05-26 Amgen Research (Munich) Gmbh Dosage regimen for administering a CD19XCD3 bispecific antibody
CN107227335A (en) * 2009-10-27 2017-10-03 安进研发（慕尼黑）股份有限公司 Dosage regimen for applying CD19xCD3 bispecific antibodies
CN102711822A (en) * 2009-10-27 2012-10-03 米克罗麦特股份公司 Dosage regimen for administering a CD19xCD3 bispecific antibody
CN105251003A (en) * 2010-10-27 2016-01-20 安进研发（慕尼黑）股份有限公司 Means and methods for treating dlbcl
CN105251003B (en) * 2010-10-27 2019-08-02 安进研发（慕尼黑）股份有限公司 Device and method for treating DLBCL
CN103533943B (en) * 2010-11-10 2018-02-13 安进研发（慕尼黑）股份有限公司 The prevention of ill-effect as caused by CD3 specific binding domains
US10130638B2 (en) 2010-11-10 2018-11-20 Amgen Research (Munich) Gmbh Prevention of adverse effects caused by CD3 specific binding domains
CN108403702A (en) * 2010-11-10 2018-08-17 安进研发（慕尼黑）股份有限公司 The prevention of ill-effect caused by CD3 specific binding domains
CN103533943A (en) * 2010-11-10 2014-01-22 安进研发（慕尼黑）股份有限公司 Prevention of adverse effects caused by cd3 specific binding domains
US11633408B2 (en) 2010-11-10 2023-04-25 Amgen Research (Munich) Gmbh Prevention of adverse effects caused by CD3 specific binding domains
CN105017422A (en) * 2014-04-30 2015-11-04 山东百因制药技术有限公司 Anti-CD3/anti-CD19 dual-specific antibody and application thereof

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