CN109912717A - In conjunction with the antibody and application thereof of CD40 - Google Patents

Abstract

The present invention provides a kind of and people CD40 specific bond isolated monoclonal antibody.The nucleic acid molecules for encoding the antibody, and expression vector, host cell and method for expressing the antibody are also provided.The present invention also provides the immunoconjugate comprising the antibody, bispecific molecule, Chimeric antigen receptor, oncolytic virus and pharmaceutical compositions, and use the treatment method of antibody of the present invention.

Description

In conjunction with the antibody and application thereof of CD40

Technical field

The present invention relates to a kind of with antibody and its preparation and use of people CD40 specific bond, especially its treatment with Purposes in the relevant human diseases of CD40, such as cancer, inflammatory disease, infectious diseases, atherosclerosis thrombus and exhale Desorption system disease.

Background technique

CD40, also known as A member of the TNF receptor family 5 or TNFR5 are a kind of cross-film costimulation albumen, It is expressed on antigen presenting cell such as B cell, macrophage and dendritic cells.The albumen and CD40L (CD154), it is a kind of main By the major ligand of T lymphocyte and the blood platelet expression activated, combination, activation antigen presenting cells excite multiple downstreams Signal path, the production including immune cell activation and proliferation and cell factor and chemotactic factor (CF) enhance cell function and exempt from Epidemic disease function (Ara A et al, (2018) Immunotargets Ther 7:55-61).

On the other hand, CD40 exists in nonimmune cell and tumour (Costello et al., (1999) Immunol Today 20 (11): 488-493；Tong et al., (2003) Cancer Gene Ther 10 (1): 1-13；Lee et Al., (2014) Curr Cancer Drug Targets 14 (7): 610-620；Ara A et al, (2018) are ibid), and Report that it is related with panimmunity disease (including auto-immune disease), atherosclerosis thrombus, cancer and respiratory disease Connection.For example, CD40/CD40L expression is raised in atheroma relevant cell.CD40 is pernicious swollen in almost all of B cell Tumor and up to 70% solid tumor in express, and in certain B cell malignant tumours CD40 Ligand combination can facilitate a variety of guarantors The expression that tumour cell is protected from the factor of Apoptosis increases (Lee et al., (1999) ProcNatIAcadSci USA 96:9136-9141).

Although effect of the CD40 in tumour generation is extremely complex, some CD40 antibody have been developed for potentially swelling Tumor treatment.CP-870,893, the IgG2 excited type CD40 antibody of the complete source of people of exploitation can activate dendritic cells, and exist Clinical efficacy (Vonderheide et al., (2007) J Clin are shown in the advanced cancer sufferer of a variety of backgrounds Oncol 25 (7): 876-883；Gladue et al., (2011) Cancer Immunol Immunother 60 (7): 1009- 1017；Beatty et al., (2013) Expert Rev Anticancer Ther 17 (2): 175-186；Vonderheide Et al., (2013) Oncoimmunology 2 (1): e23033；Nowak et al., Ann Oncol 26 (12): 2483- 2490；2015U.S.patent no.7,338,660).Dacetuzumab, also referred to as SGN-40 are Seattle Genetics The humanization IgG1 excited type CD40 antibody of exploitation shows weekly anti-tumor activity, especially with more under intravenous administration In the patient of unrestrained property large B cell lymthoma.Preclinical data also shows that Dacetuzumab and other drugs such as CD20 monoclonal antibody Synergistic effect (Lapalombella et al., (2009) Br J Haematol 144 (6): 848-855 of Rituximab； Hussein et al., (2010) Haematologica 95 (5): 845-848；De Vos et al., (2014) J He- Matol Oncol 7:44).Chi Lob 7/4, Britain's Cancer Research Center (Cancer Research UK) exploitation are fitted into IgG1 excited type antibodies against human CD 40, is carrying out initial clinical trial.11 stable diseases in 21 patients, without Partly or completely direct release (Chowdhury et al., (2014) CancerImmunolRes 2 (3): 229-240).In addition, short of money Anti- type CD40 antibody also shows antitumor action in people's Huppert's disease and chronic lymphocytic leukemia (Bensinger W et al., (2012) Br J Haemat0l.159 (1): 58-66；Mohammad Luqman et al., (2008) Blood 112:711-720).

Need CD40 antibody more with more preferable pharmacological property.

Summary of the invention

The present invention provides a kind of isolated monoclonal antibody, for example, combined with CD40 (for example, people CD40 and monkey CD40) Source of mouse, source of people, the monoclonal antibody of chimeric or humanized.Its excited type CD40 antibody that can be activation CD40 signal path.

Antibody of the invention can be used for a variety of applications, including detection CD40 albumen, treat or prevent CD40 related disease, Such as cancer, inflammatory disease, infectious diseases, atherosclerosis thrombus and respiratory disease.

Therefore, in one aspect, the present invention relates to a kind of isolated monoclonal antibody (for example, humanized antibody) or its Antigen-binding portion thereof, containing heavy chain variable region, which contains the area CDR1, the area CDR2 and the area CDR3, wherein CDR1 Area, the area CDR2 and the area CDR3 separately include (1) SEQ ID NOs:1,8 and 15；(2) SEQ ID NOs:1,9 and 15；(3)SEQ ID NOs:2,9 and 15；(4) SEQ ID NOs:3,10 and 16；(5) SEQ ID NOs:4,11 and 17；(6) SEQ ID NOs:5, 12 and 18；(7) SEQ ID NOs:6,13 and 19；Or the amino acid sequence of (8) SEQ ID NOs:7,14 and 20；Or it separately includes There is the amino acid sequence of at least 80%, 85%, 90%, 95%, 98% or 99% identity with these sequences, wherein the antibody Or its antigen-binding portion thereof is in conjunction with CD40.

In one aspect, isolated monoclonal antibody of the invention or its antigen-binding portion thereof include heavy chain variable region, Amino acid sequence comprising SEQ ID NOs:37,38,39,40,41,42,43,44,45,46,47,48,49 or 50, or comprising There is the amino acid sequence of at least 80%, 85%, 90%, 95%, 98% or 99% identity with these sequences, wherein the antibody Or its antigen-binding portion thereof is in conjunction with CD40.

In one aspect, isolated monoclonal antibody of the invention or its antigen-binding portion thereof contain light chain variable region, should Light chain variable region contains the area CDR1, the area CDR2 and the area CDR3, wherein the area CDR1, the area CDR2 and the area CDR3 separately include (1) SEQ ID NOs:21,27 and 31；Or (2) SEQ ID NOs:22,28 and 32；(3) SEQ ID NOs:23,29 and 33；(4)SEQ ID NOs:24,27 and 34；(5) SEQ ID NOs:25,27 and 35；Or the amino acid sequence of (6) SEQ ID NOs:26,30 and 36； Or the amino acid sequence that there is at least 80%, 85%, 90%, 95%, 98% or 99% identity with these sequences is separately included, Wherein the antibody or its antigen-binding portion thereof are in conjunction with CD40.

In one aspect, isolated monoclonal antibody of the invention or its antigen-binding portion thereof include light chain variable region, Comprising SEQ ID NOs:51,52,53,54,55,56,57,58,59,60 or 61, or comprising having at least with these sequences 80%, the sequence of 85%, 90%, 95%, 98% or 99% identity, wherein the antibody or its antigen-binding portion thereof and CD40 are tied It closes.

In one aspect, isolated monoclonal antibody of the invention or its antigen-binding portion thereof are comprising heavy chain variable region and gently Chain variable region respectively includes the area CDR1, the area CDR2 and the area CDR3, the wherein area heavy chain variable region CDR1, the area CDR2 and the area CDR3, light chain The variable region area CDR1, the area CDR2 and the area CDR3 separately include (1) SEQ ID NOs:1,8,15,21,27 and 31；(2)SEQ ID NOs:1,9,15,21,27 and 31；(3) SEQ ID NOs:2,9,15,21,27 and 31；(4) SEQ ID NOs:3,10,16,22, 28 and 32；(5) SEQ ID NOs:4,11,17,23,29 and 33；(6) SEQ ID NOs:5,12,18,24,27 and 34；(7)SEQ IDNOs:6,13,19,25,27 and 35；Or the amino acid sequence of (8) SEQ ID NOs:7,14,20,26,30 and 36；Or respectively Comprising having the amino acid sequence of at least 80%, 85%, 90%, 95%, 98% or 99% identity with these sequences, wherein should Antibody or its antigen-binding portion thereof are in conjunction with CD40.

In one embodiment, isolated monoclonal antibody of the invention or its antigen-binding portion thereof include weight chain variable Area and light chain variable region, the heavy chain variable region and light chain variable region separately include (1) SEQ ID NOs:37 and 51；(2)SEQ ID NOs:38 and 52；(3) SEQ ID NOs:39 and 53；(4) SEQ ID NOs:40 and 54；(5) SEQ ID NOs:41 and 55；(6) SEQ ID NOs:44 and 58；(7) SEQ ID NOs:45 and 59；(8) SEQ ID NOs:46 and 60；(9) SEQ ID NOs:46 With 61；(10) SEQ ID NOs:47 and 60；(11) SEQ ID NOs:47 and 61；(12) SEQ ID NOs:48 and 59；(13) SEQ ID NOs:49 and 60；(14) SEQ ID NOs:49 and 61；(15) SEQ ID NOs:50 and 60；Or (16) SEQ ID The amino acid sequence of NOs:50 and 61；Or separately include with these sequences have at least 80%, 85%, 90%, 95%, 98% or The amino acid sequence of 99% identity, wherein the antibody or its antigen-binding portion thereof are in conjunction with CD40.

In one embodiment, isolated monoclonal antibody of the invention or its antigen-binding portion thereof are comprising heavy chain and gently Chain, the heavy chain include heavy chain variable region and heavy chain constant region, and light chain includes light chain variable region and constant region of light chain, wherein heavy chain Variable region and light chain variable region contain above-mentioned amino acid sequence, and heavy chain constant region includes the ammonia of SEQ ID NOs:62,63 or 64 Base acid sequence, constant region of light chain include the amino acid sequence of SEQ ID NOs:65 or 66, or comprising having at least with these sequences 80%, the amino acid sequence of 85%, 90%, 95%, 98% or 99% identity, wherein the antibody or its antigen-binding portion thereof with CD40 is combined.

Antibody of the invention includes two heavy chains and two light chains in some embodiments, or by two heavy chains and two Light chain is constituted, wherein each heavy chain includes above-mentioned heavy chain constant region sequence, weight chain variabl area sequence or CDR sequence, and each light chain Include above-mentioned constant light chain sequences, light-chain variable sequence or CDR sequence.Antibody of the invention can be full length antibody, Such as IgG1, IgG2 or IgG4 hypotype.In some embodiments, antibody of the invention can be single-chain antibody, or by antibody Segment is constituted, such as 2 segment of Fab or Fab '.

Antibody of the invention or its antigen-binding portion thereof and people or monkey CD40 specific bond block or promote CD40-CD40L Interaction.Antibody of the invention is excited type CD40 antibody, activates CD40 signal path, promotes immunocyte such as dendron thin The maturation of born of the same parents.Antibody of the invention or its antigen-binding portion thereof have quite or preferably anti-in vivo with prior art CD40 antibody Tumor effect, and toxicity is suitable or lower.Tumour is no longer grown after antibody stops application, is even disappeared completely.

The present invention also provides the immunoconjugate containing antibody of the present invention or its antigen-binding portion thereof, the antibody or its antigen Bound fraction is connected with therapeutic agent such as cytotoxin.The present invention also provides contain antibody of the present invention or its antigen-binding portion thereof Bispecific molecule, the antibody or its antigen-binding portion thereof are connected with the second functional group, for example, secondary antibody, this second Functional group has the binding specificity different from antibody of the present invention or its bound fraction.On the other hand, antibody of the invention Or its antigen-binding portion thereof can be a part of Chimeric antigen receptor (CAR).Antibody of the invention or its antigen-binding portion thereof It can be encoded by oncolytic virus or be carried by oncolytic virus and used.

The present invention also provides composition, it includes antibody of the invention or its antigen-binding portion thereof or immunoconjugate or Bispecific molecule or CAR and pharmaceutically acceptable carrier.

The invention also includes the nucleic acid molecules for encoding antibody of the present invention or its antigen-binding portion thereof, and include the nucleic acid Expression vector and host cell comprising the expression vector.The present invention also provides use the host containing above-mentioned expression vector thin Born of the same parents are come the method for preparing CD40 antibody, comprising the following steps: (i) expresses antibody in host cell, and (ii) thin from host Separation antibody in born of the same parents or its culture.

On the other hand, the present invention provides a kind of method for enhancing subject immune's response, including applies and treat to subject A effective amount of antibody of the present invention or its antigen-binding portion thereof.In some embodiments, method includes application combination of the invention Object, bispecific molecule, immunoconjugate, CAR-T cell or encoding antibody or the oncolytic virus for carrying antibody.

On the other hand, the present invention, which provides, a kind of treats inflammatory disease in subject, infectious diseases, atherosclerosis The method of thrombus or respiratory disease, antibody from therapeutically effective amount to subject or antigen-binding portion of the present invention including applying Point.In some embodiments, method includes application composition of the invention, bispecific molecule, immunoconjugate, CAR-T Cell or encoding antibody or the oncolytic virus for carrying antibody.It in some embodiments, can be with antibody of the present invention or its antigen Bound fraction applies other drugs, such as anti-inflammatory agent and antimicrobial together.In some embodiments, inflammatory disease includes Auto-immune disease.

On the other hand, the method that present invention offer prevented, treated in subject or slowed down Cancerous disease, including to The antibody of the present invention or its antigen-binding portion thereof of subject's application therapeutically effective amount.Cancer can be solid tumor or non-physical knurl, Including but not limited to, B cell lymphoma, chronic lymphocytic leukemia, Huppert's disease, melanoma, enteraden cancer, pancreas Cancer, intestinal cancer, human primary gastrointestinal cancers, prostate cancer, bladder cancer, kidney, oophoroma, cervix cancer, breast cancer, lung cancer and nasopharyngeal carcinoma.Some In embodiment, this method include application composition of the invention, bispecific molecule, immunoconjugate, CAR-T cell or The oncolytic virus of coding or carrying antibody.In some embodiments, other at least one anticancrins can be anti-with the present invention Body or its antigen-binding portion thereof are applied together, for example, VISTA antibody, PD-1 antibody, PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody.In another embodiment, antibody of the invention or its antigen-binding portion thereof and cell factor (such as IL-2 And/or IL-21) or costimulation antibody (such as CD137 antibody and/or GITR antibody) apply together.In another embodiment In, antibody of the invention or its antigen-binding portion thereof can be applied together with chemotherapeutics, which can be cytotoxic agent, Such as Epi-ADM, oxaliplatin, and/or 5 FU 5 fluorouracil (5-FU).Antibody of the invention can be for example source of mouse, source of people, Chimeric or humanized antibody.

Based on the following specifically describes and embodiment, will be brighter in place of other feature and advantage of present disclosure Clear, specific descriptions and embodiment are not construed as restrictive.All documents, the Genbank quoted in this application is recorded, specially The content of benefit and public patent application is expressly included in herein by reference.

Attached drawing description

Fig. 1 shows the CD40 signal path agonist activity ranking of 108 hybridoma clones.

Fig. 2A and 2B shows CD40 antibody and acts on the CD40-CD40L promotion to interact or inhibition, wherein antibody 16A6,7B4 and 13A2 promote CD40-CD40L interaction (A) and antibody 92F6 inhibits CD40-CD40L interaction (B).

Fig. 3 shows the CD40 signal path agonist activity of CD40 antibody.

Fig. 4 A-4C show through CD86 (A), CD80 (B) and CD83 (C) dyeing and the CD40 Antibodies on Dendritic Cell that measures Mature facilitation.

Fig. 5 A and 5B show CD40 chimeric antibody to HEK293A/ people CD40 cell (A) or HEK293A/ monkey CD40 cell (B) binding force.

Fig. 6 shows the CD40 signal path agonist activity of CD40 chimeric antibody.

Fig. 7 shows the facilitation of the CD40 Antibodies on Dendritic Cell maturation measured through CD86 dyeing.

Fig. 8 A-8D shows chimeric and humanization CD40 antibody on human or the binding force of monkey CD40, wherein chimeric and humanization 13A2 antibody (A) and humanization 7B4 antibody (B) are in conjunction with people CD40, and chimeric and humanization 13A2 antibody (C) and source of people Change 7B4 antibody (D) in conjunction with monkey CD40.

Fig. 9 A and 9B show chimeric and humanization 7B4 antibody (A) and chimeric and humanization 13A2 antibody (B) CD40 believes Number access agonist activity.

Figure 10 A-10C show through CD86 (A), CD80 (B) and CD83 (C) dyeing and the CD40 antibody that measures to donor 1 The facilitation of internal dendritic cell maturation.

It is thin to dendron in 2 body of donor that Figure 11 A and 11B show the CD40 antibody measured through CD86 (A) and CD80 (B) dyeing The facilitation of born of the same parents' maturation.

Figure 12 A-12C show through CD86 (A), CD80 (B) and IL-12 (C) dyeing and CD40 is anti-in 3 body of donor that measures Facilitation of the body to dendritic cell maturation.

Figure 13 A-13N shows chimeric or humanized CD40 antibody 7B4 (A), 7B4-VH0VL0 (B), 7B4- by SPR measurement VH2VL2(C)、7B4-VH2VL3(D)、7B4-VH3VL2(E)、7B4-VH3VL3(F)、13A2(G)、13A2-VH0VL0(H)、 13A2-VH2VL2 (I), 13A2-VH2VL3 (J), 13A2-VH3VL2 (K) and 13A2-VH3VL3 (L) and reference antibody Binding affinity of the RO7009789 (M) and ADC1013 (N) to people CD40.

Figure 14 A and 14B show chimeric or humanized CD40 antibody to overall length CD40ECD or its truncate (A) and to complete The binding affinity of long CD40ECD or its mutant (B).

Figure 15 A and 15B show humanization CD40 antibody 7B4-VH2VL2 (A) and 13A2-VH3VL3 (B) to the knot of people CD40 Close specificity.

Figure 16 A, 16B and 16C show through CD86 (A), CD80 (B) and CD83 (C) dyeing and the genetic modification that measures The effect of CD40 Antibodies on Dendritic Cell maturation.

Figure 17 A-17F shows the mean tumour volume (A) of each group of application humanization CD40 antibody or control drug, and The individual that solvent group (B), 7B4VH2VL2 group (C), 13A2VH3VL3 group (D), RO7009789 group (E) and APX005 (F) are organized is swollen Knurl product.

Figure 18 shows the average animal weight of each group of application humanization CD40 antibody or control drug.

Figure 19 A and 19B show humanization CD40 antibodies on tumor infiltration CD4+T cell (A) and CD8+T cell (B) proliferation Effect.

Figure 20 shows the dyeing through CD80, CD83 and CD86 and measures humanization CD40 antibodies on tumor infiltration dendritic cells The facilitation of (CD45+CD11c+ cell) maturation.

Specific embodiment

To be best understood from the present invention, some terms are defined first.Other definition are then arranged through specific embodiment part Out.

Term " CD40 " refers to tumor necrosis factor superfamily member five.The term includes variant, allosome, homologue, straight To homologue and parallel homologue.For example, the antibody special to people CD40 can in some cases with another species such as monkey CD40 albumen cross reaction.In other embodiments, the antibody special to people's CD40 albumen can be fully to people CD40 Albumen specifically without with other species or other kinds of albumen cross reaction, or can with some other species and it is not all The CD40 albumen cross reaction of other species.

Term " people CD40 " refers to the CD40 albumen with human amino acid sequence, and human amino acid sequence is such as Genbank Accession number is the amino acid sequence (SEQ ID NO.:68) of NP_001241.1.Term " monkey CD40 " and " mouse CD40 " respectively refer to have There is the CD40 albumen of monkey and mouse sequence, such as being respectively provided with Genbank accession number is NP_001252791.1 (SEQ ID NO.:70) and Genbank accession number be NP_035741.2 (SEQ ID NO.:72) sequence.

Term " antibody " herein is intended to include full length antibody and its any antigen-binding fragment (that is, antigen-binding portion Point) or it is single-stranded.Full length antibody is the glycoprotein comprising at least two weight (H) chains and two light (L) chain, and heavy chain and light chain are by two sulphur Key connection.Each heavy chain is by heavy chain variable region (abbreviation V_H) and heavy chain constant region composition.Heavy chain constant region is made of three structural domains, That is C_H1、C_H2And C_H3.Each light chain is by light chain variable region (abbreviation V_L) and constant region of light chain composition.Constant region of light chain is by a structure Domain C_LIt constitutes.V_HAnd V_LArea can also be divided into the hypervariable region of referred to as complementary determining region (CDR), by the framework region more guarded (FR) it distinguishes and separates.Each V_HAnd V_LBe made of three CDR and four FR, from aminoterminal to c-terminus with FR1, CDR1, FR2, The sequence of CDR2, FR3, CDR3, FR4 are arranged.The variable region of heavy chain and light chain includes the binding domain with antigen interactions.Antibody Constant region can be with the combination of mediated immunity globulin and host tissue or the factor, including panimmunity system cells are (for example, effect Answer cell) and classical complement system the first component (C1q).

Term herein, " antigen-binding portion thereof " (or the referred to as antibody moiety) of antibody, refers to maintaining for antibody One or more segments of specific bond antigen (for example, CD40 albumen) ability.It has proven convenient that the antigen binding function of antibody can be with Implemented by the segment of full length antibody.The example for including the binding fragment in " antigen-binding portion thereof " of antibody includes (i) Fab segment, by V_L、V_H、C_LAnd C_mThe monovalent fragment of composition；(ii)F(ab′)₂Segment, two comprising the connection of hinge area disulphide bridges The bivalent fragment of Fab segment；(iii) by V_HAnd C_H1The Fd segment of composition；(iv) by antibody single armed V_LAnd V_HThe Fv segment of composition； (v) by V_HThe dAb segment (Ward et al., (1989) Nature 341:544-546) of composition；(vi) the complementary of separation determines Area (CDR)；And (vii) nano antibody, a kind of heavy chain variable region comprising single variable domains and two constant domains.This Outside, although two structural domain V of Fv segment_LAnd V_HEncoded by different gene, they can by recombination method via both make at For single protein chain synthetic linker and connect, wherein V_LAnd V_HIt matches to form monovalent molecule (referred to as single-stranded Fc (scFv) in area；Referring to Such as Bird et al., (1988) Science 242:423-426；And Huston et al., (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883).These single-chain antibodies, which are also intended to, to be included in term connotation.These Antibody fragment can be obtained by common technology well known by persons skilled in the art, and segment can by with complete antibody phase Same mode carries out functional screening.

The term as used herein " isolated antibody ", which refers to, is substantially free of other antibody with different antigentic specificities Antibody.For example, being substantially free of the antibody of the exoantigen of specific bond CD40 albumen with the separation antibody of CD40 albumen specific bond. But the separation antibody of specific bond people's CD40 albumen may have intersection to the CD40 albumen of other antigens such as other species Associativity.In addition, isolated antibody is substantially free of other cell materials and/or chemical substance.

Term " monoclonal antibody " or " monoclonal antibody " or " monoclonal antibody composition " refer to the antibody molecule of single molecular composition Product.Monoclonal antibody composition shows single binding specificity and affinity for defined epitope.

Term " small source of mouse antibody " refers to that variable framework and CDR region derive from the anti-of mouse germ-line immunoglobulin sequence Body.In addition, if antibody includes constant region, mouse germ-line immunoglobulin sequence is also derived from.Source of mouse antibody of the invention can With comprising not by the amino acid residue of mouse germ-line immunoglobulin sequential coding, such as pass through external random mutation or point mutation Or the mutation imported by internal somatic mutation.It is inserted however, term " source of mouse antibody " is not included in mouse Frame sequence Enter to derive from the antibody of the CDR sequence of other mammalian species.

Term " chimeric antibody " refers to the antibody got by combining inhuman exogenous genetic material and source of people inhereditary material.Or More generally, chimeric antibody refers to that combination has the inhereditary material of a species and the antibody of another species inhereditary material to person.

Term " humanized antibody " refers to from non-human species but its protein sequence has been modified to increase itself and people Naturally occur the antibody of the similarity of antibody.

The term antibody of antigen " identification " and " to the antibody of antigen-specific " herein with term " specific bond antigen Antibody " be used alternatingly.

Herein, the antibody of " specific bond people CD40 " refers to (is also possible to be other non-human species with people CD40 CD40) combine but substantially not with the protein bound antibody of non-CD40.Preferably, antibody is with " high-affinity " combination people's CD40 egg It is white, i.e. K_DValue is 5.0x10^-8M is hereinafter, it is preferred that 1.0x10^-8M is hereinafter, more preferably 5.0x 10^-9M or less.

Term " does not combine " albumen or cell to refer to substantially, not with albumen or cell combination, or not with high-affinity with It is combined, i.e. the K of binding protein or cell_DFor 1.0x 10^-6M or more, more preferable 1.0x 10^-5M or more, more preferable 1.0x 10^-4M or more, 1.0x 10^-3M or more, more preferable 1.0x 10^-2M or more.

Term " high-affinity " refers to that for the KD of antigen be 1.0x 10 for IgG antibody^-6M hereinafter, it is preferred that 5.0x 10^-8M is hereinafter, more preferably 1.0x 10^-8M or less, 5.0x 10^-9M is hereinafter, more preferably 1.0x 10^-9M or less.For other Antibody subtype, " high-affinity " combination may change.For example, " high-affinity " of IgM hypotype, which combines, refers to K_DIt is 10^-6M with Under, preferably 10^-7M is hereinafter, more preferable 10^-8M or less.

Term " K_assoc" or " K_a" refer to specific antibodies-antigen interactions association rate, and term " K_dis" or " K_d” Refer to specific antibodies-antigen interactions dissociation rate.Term " K_D" refer to dissociation constant, by K_dWith K_aThan (K_d/K_a) obtain, And it is indicated with molar concentration (M).The K of antibody_DValue can be determined by method known in field.It is preferred to determine antibody K_D's Mode is measured using surface plasma resonance instrument (SPR), it is preferable to use biological sensing system such as Biacore^TMSystem is surveyed ?.

Term " EC₅₀", it is called half-maximal effect concentration, refers to the antibody concentration for causing 50% ceiling effect.

Term " subject " includes anyone or non-human animal.Term " non-human animal " includes all vertebrates, such as Mammality and non-mammalian, such as non-human primates, sheep, dog, cat, ox, horse, chicken, amphibian animal and reptiles, although it is preferred that feeding Newborn animal, such as non-human primates, sheep, dog, cat, ox and horse.

Term " excited type CD40 antibody " used herein is to refer in conjunction with CD40 and activate or cause CD40 letter Number access is to promote the CD40 antibody of the generation of immune cell activation and proliferation and cell factor and chemotactic factor (CF).And term " antagonism type CD40 antibody " refers to blocking or inhibits that the CD40 signal path caused can be combined by CD40L.

Term " therapeutically effective amount " refer to be enough to prevent or slow down symptom relevant to disease or illness (such as cancer) and/ Or mitigate the amount of antibody of the present invention of disease severity.Therapeutically effective amount is related to treated disease, wherein art technology Personnel determine actual effective quantity in which can be convenient.

Many aspects of the invention are being described in more detail below.

CD40 antibody has to the binding specificity of people CD40 and other beneficial functional characters

Antibody of the invention specifically combines people CD40 with high-affinity, for example, K_DValue is 1x 10^-8M or less.Antibody Also there is the cross reactivity with monkey CD40, not in conjunction with mouse CD40.

Antibody of the invention is activation or causes CD40 signal path and participate in immune cell activation and proliferation and cell The excited type CD40 antibody of the generation of the factor and chemotactic factor (CF).

Antibody of the invention has quite or more preferable compared with existing CD40 antibody compared with prior art CD40 antibody Internal anti-tumor effect, toxicity is suitable or lower.After stopping administration of antibodies, tumour stops growing, and even completely eliminates.

Preferred antibody of the present invention is monoclonal antibody.In addition, antibody can be such as source of mouse, chimeric or humanization Monoclonal antibody.

CD40 monoclonal antibody

Preferred antibody of the present invention is structure and chemical characteristic monoclonal antibody described below.The V of CD40 antibody_HAmmonia Base acid sequence is SEQ ID NOs:37,38,39,40,41,42,43,44,45,46,47,48,49 or 50.The V of CD40 antibody_L Amino acid sequence is SEQ ID NOs:51,52,53,54,55,56,57,58,59,60 or 61.Heavy chain/light chain variable of antibody In region sequence table 1 listed below, some antibody V having the same_HOr VL.Preferably heavy chain constant region amino acid sequence is SEQ ID NOs:62,63 or 64, the amino acid sequence of constant region of light chain are SEQ ID NOs:65 or 66.

The V of other CD40 antibody in conjunction with people CD40_HAnd/or V_LSequence (or CDR sequence) can be with antibody of the present invention V_HAnd/or V_LSequence (or CDR sequence) " is mixed and is matched ".Preferably, work as V_HAnd V_L(or in which CDR) mixes and matches clock synchronization, Specific V_H/V_LV with centering_HSequence can be by the V of structure proximate_HSequence replaces.Similarly, preferably specific V_H/V_LWith centering V_LSequence by structure proximate V_LSequence replaces.

Therefore, in one embodiment, antibody of the invention or its antigen-binding portion thereof include:

It (a) include the heavy chain variable region for being listed in Table 1 below amino acid sequence；And

(b) comprising being listed in Table 1 below the light chain variable region of amino acid sequence or the V of another CD40 antibody_L, wherein this is anti- Body specific bond people CD40.

In another embodiment, antibody of the invention or its antigen-binding portion thereof include:

(a) CDR1, CDR2 and the CDR3 for the heavy chain variable region being listed in Table 1 below；And

(b) CDR of CDR1, CDR2 and CDR3 of the light chain variable region being listed in Table 1 below or another CD40 antibody, wherein Antibody specific bond people CD40.

In another embodiment, antibody of the invention or its antigen-binding portion thereof include the heavy chain variable region of CD40 antibody CDR2 and other in conjunction with the CDR of the antibody of people CD40, such as heavy chain variable region CDR1 and/or CDR3 and/or another CD40 be anti- Light chain variable region CDR1, CDR2 and/or CDR3 of body.

In addition, it is well known that, antibody can be individually determined to same independently of CDR1 and/or CDR2 in CDR3 structural domain in field The binding specificity of kind antigen, and can predict and be produced based on the CDR3 sequence with a variety of anti-of identical combination specificity Body.See, e.g. (2000) Klimka et al., British J.of Cancer 83 (2): 252-260；Beiboer et Al., (2000) J.Mol.Biol.296:833-849；Rader et al., Proc.Natl.Acad.Sci.U.S.A.95: 8910-8915(1998)；Barbas et al., J.Am.Chem.Soc.116:2161-2162 (1994)；Barbas et Al., (1995) Proc.Natl.Acad.Sci.U.S.A.92:2529-2533；Ditzel et al., J.Immunol.157: 739-749(1996)；Berezov et al., BIAjournal 8:Scientific Review 8 (2001)；Igarashi Et al., J.Biochem (Tokyo) 117:452-7 (1995)；Bourgeois et al., J.Virol 72:807-10 (1998)；Levi et al., Proc.Natl.Acad.Sci.U.S.A.90:4374-8 (1993)；Polymenis and Stoller, J.Immunol.152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000)；U.S.Pat.Nos.6,951,646；6,914,128；6,090,382；6,818,216；6,156,313；6,827, 925；5,833,943；5,762,905 and 5,760,185.These bibliography are fully incorporated herein by reference.

In another embodiment, antibody of the invention includes the CDR2 and at least of the heavy chain variable region of CD40 antibody The heavy chain and/or light chain variable region of the heavy chain of CD40 antibody and/or the CDR3 of light chain variable region or another CD40 antibody CDR3, wherein the antibody being capable of specific bond people CD40.It is preferred that these antibody (a) competitive bindings CD40；(b) reservation function is special Property；(c) same epitope is combined；And/or (d) there is binding affinity similar with CD40 antibody of the present invention.In another embodiment party In formula, antibody can also include the light chain variable region CDR2 of the CD40 antibody or light chain variable region CDR2 of another CD40 antibody, Wherein antibody specific bond people CD40.In another embodiment, antibody of the invention may include CD40 antibody heavy chain/ The heavy chain and/or light chain variable region CDR1 of light chain variable region CDR1 or another CD40 antibody, wherein antibody specific bond people CD40。

Conservative modification

In another embodiment, antibody of the invention includes and there is one or more guard with CD40 antibody of the present invention to repair The heavy chain and/or light-chain variable sequence or CDR1, CDR2 and CDR3 sequence of decorations.Know that some conserved sequences are modified in this field Antigen-binding will not be made to disappear.See, e.g., Brummell et al., (1993) Biochem 32:1180-8；de Wildt et al., (1997) Prot.Eng.10:835-41；Komissarov et al., (1997) J.Biol.Chem.272:26864-26870；Hall et al., (1992) J.Immunol.149:1605-12；Kelley And O ' Connell (1993) Biochem.32:6862-35；Adib-Conquy et al., (1998) Lnt.Immunol.10:341-6and Beers et al., (2000) Clin.Can.Res.6:2835-43.

Therefore, in one embodiment, antibody include heavy chain variable region and/or light chain variable region, heavy chain variable region and Light chain variable region separately includes CDR1, CDR2 and CDR3, in which:

(a) heavy chain variable region CDR1 includes the sequence and/or its conservative modification that table 1 is listed；And/or

(b) heavy chain variable region CDR1 includes the sequence and/or its conservative modification that table 1 is listed；And/or

(c) heavy chain variable region CDR3 includes the sequence and/or its conservative modification that table 1 is listed；And/or

(d) light chain variable region CDR1, and/or CDR2, and/or CDR3 includes the sequence listed of table 1 and/or it conservative is repaired Change；And

(e) antibody specific bond people CD40.

Antibody of the invention has one or more following functions features, such as to the high-affinity of people CD40, Yi Jiyin Send out the ability to the ADCC or CDC of CD40 expression cell.

In multiple embodiments, antibody can be such as source of mouse, source of people, chimeric or humanized antibody.

The term as used herein " conserved sequence modification " refers to the amino for being not significantly affected by or changing antibody binding properties Acid modification.Such conservative modification includes amino acid substitution, addition and deletion.Can by standard technique known in field, Such as the mutation that point mutation and PCR are mediated, modification is introduced into antibody of the present invention.Conserved amino acid replacement is that amino acid residue is used Amino acid residue with similar side chain is replaced.Amino acid residue group with similar side chain is known in field.These Amino acid residue group includes with basic side chain (for example, lysine, arginine, histidine), acid side-chain (for example, asparagus fern ammonia Acid, glutamic acid), uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, Cysteine, tryptophan), non-polar sidechain is (for example, alanine, valine, leucine, isoleucine, proline, phenylpropyl alcohol ammonia Acid, methionine), β-branched side chains (for example, threonine, valine, isoleucine) and beta-branched side (for example, tyrosine, Phenylalanine, tryptophan, histidine) amino acid.Therefore, one or more amino acid in the CDR region of antibody of the present invention are residual Base can be replaced with other amino acid residues of ipsilateral chain group, and Function detection pair as described herein can be used in obtained antibody Its test for carrying out reservation function (that is, above-mentioned function).

The antibody of gene modification

Antibody of the invention can be to have one or more V of CD40 antibody of the present invention_H/V_LThe antibody of sequence is used as Beginning material is prepared into the antibody of gene modification.Antibody can be by modifying one or two variable region (that is, V_HAnd/or V_L) in One or more residues of (for example, in one or more CDR regions and/or one or more framework region) carry out gene modification, To improve binding affinity and/or increase the similitude with the naturally-produced antibody of certain species.For example, framework region is through being modified into The antibody of humanization is provided.In addition, antibody gene modification can be carried out by the residue in modification constant region, such as change The effector function of antibody.

In some embodiments, CDR region implantation can be used to the variable region of gene modification antibody.Antibody mainly passes through position Amino acid residue in six heavy chains and complementary determining region of light chain (CDR) interacts with target antigen.For this Reason, amino acid residue in CDR compared with CDR outside sequence it is more various between individual antibody.Because CDR sequence is responsible for Main antibody-antigene interaction can be implanted to different characteristics by constructing the CDR sequence containing specific natural antibody The expression vector of the Frame sequence of different antibodies, to express the recombinant antibodies (Riechmann for the characteristic for simulating specific natural antibody Et al., (1998) Nature 332:323-327；Jones et al., (1986) Nature 321:522-525；Queen Et al., (1989) Proc.Natl.Acad；U.S.A.86:10029-10033；U.S.Pat.Nos.5,225,539；5,530, 101；5,585,089；5,693,762 and 6,180,370).

Therefore, another embodiment of the present invention is related to isolated monoclonal antibody or its antigen-binding portion thereof, it includes Heavy chain variable region and/or light chain variable region, heavy chain variable region include CDR1, CDR2 and the CDR3 with the above-mentioned sequence of the present invention, Light chain variable region includes CDR1, CDR2 and CDR3 with the above-mentioned sequence of the present invention.Although these antibody include Dan Ke of the present invention The V of grand antibody_HAnd V_LCDR sequence, they can contain different Frame sequences.

Such Frame sequence can be from the open DNA database for including germline antibody gene sequences or open bibliography Middle acquisition.For example, the germline DNA sequence dna for people's heavy chain and light-chain variable region gene can be in Vbase human germ line sequences' data Library (www.mrc-cpe.cam.ac.uk/vbase) and Kabat et al., (1991), ibid；Tomlinson et al., (1992) J.Mol.Biol.227:776-798；With Cox et al., obtain in (1994) Eur.J.Immunol.24:827-836 ?.It alternatively, can be in Genbank data for people's heavy chain and the germline DNA sequence dna of light-chain variable region gene It is obtained in library.For example, the Genbank accession number of the heavy chain germline sequence in following HCo7 HuMAb mouse is 1-69 (NG-- 0010109, NT--024637&BC070333), 3-33 (NG--0010109&NT--024637) and 3-7 (NG--0010109& NT--024637).As another example, the Genbank accession number of the heavy chain germline sequence below from Hco12HuMAb mouse For 1-69 (NG--0010109, NT--024637&BC070333), 5-51 (NG--0010109&NT--024637), 4-34 (NG--0010109&NT--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

By using one of the sequence similarity search method well known in the art for being known as space (gap) BLAST (Altsch μ l et al., (1997), ibid), antibody protein sequences are compared with protein sequence databank.

Preferred Frame sequence for antibody of the present invention is similar with Frame sequence used in antibody of the present invention in structure Those.V_HCDR1, CDR2 and CDR3 sequence can be implanted to and obtain the germ-line immunoglobulin gene tool of the Frame sequence Have in mutually homotactic framework region or CDR sequence can be implanted to include compared with Germline sequences have one or more In the framework region of mutation.For example, in some cases, it is beneficial that the residue in framework region, which be mutated, to keep or increase The antigen-binding of powerful antibody is (see, for example, U.S.Pat.Nos.5,530,101；5,585,089；5,693,762 and 6,180, 370)。

Another kind of variable region modification is by V_HAnd/or V_LAmino acid residue in the area CDR1, CDR2 and/or CDR3 carries out Mutation, thus one or more binding characteristics (for example, affinity) of Further aim antibody.It can carry out point mutation or PCR is situated between The mutation led is mutated to introduce, and antibody is combined for it or the influence of other function characteristic can be known in the art external Or it is evaluated in vivo detection.Preferably, conservative modification known in the art is introduced.Mutation can be amino acid substitution, add Add or lack, but preferably replaces.In addition, usually changing not more than one, two, three, four or five in CDR region Residue.

In addition, in another embodiment, the present invention provides isolated CD40 monoclonal antibody or its antigen-binding portion thereof, Comprising heavy chain variable region and light chain variable region, it includes: (a) V_HThe area CDR1, comprising sequence of the invention or one, two, Three, four or five amino acid replacement, missing or the amino acid sequence added；(b)V_HThe area CDR2 includes sequence of the invention Column or one, two, three, four or five amino acid replacement, missing or the amino acid sequence added；(c)V_HThe area CDR3, Include sequence of the invention or one, two, three, four or five amino acid replacement, missing or the amino acid sequence added Column；(d)V_LThe area CDR1, comprising sequence of the invention or one, two, three, four or five amino acid replacement, missing or The amino acid sequence of addition；(e)V_LThe area CDR2 includes sequence of the invention or one, two, three, four or five ammonia The amino acid sequence of the replacement of base acid, missing or addition；(f) V_LThe area CDR3, comprising sequence of the invention or one, two, Three, four or five amino acid replacement, missing or the amino acid sequence added.

Genetic modification antibody of the invention is included in V_HAnd/or V_LFramework residues in make gene modification for example to change Those of antibody characteristic.Typically, the modification of these frames is used to reduce the immunogenicity of antibody.For example, a kind of method be by One or more Framework residues " back mutation " are at corresponding Germline sequences.More specifically, the anti-of somatic mutation is undergone Body may include the Framework residues of the Germline sequences different from obtaining antibody.These residues can by by antibody framework sequence with The Germline sequences for obtaining antibody compare and identify.

Another kind of frame modification includes that the one or more of to framework region or even one or more CDR regions is residual Base is mutated, to remove t cell epitope, to reduce the immunogenicity that may cause of antibody.This method also referred to as " goes to exempt from Epidemic disease " has more detailed description in U.S. Patent Publication 20030153043.

In addition, as another selection except being modified in frame or CDR region, antibody of the invention can with genetic modification at It include gene modification in the area Fc, usually come the one or more functions characteristic for changing antibody, such as serum half-life, complement knot It closes, Fc receptor combines, and/or the cytotoxicity of antibody-dependant.In addition, antibody of the invention can be chemically modified (for example, Can be to the additional one or more chemical functional groups of antibody), or it is modified into its glycosylation of change, to change one of antibody Or multiple functional characteristics.

In one embodiment, C_H1Hinge area modified, change, such as increase or decrease half Guang ammonia of hinge area The quantity of sour residue.This method further describes in United States Patent (USP) 5,677,425.Change C_H1The cysteine of hinge area is residual Base, for example to promote the assembling or increase/reduction antibody stability of heavy chain light chain.

In another embodiment, the Fc hinge area of antibody is mutated, to reduce the biological half-life of antibody.More Add specifically, one or more amino acid mutations are introduced to the C of Fc hinge segment_H2-C_H3Bonding pad, so that antibody is relative to day For right Fc- hinge domain SpA is combined, there is the SpA binding force weakened.This method has in United States Patent (USP) 6,165,745 More detailed description.

In another embodiment, the glycosylation of antibody is modified.For example, deglycosylated antibody can be prepared (that is, antibody Lack glycosylation).It can change glycosylation, for example to increase antibody to the compatibility of antigen.Such saccharification modification can lead to It crosses and changes one or more glycosylation sites in antibody sequence for example to reach.For example, it is possible to make one or more amino Acid replacement, to eliminate one or more variable framework glycosylation sites, to eliminate the glycosylation of the position.Such desaccharification Baseization can increase antibody to the compatibility of antigen.See, e.g. United States Patent (USP) 5,714,350 and 6,350,861.

Furthermore, it is possible to prepare the antibody with the type of glycosylation changed, such as the low rock algae that fucosyl residues amount is reduced Aglycosyl antibodies, or with the increased antibody for dividing type GlcNac structure equally.The glycoforms of change are demonstrated to increase anti- The ADCC activity of body.It is such saccharification modification can for example, by glycosylation system change host cell in express antibody and It carries out.The cell of glycosylation system with change is in field it is known that and may be used as expressing the places of recombinant antibodies of the present invention Chief cell has the glycosylated antibody changed to prepare.For example, cell line Ms704, Ms705 and Ms709 lack fucosido Transferase gene FUT8 (α (1,6)-fucosyltransferase), thus expressed in Ms704, Ms705 and Ms709 cell line Antibody lacks fucose in its sugar.Ms704, Ms705 and Ms709FUT8-/- cell line in CHO/DG44 cell by making FUT8 gene is destroyed with two kinds of replacement carrier orientations and is prepared (referring to U.S. Patent Publication 20040110704 and Yamane- Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22).As another example, 1,176,195 EP The cell line of FUT8 gene function destruction is described, fucosyltransferase is encoded, so that expresses in the cell line is anti- Body by reducing or eliminating α -1,6 key relevant enzymes and show low fucosylation.EP 1,176,195 also illustrates a kind of thin Born of the same parents system with the lower enzymatic activity for the N-acetyl-glucosamine in the area binding antibody Fc addition fucose, or does not have There are the activity of this enzyme, such as rat myeloma cell system YB2/0 (ATCC CRL 1662).WO 03/035835 describes CHO Variant cell line, Lec13 cell have the ability that fucose is added to Asn (297)-associated sugars reduced, so that place The antibody expressed in chief cell low fucosylation (referring to Shields et al., (2002) J.Biol.Chem.277: 26733-26740).The antibody of glycosylation profile with change can also be prepared in egg, such as institute in WO 06/089231 It states.Alternatively, having the antibody of the glycosylation profile changed can prepare in plant cell such as duckweed.WO 99/54342 is public A kind of cell line is opened, genetic modification is at the glycosyl transferase of expression modified glucoprotein (for example, β (Isosorbide-5-Nitrae)-N- Acetylglucos Amine transferase I II (GnTIII)), so that the antibody expressed in genetically modified cell system shows increased to divide type GlcNac equally Structure, the ADCC activity (Umana et al., (1999) Nat.Biotech.17:176-180) for causing antibody to enhance.Or Fucosidase can be used to cut off the fucosyl residues of antibody, such as alpha-L-fucosidase in person, the fucosyl residues of antibody Enzyme removes fucosyl residues (Tarentino et al., (1975) Biochem.14:5516-23) from antibody.

Another modification of this paper antibody is Pegylation (PEGylated).Antibody can be PEGylated, such as to increase antibody Biological (for example, serum) half-life period.To keep antibody PEGylated, antibody or its segment usually with polyethylene glycol (PEG), such as PEG Reactive ester or aldehyde derivative react under conditions of making one or more PEG groups invest antibody or antibody fragment.It is preferred that Ground, it is PEGylated by anti-with the acylation reaction of reactive PEG molecule (or similar have reactive water-soluble polymer) or alkanisation Should carry out.Term " polyethylene glycol " used herein includes any type of PEG for being used to derive other albumen, such as singly (C₁-C₁₀) alkoxy-or aryloxy poly glycol or polyethylene glycol maleimide.In some embodiments, it needs PEGylated Antibody be deglycosylated antibody.The method of PEGylated albumen is in field it is known that and can be applied to antibody of the invention. See, e.g. EPO 154 316 and EP 0 401 384.

The physical characteristic of antibody

Antibody of the invention can be characterized with their a variety of physical characteristics, to detect and/or distinguish its classification.

For example, antibody can include one or more glycosylation sites in light chain or heavy chain variable region.These glycosylation positions Point may cause increased antibody immunogenicity, or due to change antigen binding and cause the antibody pK value changed (Marshall et al (1972) Annu Rev Biochem 41:673-702；Gala and Morrison(2004)J Immunol 172:5489-94；Wallick et al (1988) J Exp Med 168:1099-109；Spiro(2002) Glycobiology 12:43R-56R；Parekh et al (1985) Nature 316:452-7；Mimura et al., (2000) Mol Immunol 37:697-706).Glycosylation is known to occur in the motif containing N-X-S/T sequence.Some In the case of, preferably CD40 antibody is glycosylated not comprising variable region.This can be by selecting not include glycoylation motif in variable region Antibody or realized by the residue in mutant glycosylation region.

In the preferred embodiment, antibody does not include asparagine isomery site.The deamidation of asparagine is likely to occur In N-G or D-G sequence, it is created that different asparagicacid residue, is introduced into polypeptide chain and twist together and reduce its stability (different asparagus fern Propylhomoserin effect).

Each antibody will have unique isoelectric point (pI), be fallen within the scope of the pH of 6-9.5 substantially.The pI of IgG1 antibody is usual It falls within the scope of the pH of 7-9.5, and the pI of IgG4 antibody is fallen in substantially within the scope of the pH of 6-8.Speculate pI outside normal range (NR) Antibody may in vivo under the conditions of there are some deployed configurations and unstable.It is therefore preferable that the pI value of CD40 antibody is fallen in normally In range.This can be realized by the antibody of selection pI in the normal range or by being mutated uncharged surface residue.

Encode the nucleic acid molecules of antibody of the present invention

On the other hand, the present invention provides the heavy chain for encoding antibody of the present invention and/or light chain variable region or the nucleic acid point of CDR Son.Nucleic acid is there may be in whole cell, in cell pyrolysis liquid, or in partial purification or substantially pure form.It is marked when passing through For quasi- technology after being purified in other cellular components or other pollutants such as other cellular nucleic acids or albumen, nucleic acid is " to divide From " or " substantially pure ".Nucleic acid of the invention can be such as DNA or RNA, thereby increases and it is possible to include or may not include introne Sequence.In the preferred embodiment, nucleic acid is cDNA molecule.

The Protocols in Molecular Biology that standard can be used in nucleic acid of the invention obtains.For by hybridoma (for example, by carrying Human immunoglobulin gene transgenic mice preparation hybridoma) expression antibody, coding hybridoma preparation antibody it is light The cDNA of chain and heavy chain can be obtained by standard PCR amplification or cDNA clone technology.For (such as use phage display skill Art) antibody that is obtained from immunoglobulin gene pool, the nucleic acid for encoding this kind of antibody can collect from gene pool.

Preferred nucleic acid molecules of the present invention include the V for encoding CD40 monoclonal antibody_HAnd V_LThose of sequence or CDR.Once Obtain coding V_HAnd V_LDNA fragmentation, these DNA fragmentations can be operated further by the recombinant DNA technology of standard, Such as variable region gene is changed into full length antibody chain gene, Fab fragment gene or scFv gene.In these operations, coding V_HOr V_LDNA fragmentation and encode another DNA fragmentation of another albumen, such as antibody constant region or flexible joint operationally connect It connects.Term " being operably connected " refers to that two DNA fragmentations link together, thus the amino acid sequence of two DNA fragmentations coding Column are all in reading frame.

Encode V_HThe separation DNA in area can pass through the V that is operably connected_HCoding DNA and encoding heavy chain constant (C_H1、C_H2 And C_H3) another DNA molecular and be transformed into total length heavy chain gene.The sequence of people's weight chain constant area gene in field it is known that and DNA fragmentation including these regions can be obtained by standard PCR amplification.Heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, it is preferred that being IgG1 or IgG4 constant region.For Fab fragment heavy chain base Cause encodes V_HThe DNA in area can operationally with only encoding heavy chain C_H1Another DNA molecular of constant region connects.

Encode V_LThe separation DNA in area can pass through the V that is operably connected_LCoding DNA and coding constant region of light chain C_LIt is another DNA molecular and be transformed into full-length light chains gene.The sequence of people's light chain constant region gene is in field it is known that and including these regions DNA fragmentation can be obtained by standard PCR amplification.In the preferred embodiment, constant region of light chain can be κ and λ is constant Area.

To create scF_vGene encodes V_HAnd V_LDNA fragmentation can operationally with coding flexible joint for example encode ammonia Base acid sequence (Gly4-Ser)₃Another segment connection, thus V_HAnd V_LSequence can be used as continuous single chain protein carry out table It reaches, wherein V_HAnd V_LRegion by the flexible joint connection (see, e.g. Bird et al., (1988) Science 242: 423-426；Huston et al., (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883；McCafferty et Al., (1990) Nature 348:552-554).

The preparation of monoclonal antibody of the present invention

The body of Kohler and Milstein (1975) Nature 256:495 can be used in monoclonal antibody of the invention It is prepared by cell hydridization (hybridoma) technology.The other embodiments of preparation monoclonal antibody include the virus of bone-marrow-derived lymphocyte Or neoplastic transformation and display technique of bacteriophage.Chimeric or humanized antibody is also known in field.It is special see, e.g. the U.S. Benefit 4,816,567；5,225,539；5,530,101；5,585,089；5,693,762 and 6,180,370.

Prepare the generation of the transfectoma of monoclonal antibody of the present invention

Antibody of the invention can also use such as recombinant DNA technology combination gene transfection method, in host cell infection (such as Morrison, S. (1985) Science 229:1202) is generated in tumor.It in one embodiment, will be by standard molecule The DNA for the coded portion or full-length light chains and heavy chain that biotechnology obtains is inserted into one or more expression vectors, thus gene It is operably connected with transcription and translation regulating and controlling sequence.In this case, term " being operably connected " refers to that antibody gene connects It is connected in carrier, so that carrying intracorporal transcription and translation control sequence exercises their set regulation antibody gene transcription and translations Function.

Term " regulating and controlling sequence " includes the promoter for controlling antibody gene transcription or translation, enhancer and other expression controls Element (for example, polyadenylation signal).Such regulating and controlling sequence is in such as Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)) in There is description.The regulating and controlling sequence for being preferably used in mammalian host cell expression includes the height of guidance in mammalian cells The viral components of horizontal protein expression, such as the starting derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus Son and/or enhancer, such as adenovirus major late promoter (AdMLP) and polyomavirus.Alternatively, nonviral regulatory can be used Sequence, such as ubiquitin promoter or beta-globin promoter.In addition, Sequence composition of the controlling element by separate sources, such as SR α Promoter systems, it includes the repetitions of the long end of sequence and human T cell leukemia I type virus from SV40 early promoter (Takebe et al., (1988) Mol.Cell.Biol.8:466-472).Expression vector and expression control sequence are selected as and institute The expression host cell used is compatible.

Antibody light chain gene and antibody heavy chain gene are inserted into same or different expression vector.It is being preferably implemented In mode, variable region is and being inserted into the encoded required heavy chain constant region of hypotype and the expression vector of constant region of light chain Full length antibody gene is constructed, thus V_HWith the C in carrier_HIt is operably connected, V_LWith the C in carrier_LIt is operably connected.Or Person, recombinant expression carrier can encode the signal peptide for promoting antibody chain to secrete from host cell.Antibody chain gene can be cloned into In carrier, so that signal peptide is connected to the aminoterminal of antibody chain gene in reading frame.Signal peptide can be immunoglobulin letter Number peptide or heterologous signal peptide (that is, signal peptide from non-immunoglobulin).

In addition to antibody chain gene and regulating and controlling sequence, recombinant expression carrier of the invention can carry other sequences, such as adjust The sequence (for example, replication origin) and selectable marker gene that control carrier replicates in host cell.Selectable marker Gene can be used for selecting the host cell for having imported carrier (see, e.g., United States Patent (USP) 4,399,216；4,634,665 and 5, 179,017).For example, usually selectable marker gene assigns the host cell for having imported carrier with drug resistance, such as G418, hygromycin or methotrexate resistance.Preferred selectable marker gene includes dihyrofolate reductase (DHFR) gene (methotrexate selection/amplification for dhfr host cell) and neo gene (select) for G418.

It is thin that the expression vector of expression for light chain and heavy chain, encoding heavy chain and light chain by standard technique is transfected into host In born of the same parents.The term " transfection " of multiple forms includes a variety of technologies being usually used in Exogenous DNA transfered protokaryon or eukaryotic host cell, For example, electroporation, calcium phosphate precipitation, DEAE- dextrose transfection etc..Although expressing the present invention in protokaryon or eukaryotic host cell Antibody be theoretically it is feasible, preferred antibody is expressed in eukaryocyte, most preferably expressed in mammalian host cell, Because of eukaryocyte, especially mammalian cell is more likely to assemble and secrete and suitably folds and have immune work than prokaryotic cell The antibody of property.

Being preferably used in and expressing the mammalian host cell of recombinant antibodies of the present invention includes that (CHO is thin for Chinese hamster ovary Born of the same parents) (including the dhfr-CHO cell applied together with DHFR selectable marker, in Urlaub and Chasin, (1980) There is description in Proc.Natl.Acad.Sci.USA 77:4216-4220, DHFR selectable marker is for example Described in R.J.Kaufman and P.A.Sharp (1982) J.Mol.Biol.159:601-621), NSO myeloma cell, COS cell and SP2 cell.Especially when using NSO myeloma cell, another preferred expression system is GS gene expression system System, is recorded in WO 87/04462, WO 89/01036 and EP 338,841.When the recombinant expression carrier of encoding antibody genes imports When mammalian host cell, by the way that host cell culture is sufficient to make in host cell antibody expression or is preferably enough So that making antibody-secreting to a period of time in the culture medium of host cell growth, to prepare antibody.Egg can be used in antibody White purification process is recycled from culture medium.

Immunoconjugate

Antibody of the invention can be crosslinked with therapeutic agent, form immunoconjugate, such as antibody-drug cross-linking agent (ADC). Suitable therapeutic agent includes that cytotoxin, alkylating agent, DNA minor groove binding molecule, DNA intercalator, DNA crosslinking agent, histone are gone Inhibitor, the heat shock protein suppression of acetylase inhibitor, core output inhibitor, proteasome inhibitor, topoisomerase I or II Preparation, tyrosine kinase inhibitor, antibiotic and antimitotic agent.In the adc, antibody and therapeutic agent preferably pass through connector Crosslinking, the connector is cleavable, such as peptides connector, two sulphur class connectors or hydrazone class connector.It is highly preferred that connector is peptides connector, Such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val- Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu.ADC can such as United States Patent (USP) 7,087, 600；6,989,452；With 7,129,261；PCT Publication WO 02/096910；WO 07/038,658；WO 07/051,081；WO 07/059,404；WO 08/083,312；With WO 08/103,693；U.S. Patent Publication 20060024317；20060004081； With 20060247295 in description as prepared.

Bispecific molecule

On the other hand, the present invention relates to comprising with such as another peptide or protein of at least one other functional molecular (for example, another One antibody or receptors ligand) bispecific molecule of one or more antibody of the present invention that is connected, to generate and at least two The bispecific molecule that different binding sites or targeted molecular combine.Term " bispecific molecule " includes with three kinds or more Species specific molecule.

In embodiments, in addition to Fc binding specificity and CD40 binding specificity, bispecific molecule also has third Specificity.Third specificity can be for enhancement factor (EF), such as in conjunction with the surface protein for participating in cellular cytoxicity activity And thereby increase the molecule of the immune response for target cell.For example, enhancement factor antibody can be with cytotoxic T cell (example Such as, pass through CD2, CD3, CD8, CD28, CD4, CD40 or ICAM-1) or the combination of other immunocytes, cause enhancing is directed to target The immune response of cell.

Bispecific molecule can occur with size in a variety of forms.In one end of size spectrum, bispecific molecule is kept Conventional antibodies form, except it is with two basic change arm and in addition to each arm has not homospecificity, there are two specificity is identical for substitution tool Combination arm the case where.It is another it is extreme be bispecific molecule, by two single chain antibody fragments through peptide chain link (scFv) it constitutes, referred to as Bs (scFv)₂Construct.The bispecific molecule of intermediate sizes includes two connected by peptides connector Different F (ab) segments.The bispecific molecule of these and other forms can pass through genetic modification, somatic hybridization or chemistry It is prepared by method.See, e.g. Kufer et al, ibid；Cao and Suresh, Bioconjugate Chemistry, 9 (6), (1998) 635-644；With van Spriel et al., Immunology Today, 21 (8), 391-397 (2000).

Encoding antibody or the oncolytic virus for carrying antibody

Infects and kill cancer cell to oncolytic virus preference.Antibody of the invention is used together with oncolytic virus.In addition, compiling The oncolytic virus of code book invention antibody can be introduced into human body.

Pharmaceutical composition

On the other hand, the present invention provides a kind of pharmaceutical composition, and it includes one or more antibody of the invention, with medicine Acceptable carrier is formulated together on.Composition can optionally comprising it is one or more other pharmaceutically it is effective at Point, such as another antibody or drug, such as VISTA antibody.Pharmaceutical compositions of the invention can with for example another anticancer agent, It is administered in the conjoint therapy of another anti-inflammatory agent or antimicrobial.

Pharmaceutical compositions may include any amount of excipient.The excipient that can be used includes carrier, surface-active Agent, thickening or emulsifier, solid binder, dispersion or suspension, solubilizer, coloring agent, corrigent, coating, disintegrating agent, lubrication Agent, sweetener, preservative, isotonic agent and combinations thereof.The selection and use of appropriate excipients in Gennaro, ed., Remington:The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams& Wilkins 2003) in have introduction.

Preferably, it is (such as logical to be suitable for intravenous, intramuscular, subcutaneous, parenterally, backbone or epidermis application for pharmaceutical composition It crosses and injects or inject).Difference based on administration method, effective component can wrap in the material, sour and possible to protect it from The influence for other natural conditions for inactivating it." parenterally application " refers to the mode different from enteron aisle and local topical, usually Carried out by injection, including but not limited to intravenously, in intramuscular, intra-arterial, film, in intracapsular, socket of the eye, heart is interior, intradermal, peritonaeum Under interior, transtracheal, subcutaneous, epidermis, under intra-articular, capsule, under arachnoid, on intraspinal, endocranium and breastbone inner injection and inject. Alternatively, antibody of the invention can be applied by non-bowel outer pathway, such as external application, epidermis application or mucosal administration, such as nose Interior, oral, vagina, rectum, sublingual or local topical.

Pharmaceutical composition can be the form of aseptic aqueous solution or dispersion liquid.They can also be prepared in microemulsion, lipid Body or other be suitable for high concentration medicine ordered structure in.

The amount that the effective component of one-pack type is prepared into together with carrier material will be with treatment main body and specific application mode And become, and substantially for be generate curative effect composition amount.In percentage, which is about 0.01-'s about 99% and medicine The effective component that acceptable carriers combine on, preferably from about 0.1%- about 70% are most selected as the effective of about 1%- about 30% in advance Ingredient.

Dosage regimen is adjusted to provide optimal required reaction (for example, therapeutic response).For example, quick filling can be applied Agent, can apply over time multiple divided doses or dosage can with treatment condition the proportional reduction of criticality or It improves.Particularly advantageously, to facilitate the dosage unit form of application and dose uniformity to configure parenteral compositions.Dosage unit form Refer to and be physically separated unit, suitable for treating the single-dose of main body；Constituent parts include to calculate together with pharmaceutical carriers The effective component of the predetermined amount of curative effect needed for generating.Alternatively, antibody can be applied with sustained release agent, required application in this case Frequency reduces.

Application for antibody, dosage can be about 0.001-100mg/kg host's weight, more common for 0.01-5mg/ kg.For example, dosage can be 0.3mg/kg weight, 1mg/kg weight, 3mg/kg weight, 5mg/kg weight or 10mg/kg weight, Or within the scope of 1-10mg/kg.Illustrative therapeutic scheme is related to applying weekly once, biweekly, once in three weeks, four Mondays It is secondary, one month it is primary, 3 months it is primary or 3-6 months primary.The preferred dosage regimen of CD40 of the present invention includes intravenous application, 1mg/kg weight or 3mg/kg weight, antibody are administered with one in following administration time table: (i) every four weeks administration six Secondary, then every three months is primary；(ii) once every three weeks；(iii) 3mg/kg weight is primary, later 1mg/kg weight every three Monday It is secondary.In certain methods, it is about 25-300 μ in certain methods that dosage, which is adjusted to realize the blood concentration of about 1-1000 μ g/ml, g/ml。

The CD40 antibody of the present invention of " therapeutically effective amount " cause the reduction of disease symptoms severity, asymptomatic stage frequency and The increase of duration or to caused by susceptibility damage or incapability prevention ability.For example, for controlling with tumor subject Treat, " therapeutically effective amount " preferably, compared with not treating subject, by Tumor growth inhibition at least about 20%, more preferably inhibit At least about 40%, even more preferably inhibit at least about 60%, and more preferably inhibit at least about 80%.Therapeutically effective amount is controlled Treating antibody can reduce tumor size, or mitigate the symptom of subject, and subject can be people or another mammal.

Pharmaceutical composition can be sustained release dosage, including implant, transdermal patch and microcapsules delivery system.It can make With biodegradable, biocompatible polymer, such as ethene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, poly- original Acid esters and polylactic acid.See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical compositions can be administered through medical supply, such as (1) needlefree injection equipment is (for example, United States Patent (USP) 5,399,163；5,383,851；5,312,335；5,064,413；4,941,880；4,790,824；With 4,596,556)；(2) Microinfusion pump (United States Patent (USP) 4,487,603)；(3) transdermal drug delivery devices (United States Patent (USP) 4,486,194)；(4) equipment is injected (United States Patent (USP) 4,447,233 and 4,447,224)；(5) penetration equipment (United States Patent (USP) 4,439,196 and 4,475,196).

In some embodiments, monoclonal antibody of the invention can be formulated, to ensure suitable distribution in vivo.For example, being Ensure that treatment antibody of the invention passes through blood-brain barrier, antibody can be prepared in liposome, can also extraly include target To functional group, to enhance the selectivity conveying to specific cells or organ.See, e.g. United States Patent (USP) 4,522,811；5, 374,548；5,416,016；With 5,399,331；V.V.Ranade (1989) J.Clin.Pharmacol.29:685；Umezawa Et al., (1988) Biochem.Biophys.Res.Commun.153:1038；Bloeman et al., (1995) FEBS Lett.357:140；M.Owais et al., (1995) Antimicrob.Agents Chemother.39:180；Briscoe Et al., (1995) Am.J.Physiol.1233:134；Schreier et al., (1994) J.Biol.Chem.269: 9090；Keinanen and Laukkanen (1994) FEBS Lett.346:123；With Killion and Fidler (1994) Immunomethods 4:273.

Purposes and method of the invention

Antibody (composition, bispecific molecule and immunoconjugate) of the invention has a variety of external and outer interior applications, It is related to the treatment and/or prevention of such as cancer, inflammatory disease or infectious disease.Antibody can be applied to people experimenter, with for example Inhibit tumour growth in vivo.

In view of the ability of the inhibition tumor cell proliferation and survival of CD40 antibody of the present invention, the present invention, which provides, inhibits tested The method of growth of tumour cell in person, including applying antibody of the invention to the subject, so that tumour is raw in the subject It is long to be suppressed.It can include but is not limited to B cell lymphoma by the non-limitative example of the tumour of Antybody therapy of the present invention, chronic Lymphocytic leukemia, Huppert's disease, melanoma, enteraden cancer, cancer of pancreas, intestinal cancer, human primary gastrointestinal cancers, prostate cancer, bladder Cancer, kidney, oophoroma, cervix cancer, breast cancer, lung cancer and nasopharyngeal carcinoma, either primary carcinoma or metastatic carcinoma.In addition, refractory Or the malignant tumour of recurrence can may be inhibited with antibody of the invention.

On the other hand, the present invention, which provides, a kind of treats inflammatory disease in subject, infectious diseases, atherosclerosis The method of thrombus and respiratory disease, antibody from therapeutically effective amount to subject or antigen-binding portion of the present invention including applying Point.In some embodiments, anti-inflammatory agent, antimicrobial can be applied together with antibody of the present invention or its antigen-binding portion thereof Or other therapeutic agents.

In general, antibody of the invention can be used to enhance the immune response of subject.

These and other methods of the invention are discussed further below.

Conjoint therapy

The present invention, which provides CD40 antibody of the present invention or its antigen-binding portion thereof, can effectively inhibit subject with one or more The conjoint therapy that other antibody of middle tumour growth are applied together.In one embodiment, the present invention is provided in subject Inhibit the method for tumour growth, including applies CD40 antibody and other one or more antibody to subject, such as VISTA resists Body, LAG-3 antibody, PD-1 antibody, PD-L1 antibody and/or CTLA-4 antibody.In some embodiments, subject is people.

The activation of CD40 signal path can be in addition in conjunction with standard cancer treatments.For example, the activation of CD40 signal path can be with CTLA-4 and/or LAG-3 and/or PD-1 is blocked and chemotherapy combines.For example, chemotherapeutics can be applied together with CD40 antibody With chemotherapeutics can be cytotoxic agent.For example, Epi-ADM, oxaliplatin, and/or 5-FU, which can be administered to, receives CD40 The patient of antibody therapy.

Optionally, CD40 and other one or more antibody are (for example, CTLA-4 antibody and/or LAG-3 antibody and/or PD- 1 antibody) combination can also in conjunction with immunogenic agents, be immunized prototype agent be such as cancer cell, purifying tumour antigen (including Recombinant protein, peptide and glycan molecule) and transfection have expression immune stimulating cytokines gene cell (He et al., (2004) J.Immunol.173:4919-28).The non-limitative example for the tumor vaccine that can be used includes melanoma-associated antigen Peptide, such as gp100 peptide, MAGE antigen, Trp-2, MART1, and/or tyrosinase or transfection are with expression cell factor GM-CSF Tumour cell.

Other can be antibody combined with CD40 therapy include but is not limited to interleukin-22 (IL-2) application, radiotherapy, operation or Hormone removal.

The combination for the therapeutic agent being discussed herein can be used as the single composition in pharmaceutical acceptable carrier and be administered simultaneously, Or be administered simultaneously as separated composition, wherein each medicament is in pharmaceutical acceptable carrier.In another embodiment In, the combination of therapeutic agent can be applied sequentially.

In addition, if carry out multiple conjoint therapy application, and medicament is sequentially applied, then sequentially applying at each time point Order can invert or keep identical, and sequentially application can be combined with being administered simultaneously or any combination thereof.

The present invention is also described further by following embodiment, and embodiment should not be read as restrictive.It is all Attached drawing and all bibliography quoted in the whole text in this application, Genebank sequence, patent and public patent application all pass through The mode of reference is fully incorporated herein.

Embodiment

Embodiment 1 stablizes the building of the HEK293A cell strain of expression people, monkey or mouse CD40

(SEQ ID NOs:67,69 and 71, are separately encoded amino acid to the cDNA sequence of composite coding people, monkey or mouse CD40 Sequence SEQ ID NOs:68,70 and 72), and (English luxuriant industry in Beijing is raw through enzyme cutting clone to pLV-EGFP (2A)-Puro carrier Object Science and Technology Ltd., China) in.Obtained pLV-EGFP (2A)-Puro-CD40 and psPAX and pMD2.G plasmid is passed through The mode of liposome transfection is transfected into HEK293T cell (one hundred company, Nanjing section, China), generates slow virus, specific transfection side Method and Lipofectamine 3000 (Thermo Fisher Scientific, the U.S.) specification step are completely the same.Transfection After three days, from the cell culture medium of HEK293T cell, (DMEM culture medium (Cat#:SH30022.01, Gibco), is supplemented with 10% FBS (Cat#:FND500, Excell)) in harvest slow virus.Then with slow-virus transfection HEK293A cell, (Nanjing section one hundred is public Department, China), obtain stablizing expression people, monkey or mouse CD40 HEK293A cell (respectively HEK293A/hCD40, HEK293A/rhCD40,HEK293A/muCD40).The HEK293A cell culture of transfection is containing 0.2 μ g/ml puromycin 7 days in the DMEM+10%FBS culture medium of (Cat#:A11138-03, Gibco).The expression of people and monkey CD40 pass through flow cytometer showed Instrument uses commercially available people CD40 antibody (PE- antibodies against human CD 40, Cat#313006, Biolegend, beauty through facs analysis State).Similarly, the expression of mouse CD40 passes through commercially available mouse CD40 antibody (PE- mouse CD40 antibody, Cat# 124609, Biolegend, the U.S.) it is confirmed through FACS.

The hybridoma cell line of the preparation secretion people CD40 antibody of embodiment 2

Mouse anti human CD40 monoclonal antibody is obtained by conventional hybridization tumor integration technology, and scheme is slightly changed.

Inoculation

13 BALB/C mices (dimension tonneau China, China) by intersecting injection recombined human CD40 (ECD)-his albumen, (stick up by justice Divine Land, China, Cat:10774-H08H) and monkey CD40 (ECD)-hFc albumen (justice sticks up Divine Land, China, Cat:90097-C02H) It is immunized, specifically immune process is as shown in table 2.The bodies such as people CD40 (ECD)-his albumen and monkey CD40 (ECD)-hFc albumen use Long-pending complete Freund adjuvant (Cat#:F5881-10*10ML, Sigma, the U.S.), incomplete Freund adjuvant (Cat#: F5506-6*10ML, Sigma, the U.S.) or PBS ultrasonic emulsification.

2. immunization schedule of table

1 week after each booster immunization, 50 μ l serum are taken out of every Mice Body, detect titre through ELISA, it is specifically used heavy Group people CD40-hFc (Cat#:10774-H02H, justice stick up Divine Land, China) and monkey CD40 (ECD)-hFc (Cat#:90097-C02H, Justice sticks up Divine Land, China) it is combined test.Also people, monkey, mouse CD40 are overexpressed by using what is prepared in embodiment 1 HEK293A cell carries out titre test through FACS.

ELISA detection and FACS testing result after being reinforced based on last time, the higher 7 mouse quilts of serum titer Select the hybridoma cell line preparation for next step.

The preparation of hybridoma cell line

Hybridoma cell line is prepared by conventional hybridoma fusion techniques, scheme has been modified slightly.

Behind four days that last time is reinforced, spleen is taken out after mouse is put to death, Single cell suspensions are prepared into PBS. Splenocyte is cleaned 3 times with DMEM culture medium (Cat#:SH30243.01B, Hyclone, the U.S.).Mouse in logarithmic growth phase Myeloma cell SP2/0 (CRL-1581, ATCC, the U.S.) is used after mixing with above-mentioned isolated mouse boosting cell in 1: 4 ratio DMEM is cleaned 2 times.Cell fusion is carried out by the mode that PEG (Cat#:P7181, Sigma, the U.S.) is merged.Fused cell It is cleaned 3 times with DMEM, and is resuspended in cell growth medium (RPMI 1640+10%FBS+1X HAT).By cell suspension The bed board on 96 well culture plates, 200 μ l of every hole, 5 × 10⁴Cell is put in 37 DEG C, 5%CO by cell per well₂Cell volume training It supports case culture 7 days.At the 7th day, culture medium is changed into fresh culture (DMEM+10%FBS+1X HAT).After 2-3 days, Cell culture supernatant is drawn, screens hybridoma through ELISA and FACS.

Hybridoma cell line is screened by ELISA

The hybridoma clone in conjunction with people CD40 is screened in conjunction with detection by high-throughput ELISA first.With people CD40 In conjunction with hybridoma clone further test the ability in conjunction with monkey or mouse CD40.

ELISA is detected, employment CD40 (ECD)-his (0.5 μ g/ml, Cat#:10774-H08H, justice stick up Divine Land, in State), monkey CD40 (ECD)-hFc (0.5 μ g/ml, Cat#:90097-C02H, justice stick up Divine Land, China) or mouse CD40-his (0.5 μ g/ml, Cat#:50324-M03H, justice sticks up Divine Land, Chinese) to 96 hole elisa plate bed boards, the 100 every holes μ l, ambient temperature overnight. Elisa plate is cleaned 3 times with PBST (PBS+0.05% polysorbas20) solution, with 200 μ l confining liquid (PBS+1%BSA+1% blood of goats + 0.05% polysorbas20 clearly) room temperature closing 2 hours, it is cleaned 3 times with PBST solution.Hybridoma Cell Culture base supernatant dilution After (PBS+1%BSA ,+0.01% polysorbas20 of+1% lowlenthal serum) dilutes 10 times, draws 100 μ l and sample detection hole is added.Room temperature After being incubated for 1 hour, plate is cleaned 3 times with PBST.100 mountain μ l sheep anti mouse Fc-HRP (1: 5000, Cat#:A9309- are added in every hole 1ml, Sigma, the U.S.), it is incubated at room temperature 1 hour, PBST is cleaned 3 times, and 80 μ l TMB colour developing is added.80 μ l are added after 5-10 minutes 0.16M sulfuric acid color development stopping, with microplate reader (SpectraMaxR i3X, Molec μ lar Devies, the U.S.) detect OD450 Readings

Through above-mentioned ELISA, 234 hybridoma cell lines for having specific bond power with people and monkey CD40 are filtered out.

Screening hybridoma cell line is detected by FACS

234 hybridoma cell lines filtered out are further tested with people, monkey or the mouse of itself and the expression of HEK293A cell The binding ability of CD40.First by 10⁵Preparation HEK293A/ people CD40 cell, HEK293A/ monkey CD40 in a embodiment 1 is thin Born of the same parents or HEK293A/ mouse CD40 cell are added in each detection hole of 96 orifice plates.Hybridoma culture supernatant with dilution (PBS, Containing 1%BSA, 1% lowlenthal serum, 0.01% polysorbas20) dilution 10 times be added to sample detection hole, the 100 every holes μ l.4 DEG C are incubated for 1 After a hour, cleaned 3 times with FACS washing lotion (PBS+1%BSA+0.01% polysorbas20).Later, cell is resuspended with FACS washing lotion, It is added 500 times 4 degree of diluted APC goat anti-mouse IgG secondary antibody (Cat#:405308, BioLegen, the U.S.) to be incubated for 1 hour, plate Upper FACS detector (BD) detects cell fluorescence after cleaning 3 times with PBS.

It is screened based on above-mentioned FACS, obtains 162 to HEK293A/ people CD40 cell and HEK293A/ monkey CD40 cell There is high-bond without the hybridoma clone with HEK-293A/ mouse CD40 cell combination.

Generate the subclone of the hybridoma of CD40 antibody

2 wheel subclones are carried out to above-mentioned 162 hybridoma clones.In subcloning procedures, multiple Asias of each clone are selected It clones (n > 3), and carries out feature confirmation through above-mentioned ELISA and FACS detection.The subclone obtained through the step is determined as list Cloned hybridoma cell system.108 subclones that high-bond is showed to people and monkey CD40 are finally obtained, it is each to be subcloned From different original female clones.

Hybridoma cell line is screened by HEK-Blue Activity determination

By above-mentioned 108 screened subclone bed board in 96 orifice plates, and cultivate 5 days.It collects supernatant and carries out HEK- Blue Activity determination, to identify the CD40 antibody for having agonist activity to people's CD40 signal path.

By infecting HEK-Blue with the slow virus (preparation such as embodiment 1) of expression people CD40 (SEQ ID NO.:68) Null 1_v cell (InvivoGen, the U.S.) expresses the HEK-Blue reporter cell lines (HEK- of the fusion protein to construct Blue/CD40), then with the puromycin of 10 μ g/ml pressurization screening is carried out.

HEK-Blue/CD40 reporting system is detected, by 4 × 10⁴A HEK-Blue/CD40 cell is resuspended in 200 μ l Cell culture medium (DMEM culture medium (Hyclone, USA, Cat#:SH30243.01)+10%FBS (Excell, China, Cat#: FND500)+100 μ g/ml Normocin of+10 μ g/ml puromycin (GIBCO, USA, Cat#:A11138-03)^TM (Invivogen, USA, Cat#:ant-nr-2)+100 μ g/ml bleomycin (Invivogen, USA, Cat#:ant-Zn-5) In, in 96 orifice plate bed boards, and in 37 DEG C of overnight incubations.Second day, 200 μ l DMEM culture mediums were added in each hole, replaced former culture Base.After culture 7 hours, DMEM culture medium is replaced with HEK-Blue detection buffer (Invivogen, US, Cat#:hb-det3), The 100 every holes μ L, and 100 μ L doma supernatants are added in every hole.Obtained mixture is in 37 DEG C, 5%CO₂Lower incubation, until aobvious It is blue out.With the readings at SpectraMax microplate reader (Molec μ lar Devices, the U.S.) measurement OD630.It is wherein negative right According to for HEL antibody (LifeTein, LLC, US, Cat.#:LT12031).RO7009789 is (a kind of to be disclosed according to US7338660B2 Amino acid sequence preparation and have human IgG2/κ constant region agonistic antibody) and CD40L (Cat#:10239-H08E, justice are stuck up Divine Land, China, the native ligand and activity factor of CD40) it is used as positive control.

As shown in Figure 1,38 clones show different degrees of CD40 agonist activity.

The purifying of 3 mouse CD40 monoclonal antibody of embodiment

It is detected based on above-mentioned HEK-Blue, picking 20 there is the active clone of high HEK-Blue (to be detailed in following table altogether 3) it, conducts further research.The monoclonal mouse antibody of clone selected by 20 is purified first.Briefly, each The hybridoma of subclone is grown in T175 Tissue Culture Flask, each culture bottle fresh serum free containing 100ml hybridoma training Support base (Gibco, the U.S., Cat#:12045-076) and 1%HT replenisher (Gibco, Cat#:11067-030).Cell is 37 DEG C, 5%CO₂Incubator in cultivate 10 days.Culture is collected, 3500rpm is centrifuged 5 minutes, and passes through 0.22 μm of membrane filtration Remove cell fragment.Monoclonal antibody is enriched with by the albumen-A affinity column (Cat#:17040501, GE, the U.S.) pre-equilibrated Purifying.It is eluted afterwards with elution buffer (20mM citric acid, pH3.0-pH3.5).Later, antibody is stored in PBS (pH 7.0) in, and antibody concentration is detected by NanoDrop.

By using K and λ-mouse fast typing kit (Thermal, the U.S., Cat#:26179) and mouse monoclonal Antibody typing reagent (Sigma, the U.S., Cat#:IS02-1KT) determines the hypotype of antibody purification, and detecting step and kit say It is consistent in bright book.The hypotype and expression titre of selected 20 clonal antibodies are summarised in table 3.

The hypotype and expression titre of table 3.CD40 antibody

Clone	Hypotype	It expresses titre (mg/1)
			13A2	1/ κ of mouse IgG	24.744
16A6	1/ κ of mouse IgG	31.111
			29A10	1/ κ of mouse IgG	33.889
7B4	1/ κ of mouse IgG	18.667
			9A7	1/ κ of mouse IgG	7.778
19H4	1/ κ of mouse IgG	10.000
			37G10	1/ κ of mouse IgG	65.333
35C9	1/ κ of mouse IgG	11.667
			16F4	Mouse IgG 2b/ κ	14.000
50F6	1/ κ of mouse IgG	12.778
			77D9	1/ κ of mouse IgG	12.22
79D7	1/ κ of mouse IgG	20.39
			142F7	1/ κ of mouse IgG	17.22
89D11	1/ κ of mouse IgG	95.73
			91E4	Mouse IgG 2a/ κ	4.47
101C12	1/ κ of mouse IgG	18.94
			92F6	1/ κ of mouse IgG	39.97
82D3	Mouse IgG 2a/ κ	16.27
			23B8	Mouse IgG 2a/ κ	32.33
51F7	1/ κ of mouse IgG	1.44

The mouse CD40 monoclonal antibody that embodiment 4 purifies is in conjunction with people and monkey CD40

The mouse CD40 monoclonal antibody of purifying determines itself and recombined human, monkey or mouse by ELISA detection first The binding affinity of CD40 albumen.

ELISA detection plate is with 4 DEG C of 500ng/m1 people CD40 (ECD)-his (justice sticks up Divine Land, China, Cat:10774-H08H) Coating is overnight.2 are closed at room temperature with 200 μ l confining liquids (+0.05% polysorbas20 of PBS+1%BSA+1% lowlenthal serum) in each hole Hour, the CD40 antibody (40 μ g/ml of maximum concentration) of 100 μ l gradient dilutions is then added, is incubated at room temperature 1 hour.Plate PBST (PBS+0.05% polysorbas20) be added after washing 3 times 5000 times of diluted goat anti-mouse IgG-HRP (Simga, the U.S., Cat#: A9309-1ml), it is incubated at room temperature 1 hour.Ultra-TMB (BD, the U.S., Cat#no.:555214) room temperature of plate Fresh Colour developing 5 minutes.SpectraMax is used later^RI3X (Molecular Devies, the U.S.) is in 450nm readings.

20 CD40 monoclonal antibodies to the cross-species reactivity of monkey or mouse CD40 further pass through Salmonella into Row test.Specifically, by 500ng/ml monkey CD40 (ECD)-hFc albumen (justice sticks up Divine Land, China, Cat:90097-C02H) or small Mouse CD40-hFc (justice sticks up Divine Land, China, Cat:50324-M03H) is coated in 96 hole elisa plates, and with 100 μ l gradient dilutions CD40 antibody (40 μ g/m1 of maximum concentration) jointly be incubated for.Later using HRP- goat anti-mouse IgG (Sigma, the U.S., Cat#:A9309-lml).CD4 antibody RO7009789 and ADC1013 (the amino acid sequence according to disclosed in US2016/0311916A1 Column preparation, and there is human IgG1/κ constant region) it is used as reference.

The EC of above-mentioned binding test_s0Value is summarized in table 4.It can be seen that in 20 kinds of antibody, in addition to 51F7, and monkey CD40 cross reaction without with mouse CD40 cross reaction.

Binding force of the 4. 20 kinds of mouse CD40 monoclonal antibodies of table to people, monkey or mouse CD40

5 mouse CD40 monoclonal antibody of embodiment is in conjunction with people and monkey CD40 that HEK293A cell is expressed

To further determine that CD40 antibody whether in conjunction with the people of HEK293A cell expression, monkey or mouse CD40, makes respectively FACS cell combination detection is carried out with the HEK293A cell (see embodiment 1) for being overexpressed people, monkey or mouse CD40 is stablized.Simply For, by 10⁵A HEK293A cell bed board on 96 orifice plates, and CD40 antibody (the 40 μ g/ of maximum concentration of gradient dilution is added ml).After 4 DEG C of 1 hours of incubation, plate is cleaned 3 times with PBST.Later, 500 times of diluted APC- goat anti-mouse IgGs are added (BioLegen, the U.S., Cat#:405308).After 4 DEG C are incubated for 1 hour, cell is cleaned 3 times with PBS, is then detected using FACS Instrument (BD) monitors cell fluorescence.

As shown in table 5, all 20 mouse CD40 monoclonal antibodies show the high-bond with people and monkey CD40, but not In conjunction with mouse CD40 (data are not shown).

The binding affinity of table 5.CD40 antibody and people and monkey CD40

6 mouse CD40 antibody of embodiment inhibits or promotes the interaction between people CD40-CD40L

It is further tested to the CD40 antibody of purifying and hinders or promote ability of the human CD 40 L in conjunction with people CD40.Simply For, 96 hole elisa plates were coated with 4 DEG C of the human CD 40 L (Cat#:10239-H08E, justice stick up Divine Land, China) of 500ng/ml Night.Later, plate is closed 2 hours with 200 μ l confining liquid (PBS+2%BSA) room temperatures.CD40 antibody (the maximum concentration of gradient dilution It mixes for 40 μ g/ml) and 2 μ g/ml people CD40-hFc (Cat#:10774-H02H, justice stick up Divine Land), and is incubated for 1 hour in 37 DEG C, Detection hole is then added in obtained mixture, is incubated at room temperature 1 hour.Plate is washed 3 times with PBST (PBS+0.05% polysorbas20), is added 5000 times of diluted anti-human igg FC-HRP (Simga, the U.S., Cat#:A0170-1ML) are incubated at room temperature 1 hour.Plate PBST Washing lotion is washed 3 times, and 80 μ l TMB colour developing is added in each hole.After 5-10 minutes, 80 μ l 0.16M sulfuric acid are added to terminate reaction, Zhi Houyong SpectraMaxR i3X (Molec μ lar Devies, the U.S.) carries out OD450 readings.

Ironically, statistics indicate that, 6 kinds of mouse antibodies (13A2,16A6,7B4,50F6,142F7 and 101C12) promote People CD40-CD40L interaction, and there are 3 kinds of antibody (23B8,92F6,82D3) to hinder CD40-CD40L interaction, it is remaining Several antibody combine CD40-CD40L and do not act on significantly.4 representative clones as the result is shown in Figures 2 A and 2 B.

7 mouse CD40 antibody of embodiment activates CD40 signal path activity

To determine whether selected mouse CD40 antibody has CD40 signal path agonist activity, HEK-Blue activity is carried out Detection.Briefly, the HEK-Blue/CD40 cell incubation prepared by embodiment 2 DMEM culture medium (Hyclone, the U.S., Cat#:SH30243.01 in), culture medium also contains 10%FBS (Excell, China, Cat#:FND500), 10 μ g/ml purine Mycin (GIBCO, the U.S., Cat#:A11138-03), 100 μ g/mlNormocin^TM(Invivogen, the U.S., Cat#:ant-nr- 2), 100 μ g/ml bleomycin (Invivogen, the U.S., Cat#:ant-Zn-5).By 4 × 10⁴A HEK-Blue/CD40 cell It assigns in 200 μ l cell culture mediums of 96 orifice plates, 37 DEG C of overnight incubations (about 12 hours).With 200 μ l fresh DMEM mediums Former culture medium is replaced, culture 7 hours is continued.Later, the DMEM culture medium in each hole 100 μ l HEK Blue detection is substituted for delay Fliud flushing (Invivogen, U.S. Cat#:hb-det3；), wherein (concentration range is from 100 μ g/ for the CD40 antibody containing various concentration Ml to 0.01ng/ml).Cell continues to cultivate at 37 DEG C, until showing blue.Use SpectraMaxR i3X microplate reader (Molec μ lar Devices, the U.S.) detects OD630 readings.

EC₅₀Value is summarised in table 6, and the curve of representative monoclonal antibody is shown in Fig. 3.As can be seen that this 20 kinds of antibody are in function Different degrees of agonist activity can be shown in property HEK-Blue detection, show that CD40 downstream signaling pathway can be stimulated.

The agonist activity of table 6.CD40 antibody

Antibody	HEK-Blue EC50(ng/mL)
		RO7009789	23.26
ADC1013	1034
		13A2	12.61
16A6	11.37
		7B4	26.14
50F6	42.16
		101C12	175.2
77D9	22.26
		35C9	86.08
37G3	146.1
		79D7	55.29
142F7	64.77
		89D11	91.06
91E4	152.7
		82D3	285.4
23B8	3373
		51F7	6173
92F6	263.7
		19H4	92.79
16F4	252.1
		29A10	139.8
9A7	61.96

The competition of 8 epitope of embodiment

The antigen binding epitope competition between antibody is detected by the method for competitive ELISA.Briefly, 96 hole ELISA Detection plate is coated with the R07009789 or ADC1013 of 5 μ g/ml, and 4 DEG C overnight.200 μ l confining liquid (PBS+1%BSA of this some holes + 0.05% polysorbas20 of+1% lowlenthal serum) room temperature closing 2 hours.0.5 μ g/ml people CD40 (ECD)-his albumen (Cat#: 10774-H08H, justice stick up Divine Land, China) it is added in detection plate, room temperature continues to be incubated for 1 hour.Plate is cleaned 3 times with PBST, is added 1 The antibody of μ g/ml purifying, is incubated at room temperature 1 hour.20000 times of diluted anti-mouse Fc- are added after washing 3 times with PBST in elisa plate HRP (Cat#:A9309-1MC, Simga, the U.S.) is incubated at room temperature 1 hour.Continue after washing 3 times with PBST washing lotion, uses Fresh Ultra-TMB (Huzhou Yingchuang, China, Cat#:TMB-S-003) color development at room temperature 5 minutes, be used in combination SpectraMax microplate reader (Molecular Devices；US；SpectraMaxR i3X) in 450nm progress readings.

As a result it is summarised in table 7.7 kinds of mouse antibodies (101C12,142F7,89D11,13A2,16A6,7B4 and 50F6) with There are epitope competitions for two kinds of reference antibodies, and antibody 9A7,92F6,19H4,16F4 and 51F7 be not with any reference antibody that there are tables Position competition.There are epitope competitions for one of remaining several antibody and two reference antibodies.

The epitope competition of 7. competitive ELISA of table test

+: there is competition；: there is no competitions

9 excited type CD40 antibody of embodiment promotes dendritic cell maturation

Dendritic cell maturation experiment is carried out, to further confirm that the agonist activity of mouse CD40 antibody.Briefly, it comes from PBMC in one Healthy People donor blood sample is collected by way of gradient density centrifugal, and is resuspended in RPMI1640 culture medium In, 37 DEG C are cultivated 2 hours, adherent cell collecting, i.e. mononuclearcell.Above-mentioned cell culture in contain 100ng/ml recombined human GM-CSF(R&D；The U.S.；Cat.7954-GM), 100ng/ml recombined human IL-4 (R&D；The U.S.；) and 10% Cat#:6507-IL In the RPMI1640 culture medium of FBS.Culture medium is partly changed into liquid after 3 days, after cell culture 5 days, the CD40 of various concentration is anti- Body (10ug/ml or 1ug/ml) and control antibodies are added in cell culture and continue culture 48 hours.Use mouse anti human CD83(BD；USA；Cat#:556910), PE mouse anti human CD86 (BD；USA；) and BV650 mouse anti human Cat#:555658 CD80(BD；USA；Cat#:564158) dendritic cells Activation marker is dyed, and passes through flow cytomery.

Representative antibodies as the result is shown in figs. 4 a-4 c.Compared with Hel control, mouse CD40 antibody 16A6,29A10, 7B4 and 13A2 increases the expression of CD86 (biomarker of mature dendritic cell), and antibody 16A6,29A10,7B4 and 13A2 has significantly raised the expression of CD80 and CD83 (the two is costimulatory molecules).

Embodiment 10 is fitted into the expression and purifying of CD40 antibody

Choosing 8 antibody, ((13A2,16A6,7B4,29A10,92F6,77D9,50F6 and 142F7) is further ground Study carefully.The clone for carrying out variable region sequences to the hybridoma of this 8 antibody by RT-PCR method first, using being mentioned in document And primer (Juste, Muzard , &Billiald, (2006), Anal Biochem., 1；349 (1): 159-61).Passing through will (amino acid sequence of heavy chain constant region and constant region of light chain is listed in respectively for encoding variable regions and respective human IgG2/κ constant region SEQ ID NOs:63 and sequence 65) are inserted into the restriction enzyme site XhoI/ in (Invitrogen, the U.S.) pCDNA3.1 BamHI carrys out construction of expression vector.The amino acid SEQ ID coding of variable region is summarised in table 1.

By the expression vector transfection HEK-293F cell (Cobioer, China) of above-mentioned acquisition.Specifically, HEK-293F Cell is in Free Style^TMCulture in 293 expression culture medium (Gibco, Cat#:12338-018), and use polyethyleneimine (PEI) mode transfected transfects each expression vector to cell, and the ratio of DNA and PEI are 1:3, in every milliliter of cell culture fluid The amount that DNA is added is 1.5 μ g.HEK-293F cell after transfection is in 37 DEG C, 5%CO₂Incubator in 120RPM revolving speed training It supports.After 10-12 days, cells and supernatant is collected, according to the method and step monoclonal antibody purification of embodiment 3.

Embodiment 11CD40 chimeric mAb is in conjunction with the people that HEK293A cell is expressed or monkey CD40

According to the method and step of embodiment 5, the HEK293A/ people CD40 of they and the preparation of embodiment 1 is detected to chimeric antibody The binding force of cell, HEK293A/ monkey CD40 cell and HEK293A/ mouse CD40 cell.RO7009789 and ADC1013 is anti- Body is as positive control

As shown in Figure 5 A and 5B, chimeric antibody and people and monkey CD40 have high-affinity, without (the number in conjunction with mouse CD40 According to being not shown).

Embodiment 12CD40 chimeric antibody has CD40 signal path excitability and promotes dendritic cell maturation

According to the method and step of embodiment 7 and embodiment 9, detected by HEK-Blue detection and dendron maturation, to chimeric Antibody further detects its activation effect to CD40 signal path.RO7009789, APX005 and/or ADC1013 are used as the positive Control antibodies, wherein APX005 has human IgG1/κ constant region, prepares according to the amino acid sequence in WO2014/070934A1.

As shown in fig. 6,8 chimeric antibodies show functional activity similar with its mother's monoclonal antibody.Fig. 7 is shown, all tests Chimeric antibody can raise the expression of CD86 albumen on dendritic cells (biomarker of mature dendritic cell), show that its is right The promotion of dendritic cell maturation.

Embodiment 13CD40 antibody it is humanization modified

Based on above-mentioned relevant function test, two antibody, 7B4 and 13A2 are selected, is carried out humanization modified and further Research.The humanization modified of small source of mouse antibody is carried out by complementary determining region (CDR) grafting (United States Patent (USP) 5,225,539), Specific method is as detailed below.

For the humanization acceptor framework for selecting source of mouse antibody 7B4 and 13A2, by the light chain and heavy chain variable region of 7B4 and 13A2 Human immunoglobulin gene's database (http://www.ncbi.nlm.nih.gov/igblast/) of sequence and the website NCBI It is compared.It selects with the homologous highest ethnic group system IGVH and IGVK of degree of 7B4 and 13A2 as humanization modified frame.It is selected The light chain germline receptor sequence selected is people IGKV2-30*02, and selected heavy chain germline receptor sequence is people IGHV4-28*06, Specifically it is shown in Table 8.

Three dimensional structure simulation is carried out to the variable domains of 7B4 and 13A2, it may be for maintaining CDR cyclic structure with determination The crucial framework amino acid residues to play an important role, to design the back mutation of humanized antibody.In simple terms, selected structure Template has L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 of same type with 7B4 and 13A2 respectively Cyclic structure.Using selected stay in place form, structure and replacing small mouse framework with human germline heavy and light chain framework sequences Build the structural model of humanization 7B4 and 13A2.Then carry out three dimensional joint element, with identify may to maintain CDR ring-type knot Structure or heavy chain connect the crucial framework amino acid residues to play an important role with light chain.When source of mouse antibody framework and ethnic group system receptor frame Frame retains ethnic group system amino acid residue when possessing identical amino acid residue on a certain site.On the other hand, when source of mouse frame From ethnic group system acceptor framework when possessing different amino acid residues on a certain site, which is evaluated by structural simulation Importance.If it find that certain amino acid residue and CDR region residue in ethnic group system acceptor framework interact and to CDR residue Have an impact, then the residue will back mutation be source of mouse residue.

Table 8. is used for the stay in place form of antibody structure simulation

Antibody chain	The PDB of formwork structure is encoded	Sequence identity	Sequence similarity
				13A2 heavy chain	5E2T	71%	83%
13A2 light chain	1DLF	84%	92%
				7B4 heavy chain	5E2T	87%	90%
7B4 light chain	1DLF	87%	95%

On the basis of above structure modeling, 13A2 heavy chain identify 5 potential back mutations (I49M, V68I, M70I, K44N, G45K), light chain identifies 5 potential back mutations (M4L, R51L, F76L, Y92F, Q105S).7B4 heavy chain 5 potential back mutations (I49M, V68I, M70I, K44N, G45K) are identified, light chain identifies 4 potential back mutations (M4L、R51L、Y92F、Q105S)。

As shown in table 1, for 13A2, three humanization heavy chain variable regions and three humanization light chain variables are designed altogether 5 humanized antibodies are obtained in area.Similarly, for 7B4, three humanization heavy chain variable regions and three source of people are designed altogether Change light chain variable region, 5 humanized antibodies are obtained.

Sequence and encoding humanized light chain variable of the composite coding humanized heavy chain variable region plus human IgG2's constant region Area adds the sequence of people K constant region, and the amino acid sequence of heavy chain constant region and constant region of light chain is listed in SEQ ID NOs:63 respectively PCDNA3.1 (+) expression vector (Invitrogen, beauty are cloned into 65, and using BamH I and Xho I restriction enzyme site State) in.All expression buildings are confirmed through sequencing.With heavy chain expression vector and light chain expression vector transfection HEK293F expression system It unites in (Invitrogen, the U.S.), and 10 humanization CD40 antibody of transient expression (7B4 antibody 5,13A2 antibody 5), method Step is as described in Example 10.Humanized antibody is purified according to described in embodiment 3.

The chimeric characteristic test with humanization CD40 antibody of embodiment 14

According to the method and step in embodiment 5, itself and HEK193A/ are further tested to chimeric and humanization CD40 antibody The binding force of people CD40 and HEK293A/ monkey CD40.It is anti-to chimeric and humanization CD40 by HEK-Blue referring to embodiment 7 Body further detects its activating force to CD40 signal path.According to the method and step in embodiment 9, to humanized antibody into one Pacing tries its effect for promoting dendritic cell maturation, and dendritic cells derive from the blood sample of 3 different Healthy People donors.Dendron is thin The secretion situation of IL-12 (p40) in born of the same parents is detected with IL-12 (p40) detection kit (BD, US, Cat#:551116), specific real It is completely the same with specification to test step.

As shown in Fig. 8 A-8D, 9A-9B, 10A-10C (donor 1), 11A-11B (donor 2) and 12A-12C (donor 3), All people source antibody with people and monkey OX40 protein binding, not in conjunction with mouse OX40, can activate CD40 signal path, Promote dendritic cell maturation, and 13A2-VH3VL2,13A2-VH3VL3 and 7B4VH2VL2 show highest combination, excitement and Functional activity.

Embodiment 15. is fitted into and the affinity of humanization CD-40 antibody and people CD40

Chimeric or humanized CD40 antibody is quantitative determined by BIAcoreTM 8K (GE Life Sciences, the U.S.) To the binding affinity of people CD40.

Specifically, by people CD40 (the ECD)-his albumen of 100-200RU (reacton) (justice sticks up Divine Land, China, Cat: It 10774-H08H) is coupled in CM5 biochip (Cat#:BR-1005-30, GE Life Sciences, the U.S.), then uses 1M ethylaminoethanol closes chip unreacted group.The antibody of gradient dilution (concentration from 0.3 μM to 10 μM) is injected into SPR reaction solution In (HBS-EP buffer, pH7.4, Cat#:BR-1006-69, GE Life Sciences, the U.S.), speed control is in 30 μ L/ Minute.When the binding force of antibody calculates, the RU of blank control wells is reduced.Association rate (ka) and dissociation rate (kd) use BIA The formula of 1: 1 pairing model in assessment software is calculated.Equilibrium dissociation constant K_DPass through k_d/k_aIt is calculated.SPR is determined Antibody combination dissociation curve as shown in Figure 13 A-13N, table 9 shows the binding affinity number of chimeric antibody and humanized antibody According to.RO7009789 and ADC1013 antibody is as positive control.

The binding affinity of table 9.CD40 antibody on human CD40

Antibody	ka	kd	KD
				RO700789	1.07E+5	1.83E-4	1.71E-9
ADC1013	1.2E+6	3.48E-2	2.9E-08
				13A2	4.84E+05	1.73E-03	3.58E-09
13A2-VH0VL0	3.96E+05	1.14E-02	2.88E-08
				13A2-VH2VL2	8.23E+05	1.71E-03	2.08E-09
13A2-VH2VL3	7.35E+05	2.61E-03	3.55E-09
				13A2-VH3VL2	8.25E+5	2.42E-3	2.93E-09
13A2-VH3VL3	5.98E+5	4.59E-3	7.68E-09
				7B4	1.00E+06	3.6E-03	3.6E-09
7B4-VH0VL0	2.75E+06	6.8E-03	2.47E-09
				7B4-VH2VL2	2E+06	5.81E-03	2.91E-09
7B4-VH2VL3	1.78E+06	6.39E-03	3.6E-09
				784-VH3VL2	1.46E+06	6.15E-03	4.2E-09
784-VH3VL3	2.13E+06	4.88E-03	2.99E-09

The chimeric antigen binding Epitope Identification with humanization CD40 antibody of embodiment 16

Pass through the chimeric antigen binding epitope with humanization CD40 antibody of ELISA test.

The extracellular region (ECD) of CD40 include 4 cysteine rich structural domains (CRD), be respectively designated as CRD1, CRD2, CRD3 and CRD4.Based on the structure of CD40 ECD, overall length CD40 ECD, five CD40 ECD truncates and four are constructed CD40 ECD mutant, the information of these recombinant proteins are shown in following table 10.These albumen are connected with signal peptide in N-terminal (SEQ ID NO.:83) secretes to protein expression, is connected with mFc label (SEQ ID NO.:84) in C-terminal, examine for ELISA It surveys.The DNA sequence dna of these recombinant proteins of composite coding is simultaneously cloned into pcDNA3.1 carrier.Recombinant protein expression and purifying basis Method and step in embodiment 10 carries out.ELISA detection is carried out according to method and step as described in example 2, to assess monoclonal antibody pair The binding force of recombinant C D40 albumen.

As shown in Figure 14 A, all antibody shows not in conjunction with truncate in conjunction with overall length CD40 ECD, and CRD1 structural domain participates in antibody combination, and more important to antibody combination.Figure 14 B is shown, 13A2 chimeric antibody, three kinds of people Source antibody and ADC1013 show that this five kinds of antibody have the same or similar combination epitope not in conjunction with CD40 mutant 2-4. RO7009789 is not combined with mutant 1,2 or 4, and APX005 is not combined with mutant 2 or 4.

Table 10.CD40ECD truncate and mutant

Note: CRD Δ 1 indicates truncated CRD1 structural domain

17 humanization CD40 antibody of embodiment and people CD40 specific bond

The binding specificity of CD40 is examined according to method and step as described in example 2 by ELISA method for detection antibody Survey the binding force of humanized antibody and people CD40 and higher other members of TNFRSF family of sequence homology.

Specifically, having detected humanized antibody and people CD40 (ECD)-his (TNFRSF4, Cat#:10774-H08H, justice Stick up Divine Land, China), people OX40-his (TNFRSF5, Cat#:10481-H08H, justice stick up Divine Land, China), people HVEM-mFc (TNFRSF14, Cat#:HVM-H5255, ACRO, China), people 4-1BB (TNFRSF9, Cat#:41B-H522a, ACRO, in State), people NGFR (TNFRSF16, Cat#:13184-H08H, justice stick up Divine Land, China), people DR6 (TNFRSF21, Cat#:10175- H08H, justice stick up Divine Land, China) and people RANK (TNFRSF11, Cat#:RAL-H5240, ACRO, justice stick up Divine Land, China) knot Close affinity.

As shown in figs. 15a and 15b, 13A2VH3VL3 and 7B4VH2VL2 not with people OX40 (TNFRSF4), HVEM (TNFRSF14), 4-1BB (TNFRSF9), NGFR (TNFRSF16), DR6 (TNFRSF21) or RANK (TNFRSF11) are combined, table The binding specificity of bright 13A2VH3VL3 and 7B4VH2VL2 antibody and people CD40.

The CD40 antibody of 18 gene modification of embodiment has better agonist activity

Studies have shown that the best biology and antitumous effect of excited type CD40 antibody need the common work of Fc receptor (FcR) With (Richman and Vonderheide, (2014) Cancer Immunol Res 2 (1): 19-26).Therefore, CD40 is resisted Body is prepared into weight/light chain variable region and human IgG1/κ constant region with 13A2VH3VL3 or 7B4VH2VL2, and human IgG1 is permanent Determining zone has S267E and L328F mutation (mutation IgG1 amino acid constant region sequence such as SEQ ID NO.:64).Obtained antibody It is respectively designated as 13A2-VH3VL3-IgG1 (SE/LF) and 7B4-VH2VL2-IgG1 (SE/LF), and according to the side in embodiment 9 Method step further tests its activity for promoting dendritic cell maturation.RO7009789, ADC1013 and APX005 are used as positive right According to Hel is used as negative control.

As shown in Figure 16 A, 16B and 16C, compared with parental antibody, 13A2-VH3VL3-IgG1 (SE/LF) and 7B4- VH2VL2-IgG1 (SE/LF) all shows higher agonist activity, and 13A2-VH3VL3- in terms of promoting dendritic cell maturation IgG1 (SE/LF) has most highly active in all test antibodies.

19 humanized antibody of embodiment has internal antitumous effect

To the 1/ κ constant region (mouse of weight/light chain variable region and mouse IgG with 13A2VH3VL3 or 7B4VH2VL2 The amino acid sequence of IgG1/ κ constant region is as shown in SEQ ID NOs:62 and 66) CD40 antibody internal anti-tumor activity into Row research, used animal model by transgenic mice to CD40 target spot humanization (GemPharmatech Co.Ltd, China) it is implanted into MC38 mouse enteraden cancer and establishes.Antibody reinforces antibody in mouse model using 1/ κ constant region of mouse IgG Fc function.

Each mouse is at the 0th day in side abdominal part hypodermic 1 × 10⁶MC38 cell.80mm is grown to tumour³When, random point At five groups, every group 8.Mouse was in the 4th, 7,11,14,18 and 21 day intraperitoneal injection 13A2-VH3VL3,7B4-VH2VL2, control Antibody (RO7009789 or APX005) or PBS, 10mg/kg/ days, wherein RO7009789 and APX005, which is transformed into, had such as 1/ κ constant region of mouse IgG shown in SEQ ID NOs:62 and 66.

The variation of tracking mouse weight and tumor size at any time.Measure the long side (D) of tumour and short every other day with vernier caliper Side (d), and pass through formula TV=0.5x D x d²Calculate gross tumor volume.Reach 3.5cm in antibody group tumour³Preceding stopping experiment. Tumor volume difference is determined with one-way analysis of variance.

At the 25th day, each group took 4 biggish mouse of tumour to analyze for T cell, and it is big that other mouse continue tumour Small measurement.Mouse for T cell analysis takes tumour to be placed in the Hanks buffer of clostridiopetidase A immediately after execution.With Tumor tissues are cut into small pieces by scissors, and the tumor tissues shredded are continued to be incubated in the Hanks buffer containing clostridiopetidase A, 37 It is DEG C light and slow to shake 30 minutes.Later, 10ml RPMI 1640+10%FBS is added to each sample, terminates collagenase activity, keep Immunocyte vigor.It by sample by 70 μm of cell filter membranes (Corning, Cat#:352350), is filtered, and is placed on new In centrifuge tube.It is resuspended in PBSF buffer (PBS+2%FBS) after sample centrifugation, cell density is 1 × 10⁷Cell/ml.Sample Product are cleaned 2 times with PBSF, are then divided into two parts, thereto a CD45 antibody (Brilliant that fluorescent marker is added Violet 785^TMMouse CD45 antibody, Biolegend, US, Cat#:103149), CD8 antibody (APC mouse CD8a antibody, Biolegend, US, Cat#:100712), CD3 antibody (FITC mouse CD3 antibody, Biolegend, US, Cat#:100203) and CD4 antibody (PerCP mouse CD4 antibody, Biolegend, US, Cat#:100432) mixture, to other a addition fluorescence mark CD11c antibody (APC mouse CD8a antibody, Biolegend, US, Cat#:100712), CD80 antibody (the APC mouse CD11c of note Antibody, Biolegend, US, Cat#:117310), CD83 antibody (E/Cv7 mouse CD83 antibody, Biolegend, US, Cat#: And CD86 antibody (FITC mouse CD86 antibody, Biolegend, US, Cat#:105005) mixture 121518).Resulting mixing 4 DEG C of incubation half an hours of object.Cell is cleaned 2 times with PBSF, is then analyzed through FACS instrument (BD).

As shown in Figure 17 A-17F, compared with negative control group, the treatment of CD40 antibody slows down or inhibits significantly tumour raw It is long, although individual reaction is different.In 7B4VH2VL2 and APX005 group, Tumor growth inhibition is observed in all mouse, 13A2VH3VL3 and RO7009789 group, most of mouse show as Tumor growth inhibition.At the 28th day, applied in 7B4VH2VL2 With in group, 4 intracorporal cases of complete remission of remaining mouse, in APX005 group, 2 intracorporal tumours of mouse in 4 are complete It totally disappeared mistake.

CD40 Antybody therapy may be because that antibody toxicity problem causes mouse weight to decline.As shown in Figure 18 and table 11, button Except (tumour was about 1000mm to tumor weight on 25th³, 1.2g) after, 13A2VH3VL3 processing group mouse the 25th day weight and just Initial body is increased slightly again, without such as RO7009789 processing bring significantly weight loss the problem of, and compare 7B4VH2VL2 and APX005, it is smaller for weight influence, show that the toxicity of 13A2VH3VL3 antibody is lower.

The weight (mean value ± SE (g), every group of 8 mouse) of 11. each group mouse of table

Group/number of days

4

6

8

11

14

18

21

25

13A2-VH3VL3

22.9±0.5

20.6±0.5

22.5±0.5

24.1±0.5

24.7±0.5

25.2±0.5

24.7±0.5

25.4±0.8

7B4-VH2VL2

23.0±0.3

21.2±0.2

23.4±0.2

23.8±0.2

22.7±0.5

24.4±0.2

22.2±0.5

22.9±0.5

RO7009789

23.9±0.3

21.5±0.4

22.9±0.4

22.6±0.3

22.2±0.4

22.1±0.5

21.8±0.4

19.6±0.5

APX005

22.6±0.3

20.8±0.3

22.9±0.3

23.1±0.4

23.4±0.6

24.1±0.4

21.5±0.6

22.0±0.7

PBS

23.3±0.4

23.2±0.3

24.2±0.4

23.7±0.3

25.1±0.4

26.5±0.4

27.8±0.5

30.7±0.6

Figure 19 A and 19B are shown, and antibody 7B4VH2VL2 has been obviously improved CD45⁺CD45 in cell⁺CD3⁺CD8⁺Cell and CD45⁺CD3⁺CD4⁺The percentage of cell.CD45⁺CD3⁺CD8⁺The percentage of cell also rises in 13A2VH3VL3 processing group. In addition, Figure 20 shows that 7B4VH2VL2 processing significantly increases tumor-infiltrated dendritic cells (CD45⁺CD11c⁺Cell) in CD80 It is expressed with CD83, shows that it is promoting the stronger agonist activity in dendritic cell maturation.

Although the present invention has combined one or more embodiments to be described, it should be appreciated that the present invention is unlimited In these embodiments, and foregoing description be intended to it is included in the spirit and scope of the appended claims it is every other can Selection form, modification and equivalent.All references recited herein are fully incorporated herein by reference.

Sequence table

<110>Beijing Tian-Guang Biotechnology Co., Ltd. Is

<120>antibody and application thereof of CD40 is combined

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<220>

<223> 13A2/7B4-HV-CDR1

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Thr Asn Tyr Tyr Trp Asn

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<220>

<223> 16A6-HV-CDR1

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Thr Asn Tyr His Trp Asn

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<220>

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Ser His Tyr Tyr Met Tyr

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Asn Tyr Leu Ile Glu

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<223> 29A10-HV-CDR2

<400> 10

Thr Ile Ser Asp Ala Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-HV-CDR2

<400> 11

Arg Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 12

<211> 19

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-HV-CDR2

<400> 12

Glu Val Ser Gly Ser Gly Tyr Tyr Thr Tyr Tyr Pro Asp Thr Val Thr

1 5 10 15

Gly Arg Phe

<210> 13

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-HV-CDR2

<400> 13

Arg Ile Ser Pro Gly Ser Gly Ser Thr His Tyr Asn Glu Met Phe Lys

1 5 10 15

Gly

<210> 14

<211> 15

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-HV-CDR2

<400> 14

Asn Pro Gly Thr Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys Asp

1 5 10 15

<210> 15

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> 13A2/7B4/16A6-HV-CDR3

<400> 15

Leu Asp Tyr

1

<210> 16

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-HV-CDR3

<400> 16

Gly Gly Tyr Trp Phe Phe Asp Val

1 5

<210> 17

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-HV-CDR3

<400> 17

Trp Gly Tyr Asp Trp Tyr Phe Asp Val

1 5

<210> 18

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-HV-CDR3

<400> 18

Arg Ala Tyr

1

<210> 19

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-HV-CDR3

<400> 19

Asn Asp Tyr

1

<210> 20

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-HV-CDR3

<400> 20

Gly Gly Ser Gly Phe Ala Tyr

1 5

<210> 21

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223> 13A2/7B4/16A6-LV-CDR1

<400> 21

Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn Gly Asn Thr Phe Leu Asn

1 5 10 15

<210> 22

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-LV-CDR1

<400> 22

Glu Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 23

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-LV-CDR1

<400> 23

Ser Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 24

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-LV-CDR1

<400> 24

Arg Ser Ser Gln Ser Ile Val Leu Thr Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 25

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-LV-CDR1

<400> 25

Arg Ser Ser Gln Ser Ile Val Asn Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 26

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-LV-CDR1

<400> 26

Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

1 5 10

<210> 27

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 13A2/7B4/16A6/77D9/50F6-LV-CDR2

<400> 27

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 28

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-LV-CDR2

<400> 28

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 29

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-LV-CDR2

<400> 29

Thr Thr Ala Asn Leu Ala Ser

1 5

<210> 30

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-LV-CDR2

<400> 30

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 31

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 13A2/7B4/16A6-LV-CDR3

<400> 31

Leu Gln Val Thr His Val Pro Phe Thr

1 5

<210> 32

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-LV-CDR3

<400> 32

Gln Gln Tyr Tyr Arg Ser Pro Leu Thr

1 5

<210> 33

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-LV-CDR3

<400> 33

Gln Gln Arg Ser Asn Tyr Pro Phe Thr

1 5

<210> 34

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-LV-CDR3

<400> 34

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 35

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-LV-CDR3

<400> 35

Phe Gln Gly Ser His Val Pro Leu Thr

1 5

<210> 36

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-LV-CDR3

<400> 36

Gln Gln Gly Asn Thr Leu Pro

1 5

<210> 37

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-HV

<400> 37

Glu Val Lys Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 38

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 7B4-HV

<400> 38

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 39

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 16A6-HV

<400> 39

Glu Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr His Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 40

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-HV

<400> 40

Gln Val Lys Leu Glu Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Asp Ala Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Asp Asp Thr Ala Met Tyr Phe Cys

85 90 95

Ala Arg Thr Tyr Tyr Arg Gly Asp Gly Gly Tyr Trp Phe Phe Asp Val

100 105 110

Trp Gly Ala Gly Thr Ala Val Thr Val Ser Ser

115 120

<210> 41

<211> 118

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-HV

<400> 41

Gln Val Gln Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Gly Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 42

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-HV

<400> 42

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ser Pro Gly Glu Arg Leu Glu Trp Val

35 40 45

Ala Glu Val Ser Gly Ser Gly Tyr Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr

65 70 75 80

Leu Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Ser Arg Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

100 105 110

<210> 43

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-HV

<400> 43

Gln Val Gln Leu Glu Gln Ser Gly Asp Asp Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Ser Pro Gly Ser Gly Ser Thr His Tyr Asn Glu Met Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Ser Leu Ser Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Thr Arg Asn Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

100 105 110

<210> 44

<211> 116

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-HV

<400> 44

Glu Val Gln Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Gly Ile Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Thr Gly Gly Thr Asn Tyr Asn Glu 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 Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 45

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH0VL0-HV

<400> 45

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 46

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH2VL2/VH2VL3-HV

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 47

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH3VL2/VH3VL3-HV

<400> 47

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 48

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 7B4-VH0VL0-HV

<400> 48

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 49

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 7B4-VH2VL2/VH2VL3-HV

<400> 49

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 50

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 7B4-VH3VL2/7B4-VH3VL3-HV

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 51

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-LV

<400> 51

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 52

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 7B4-LV

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 53

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 16A6-LV

<400> 53

Asp Ile Val Leu Thr Gln Ser Thr Leu Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Leu Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 54

<211> 113

<212> PRT

<213>artificial sequence

<220>

<223> 29A10-LV

<400> 54

Asp Ile Val Ile Thr Gln Ser Thr Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Glu Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Arg Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 55

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223> 92F6-LV

<400> 55

Asp Ile Val Ile Thr Gln Ser Thr Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Thr Thr Ala Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 56

<211> 113

<212> PRT

<213>artificial sequence

<220>

<223> 77D9-LV

<400> 56

Asp Ile Val Met Thr Gln Ser Pro Thr Leu Ser Leu Pro Val Ser Leu

1 5 10 15

Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Leu

20 25 30

Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln

85 90 95

Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 57

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 50F6-LV

<400> 57

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 58

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223> 142F7-LV

<400> 58

Asp Ile Val Leu Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 59

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH0VL0/7B4-VH0VL0-LV

<400> 59

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 60

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH2VL2/VH3VL2/7B4-VH2VL2/VH3VL2-LV

<400> 60

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223> 13A2-VH2VL3/VH3VL3/7B4-VH2VL3/VH3VL3-LV

<400> 61

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 62

<211> 324

<212> PRT

<213>artificial sequence

<220>

<223>1 heavy chain constant region of mouse IgG

<400> 62

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 63

<211> 326

<212> PRT

<213>artificial sequence

<220>

<223>human IgG2's heavy chain constant region

<400> 63

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 64

<211> 330

<212> PRT

<213>artificial sequence

<220>

<223>human IgG1's heavy chain constant region is mutated containing S267E and L328F

<400> 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 65

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>people Κ constant region of light chain

<400> 65

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 66

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>mouse Κ constant region of light chain

<400> 66

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 67

<211> 834

<212> DNA

<213>homo sapiens (Homo sapiens)

<400> 67

atggttcgtc tgcctctgca gtgcgtcctc tggggctgct tgctgaccgc tgtccatcca 60

gaaccaccca ctgcatgcag agaaaaacag tacctaataa acagtcagtg ctgttctttg 120

tgccagccag gacagaaact ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt 180

ccttgcggtg aaagcgaatt cctagacacc tggaacagag agacacactg ccaccagcac 240

aaatactgcg accccaacct agggcttcgg gtccagcaga agggcacctc agaaacagac 300

accatctgca cctgtgaaga aggctggcac tgtacgagtg aggcctgtga gagctgtgtc 360

ctgcaccgct catgctcgcc cggctttggg gtcaagcaga ttgctacagg ggtttctgat 420

accatctgcg agccctgccc agtcggcttc ttctccaatg tgtcatctgc tttcgaaaaa 480

tgtcaccctt ggacaagctg tgagaccaaa gacctggttg tgcaacaggc aggcacaaac 540

aagactgatg ttgtctgtgg tccccaggat cggctgagag ccctggtggt gatccccatc 600

atcttcggga tcctgtttgc catcctcttg gtgctggtct ttatcaaaaa ggtggccaag 660

aagccaacca ataaggcccc ccaccccaag caggaacccc aggagatcaa ttttcccgac 720

gatcttcctg gctccaacac tgctgctcca gtgcaggaga ctttacatgg atgccaaccg 780

gtcacccagg aggatggcaa agagagtcgc atctcagtgc aggagagaca gtga 834

<210> 68

<211> 277

<212> PRT

<213>homo sapiens

<400> 68

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile

195 200 205

Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn

210 215 220

Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225 230 235 240

Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His

245 250 255

Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser

260 265 270

Val Gln Glu Arg Gln

275

<210> 69

<211> 837

<212> PRT

<213>macaque (Macaca mulatta)

<400> 69

Ala Thr Gly Gly Thr Thr Cys Gly Thr Cys Thr Gly Cys Cys Thr Cys

1 5 10 15

Thr Gly Cys Ala Gly Thr Gly Cys Gly Thr Cys Cys Thr Cys Thr Gly

20 25 30

Gly Gly Gly Cys Thr Gly Cys Thr Thr Gly Cys Thr Gly Ala Cys Cys

35 40 45

Gly Cys Thr Gly Thr Cys Thr Ala Thr Cys Cys Ala Gly Ala Ala Cys

50 55 60

Cys Ala Cys Cys Cys Ala Cys Thr Gly Cys Ala Thr Gly Cys Ala Gly

65 70 75 80

Ala Gly Ala Ala Ala Ala Ala Cys Ala Gly Thr Ala Cys Cys Thr Ala

85 90 95

Ala Thr Ala Ala Ala Cys Ala Gly Thr Cys Ala Gly Thr Gly Cys Thr

100 105 110

Gly Thr Thr Cys Thr Thr Thr Gly Thr Gly Cys Cys Ala Gly Cys Cys

115 120 125

Ala Gly Gly Ala Cys Ala Gly Ala Ala Ala Cys Thr Gly Gly Thr Gly

130 135 140

Ala Gly Thr Gly Ala Cys Thr Gly Cys Ala Cys Ala Gly Ala Gly Thr

145 150 155 160

Thr Cys Ala Cys Cys Gly Ala Ala Ala Cys Ala Gly Ala Ala Thr Gly

165 170 175

Cys Cys Thr Thr Cys Cys Thr Thr Gly Cys Ala Gly Thr Gly Ala Ala

180 185 190

Ala Gly Cys Gly Ala Ala Thr Thr Cys Cys Thr Ala Gly Ala Cys Ala

195 200 205

Cys Cys Thr Gly Gly Ala Ala Thr Ala Gly Ala Gly Ala Gly Ala Cys

210 215 220

Ala Cys Gly Cys Thr Gly Cys Cys Ala Cys Cys Ala Gly Cys Ala Cys

225 230 235 240

Ala Ala Ala Thr Ala Cys Thr Gly Cys Gly Ala Cys Cys Cys Cys Ala

245 250 255

Ala Cys Cys Thr Ala Gly Gly Gly Cys Thr Thr Cys Gly Gly Gly Thr

260 265 270

Cys Cys Ala Gly Cys Ala Gly Ala Ala Gly Gly Gly Cys Ala Cys Cys

275 280 285

Thr Cys Ala Gly Ala Ala Ala Cys Ala Gly Ala Cys Ala Cys Cys Ala

290 295 300

Thr Cys Thr Gly Cys Ala Cys Cys Thr Gly Thr Gly Ala Ala Gly Ala

305 310 315 320

Ala Gly Gly Cys Cys Thr Gly Cys Ala Cys Thr Gly Thr Ala Thr Gly

325 330 335

Ala Gly Thr Gly Ala Gly Thr Cys Cys Thr Gly Thr Gly Ala Gly Ala

340 345 350

Gly Cys Thr Gly Thr Gly Thr Cys Cys Cys Gly Cys Ala Cys Cys Gly

355 360 365

Cys Thr Cys Ala Thr Gly Cys Thr Thr Gly Cys Cys Thr Gly Gly Cys

370 375 380

Thr Thr Thr Gly Gly Gly Gly Thr Cys Ala Ala Gly Cys Ala Gly Ala

385 390 395 400

Thr Thr Gly Cys Thr Ala Cys Ala Gly Gly Gly Gly Thr Thr Thr Cys

405 410 415

Thr Gly Ala Thr Ala Cys Cys Ala Thr Cys Thr Gly Thr Gly Ala Gly

420 425 430

Cys Cys Cys Thr Gly Cys Cys Cys Gly Gly Thr Cys Gly Gly Cys Thr

435 440 445

Thr Cys Thr Thr Cys Thr Cys Cys Ala Ala Thr Gly Thr Gly Thr Cys

450 455 460

Ala Thr Cys Thr Gly Cys Thr Thr Thr Thr Gly Ala Ala Ala Ala Gly

465 470 475 480

Thr Gly Thr Cys Gly Cys Cys Cys Thr Thr Gly Gly Ala Cys Ala Ala

485 490 495

Gly Cys Thr Gly Thr Gly Ala Gly Ala Cys Cys Ala Ala Ala Gly Ala

500 505 510

Cys Cys Thr Gly Gly Thr Thr Gly Thr Gly Cys Ala Ala Cys Ala Gly

515 520 525

Gly Cys Ala Gly Gly Cys Ala Cys Ala Ala Ala Cys Ala Ala Gly Ala

530 535 540

Cys Thr Gly Ala Thr Gly Thr Thr Gly Thr Cys Thr Gly Thr Gly Gly

545 550 555 560

Thr Cys Cys Cys Cys Ala Gly Gly Ala Thr Cys Gly Gly Cys Ala Gly

565 570 575

Ala Gly Ala Gly Cys Cys Cys Thr Gly Gly Thr Gly Gly Thr Gly Ala

580 585 590

Thr Cys Cys Cys Cys Ala Thr Cys Thr Gly Cys Thr Thr Gly Gly Gly

595 600 605

Gly Ala Thr Cys Cys Thr Gly Thr Thr Thr Gly Thr Cys Ala Thr Cys

610 615 620

Cys Thr Cys Cys Thr Cys Thr Thr Gly Gly Thr Gly Cys Thr Gly Gly

625 630 635 640

Thr Cys Thr Thr Thr Ala Thr Cys Ala Ala Ala Ala Ala Gly Gly Thr

645 650 655

Gly Gly Cys Cys Ala Ala Gly Ala Ala Gly Cys Cys Ala Ala Ala Cys

660 665 670

Gly Ala Thr Ala Ala Gly Gly Cys Cys Cys Cys Cys Cys Ala Cys Cys

675 680 685

Cys Cys Ala Ala Gly Cys Ala Gly Gly Ala Ala Cys Cys Cys Cys Ala

690 695 700

Gly Gly Ala Gly Ala Thr Cys Ala Ala Thr Thr Thr Thr Cys Thr Gly

705 710 715 720

Gly Ala Cys Gly Ala Thr Cys Thr Thr Cys Cys Thr Gly Gly Cys Thr

725 730 735

Cys Cys Ala Ala Cys Cys Cys Thr Gly Cys Cys Gly Cys Thr Cys Cys

740 745 750

Ala Gly Thr Gly Cys Ala Gly Gly Ala Gly Ala Cys Thr Thr Thr Ala

755 760 765

Cys Ala Thr Gly Gly Ala Thr Gly Cys Cys Ala Ala Cys Cys Ala Gly

770 775 780

Thr Cys Ala Cys Cys Cys Ala Gly Gly Ala Gly Gly Ala Thr Gly Gly

785 790 795 800

Cys Ala Ala Ala Gly Ala Gly Ala Gly Thr Cys Gly Cys Ala Thr Cys

805 810 815

Thr Cys Ala Gly Thr Gly Cys Ala Gly Gly Ala Gly Ala Gly Ala Cys

820 825 830

Ala Gly Thr Gly Ala

835

<210> 70

<211> 278

<212> PRT

<213>macaque

<400> 70

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val Tyr Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Ser Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Met

100 105 110

Ser Glu Ser Cys Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys Arg Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Cys Leu Gly Ile Leu Phe Val Ile

195 200 205

Leu Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Asn

210 215 220

Asp Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Leu

225 230 235 240

Asp Asp Leu Pro Gly Ser Asn Pro Ala Ala Pro Val Gln Glu Thr Leu

245 250 255

His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

260 265 270

Ser Val Gln Glu Arg Gln

275

<210> 71

<211> 869

<212> DNA

<213>mouse (Mus musculus)

<400> 71

atggtgtctt tgcctcggct gtgcgcgcta tggggctgct tgttgacagc ggtccatcta 60

gggcagtgtg ttacgtgcag tgacaaacag tacctccacg atggccagtg ctgtgatttg 120

tgccagccag gaagccgact gacaagccac tgcacagctc ttgagaagac ccaatgccac 180

ccatgtgact caggcgaatt ctcagcccag tggaacaggg agattcgctg tcaccagcac 240

agacactgtg aacccaatca agggcttcgg gttaagaagg agggcaccgc agaatcagac 300

actgtctgta cctgtaagga aggacaacac tgcaccagca aggattggag gcatgtgctc 360

agcacacgcc ctgtatccct ggctttggag ttatggagat ggccactgag accactgata 420

ccgtctgtca tccctgccca gtcggcttct tctccaatca gtcatcactt ttcgaaaagt 480

gttatccctg gacaagctgt gaggataaga acttggaggt cctacagaaa ggaacgagtc 540

agactaatgt catctgtggt ttaaagtccc ggatgcgagc cctgctggtc attcctgtcg 600

tgatgggcat cctcatcacc attttcgggg tgtttctcta tatcaaaaag gtggtcaaga 660

aaccaaagga taatgagatc ttaccccctg cggctcgacg gcaagatccc caggagatgg 720

aagattatcc cggtcataac accgctgctc cagtgcagga gacgctgcac gggtgtcagc 780

ctgtcacaca ggaggatggt aaagagagtc gcatctcagt gcaggagcgg caggtgacag 840

acagcatagc cttgaggccc ctggtctga 869

<210> 72

<211> 289

<212> PRT

<213>mouse

<400> 72

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu Gly Gln Cys Val Thr Cys Ser Asp Lys Gln Tyr Leu

20 25 30

His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr

35 40 45

Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys Asp Ser

50 55 60

Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His Gln His

65 70 75 80

Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr

85 90 95

Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr

100 105 110

Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly

115 120 125

Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys

145 150 155 160

Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln

165 170 175

Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met

180 185 190

Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile Leu Ile Thr Ile

195 200 205

Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys Lys Pro Lys Asp

210 215 220

Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg Gln Asp Pro Gln Glu Met

225 230 235 240

Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val Gln Glu Thr Leu

245 250 255

His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

260 265 270

Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala Leu Arg Pro Leu

275 280 285

Val

<210> 73

<211> 173

<212> PRT

<213>artificial sequence

<220>

<223>overall length OD40 ECD

<400> 73

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 74

<211> 133

<212> PRT

<213>artificial sequence

<220>

<223>truncate 1

<400> 74

Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His

1 5 10 15

Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln

20 25 30

Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly

35 40 45

Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser

50 55 60

Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp

65 70 75 80

Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser

85 90 95

Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu

100 105 110

Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro

115 120 125

Gln Asp Arg Leu Arg

130

<210> 75

<211> 90

<212> PRT

<213>artificial sequence

<220>

<223>truncate 2

<400> 75

Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys

1 5 10 15

Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala

20 25 30

Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe

35 40 45

Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys

50 55 60

Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp

65 70 75 80

Val Val Cys Gly Pro Gln Asp Arg Leu Arg

85 90

<210> 76

<211> 50

<212> PRT

<213>artificial sequence

<220>

<223>truncate 3

<400> 76

Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu

1 5 10 15

Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln

20 25 30

Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg

35 40 45

Leu Arg

50

<210> 77

<211> 156

<212> PRT

<213>artificial sequence

<220>

<223>truncate 4

<400> 77

Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu

1 5 10 15

Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp

20 25 30

Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro

35 40 45

Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr

50 55 60

Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu

65 70 75 80

Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln

85 90 95

Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly

100 105 110

Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr

115 120 125

Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys

130 135 140

Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

145 150 155

<210> 78

<211> 150

<212> PRT

<213>artificial sequence

<220>

<223>truncate 5

<400> 78

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr

20 25 30

His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val

35 40 45

Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu

50 55 60

Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg

65 70 75 80

Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser

85 90 95

Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser

100 105 110

Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp

115 120 125

Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly

130 135 140

Pro Gln Asp Arg Leu Arg

145 150

<210> 79

<211> 173

<212> PRT

<213>artificial sequence

<220>

<223>mutant 1

<400> 79

Glu Pro Pro Thr Ala Cys Ala Ala Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 80

<211> 173

<212> PRT

<213>artificial sequence

<220>

<223>mutant 2

<400> 80

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Ala

20 25 30

Ala Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 81

<211> 173

<212> PRT

<213>artificial sequence

<220>

<223>mutant 3

<400> 81

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Ala Ala Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 82

<211> 173

<212> PRT

<213>artificial sequence

<220>

<223>mutant 4

<400> 82

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Ala Ala Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 83

<211> 19

<212> PRT

<213>artificial sequence

<220>

<223>signal peptide

<400> 83

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 84

<211> 324

<212> PRT

<213>artificial sequence

<220>

<223>mFc- label

<400> 84

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

Claims (18)

1. a kind of isolated monoclonal antibody or its antigen-binding portion thereof, include heavy chain variable region, which includes The area CDR1, the area CDR2 and the area CDR3, wherein the area CDR1, the area CDR2 and the area CDR3 separately include (1) SEQ ID NOs:1,8 and 15；(2) SEQ ID NOs:1,9 and 15；(3) SEQ ID NOs:2,9 and 15；(4) SEQ ID NOs:3,10 and 16；(5)SEQ ID NOs:4,11 and 17；(6) SEQ ID NOs:5,12 and 18；(7) SEQ ID NOs:6,13 and 19；Or (8) SEQ ID The amino acid sequence of NOs:7,14 and 20, wherein the antibody or its antigen-binding portion thereof are in conjunction with CD40.

2. isolated antibody according to claim 1 or its antigen-binding portion thereof, wherein the heavy chain variable region includes SEQ The amino acid sequence of ID NOs:37,38,39,40,41,42,43,44,45,46,47,48,49 or 50.

3. isolated antibody according to claim 1 or its antigen-binding portion thereof include light chain variable region, the light chain variable Area includes the area CDR1, the area CDR2 and the area CDR3, and wherein the area CDR1, the area CDR2 and the area CDR3 separately include (1) SEQ ID NOs: 21,27 and 31；(2) SEQ ID NOs:22,28 and 32；(3) SEQ IDNOs:23,29 and 33；(4) SEQ ID NOs:24,27 With 34；(5) SEQ ID NOs:25,27 and 35；Or the amino acid sequence of (6) SEQ ID NOs:26,30 and 36.

4. isolated antibody according to claim 1 or its antigen-binding portion thereof are comprising light chain variable region, this is light Chain variable region includes the amino acid sequence of SEQ ID NOs:51,52,53,54,55,56,57,58,59,60 or 61.

5. isolated antibody according to claim 1 or its antigen-binding portion thereof include light chain variable region, wherein described heavy Chain variable region and the light chain variable region separately include (1) SEQ ID NOs:37 and 51；(2) SEQ ID NOs:38 and 52；(3) SEQ ID NOs:39 and 53；(4) SEQ ID NOs:40 and 54；(5) SEQ ID NOs:41 and 55；(6) SEQ ID NOs:44 With 58；(7) SEQ ID NOs:45 and 59；(8) SEQ ID NOs:46 and 60；(9) SEQ ID NOs:46 and 61；(10)SEQ ID NOs:47 and 60；(11) SEQ ID NOs:47 and 61；(12) SEQ ID NOs:48 and 59；(13) SEQ ID NOs:49 and 60；(14) SEQ ID NOs:49 and 61；(15) SEQ ID NOs:50 and 60；Or the amino of (16) SEQ ID NOs:50 and 61 Acid sequence.

6. isolated antibody according to claim 1 or its antigen-binding portion thereof, permanent comprising heavy chain constant region and/or light chain Area is determined, wherein the heavy chain constant region includes the amino acid sequence of SEQ ID NOs:62,63 or 64, the light chain variable region packet The amino acid sequence of the NOs:65 or 66 of ID containing SEQ.

7. isolated antibody according to claim 1 or its antigen-binding portion thereof, (a) is in conjunction with people or monkey CD40；(b) Not in conjunction with mouse CD40；(c) hinder or promote CD40-CD40L interaction；(d) CD40 signal path is activated；(e) promote Dendritic cell maturation；And/or (f) promote CD8+ and/or CD4+T cell Proliferation.

8. isolated antibody according to claim 1 or its antigen-binding portion thereof are source of mouse, source of people, chimeric or source of people Change antibody.

9. isolated antibody according to claim 1 or its antigen-binding portion thereof are IgG1, IgG2 or IgG4 hypotype.

10. a kind of bispecific molecule, immunoconjugate, Chimeric antigen receptor, genetic modification T cell receptor or oncolytic virus, Include isolated antibody of any of claims 1-9 or its antigen-binding portion thereof.

11. a kind of composition, comprising isolated antibody of any of claims 1-9 or its antigen-binding portion thereof, with And pharmaceutically acceptable carrier.

12. composition according to claim 11 also includes secondary antibody, and/or cell factor.

13. composition according to claim 12, wherein the secondary antibody is selected from VISTA antibody, PD-1 antibody, PD- L1 antibody, LAG-3 antibody, TIM-3 antibody, STAT3 antibody and ROR1 antibody.

14. composition according to claim 11, wherein the cell factor is GM-CSF and/or IL-4.

15. a kind of nucleic acid encodes isolated antibody of any of claims 1-9 or its antigen-binding portion thereof.

16. a kind of expression vector or host cell include the nucleic acid described in claim 15.

17. in isolated antibody of any of claims 1-9 or its antigen-binding portion thereof or claim 11-14 Described in any item compositions are in preparation treating cancer, inflammatory disease, infectious diseases, atherosclerosis thrombus or breathing Purposes in the drug of systemic disease.

18. purposes according to claim 17, wherein the cancer is selected from B cell lymphoma, the white blood of chronic lymphocytic Disease, Huppert's disease, melanoma, enteraden cancer, cancer of pancreas, intestinal cancer, human primary gastrointestinal cancers, prostate cancer, bladder cancer, kidney, ovary Cancer, cervix cancer, breast cancer, lung cancer and nasopharyngeal carcinoma.