CN110003338A - Anti- OX40 antibody and its application - Google Patents

Abstract

This application provides anti-OX40 (CD134) antibody of recombination and application thereof.Anti- OX40 (CD134) antibody of the application can be specifically bound with mammal OX40, it is therefore preferred to have the combination of OX40 and its ligand can be blocked to the high-affinity of people and/or primate OX40 albumen, under a certain concentration, adjust receptor active, the activation for stimulating T cell and proliferation and other effects.The antibody of the application can be used for treating OX40 related disease, such as the relevant tumour of OX40.

Description

Anti- OX40 antibody and its application

Technical field

This application involves antibody arts, more specifically, this application involves the antibody of anti-OX40 and its applications.

Background technique

The concept that immune system identified and removed cancer cell was proposed before more than 100 years earliest, in different type cancer T cell be can detecte in disease patient blood to the reactivity of cancer associated antigens.In effective antitumour immunologic process, T Executor of the cell as core, the antigen recognizing signal mediated first by T cell receptor (T cell receptor, TCR) swash It is living, while numerous costimulatory signals and coinhibitory signals fine-tunes the intensity and quality of t cell responses, these inhibit signal As immunologic test point.Under physiological conditions, costimulatory molecules and immunologic test point molecule keep balancing, to utmostly subtract Few damage for normal surrounding tissue maintains the tolerance to autologous tissue, avoids autoimmune response.And tumour cell can be with It is abnormal to raise Co inhibitor and its associated ligands by this mechanism, inhibit t cell activation, to escape immunologic cytotoxicity.For The blocking of immunologic test point is one of the available strategy of enhancing t cell activation, and the hot topic of anti-tumor drug exploitation in recent years Target spot.Up to the present, it is granted in the U.S. to have 3 immunologic test point inhibitor, for treating kinds cancer type, target spot point It is not CTLA-4 and PD-1/PD-L1.

Research shows that CTLA-4 and PD-1 plays immunosuppressive action during t cell activation, to inhibit T cell pair The immunologic cytotoxicity function of tumour cell；And this immune suppression can be released for the blocking monoclonal antibody of the two target spots System restores T cell antineoplastic immune function.In addition to suppressive immunologic test point, there are also a kind of activated form immunologic test points, just by Gradually become the new target drone of medicament research and development.

Activated form immunologic test point molecule refers mainly to costimulatory signal molecule-T cell costimulation receptor of T cell activation, It belongs to Tumor Necrosis Factor Receptors (tumor necrosis factor receptor, TNFR) family, and effect is to adjust T Proliferation, activation and the differentiation reaction of cell, including OX40 (TNFRSF4), CD40 (TNFRSF5), 4-1BB (T cell Antigen4-1BB homologue, CD137) and GITR (Glucocorticoid-induced TNFR-related Protein, GITR) etc..

OX40, also known as CD134, ACT45 or TNFRSF4, belong to a member of TNFR superfamily, and gene is located at people No. 1 dye On colour solid, the I type transmembrane glycoprotein of 50kD a kind of is encoded.Extracellular region has 191 amino acid, contain three it is complete and one It is a slightly shorter to be rich in Cysteine domains (CRD).OX40 is mainly in the effector T cell of activation (Teff) and regulatory T cells (Treg) it is expressed on, is the CD4 of activation⁺T、CD8⁺The activation receptor on T cell surface, is currently in referred to as that " T cell is pierced altogether Swash " forward position in field, while also being expressed on NKT cell, NK cell and neutrophil cell.OX40 signal can activate downstream NF- κ B, PI3K and PKB access, the sustained activation of these accesses is finally able to extend the time-to-live of T cell, and it is thin to extend T Born of the same parents' memory, promotes the cellkilling capacity of T cell；In addition, OX40 can also by inhibit regulatory T cells (Treg) differentiation and Activity improves the immunosuppressive action in tumor microenvironment, further enhances the function of effector T cell.

Therefore, it includes the related diseases such as late malignant tumour that exploitation OX40 targeting antibodies, which will be expected to be used for treatment tumour,.

Summary of the invention

In a first aspect, this application provides a kind of antibody or its antigen-binding portion thereof for specifically binding OX40, it includes Heavy chain variable region, the heavy chain variable region include HCDR1, HCDR2 and/or HCDR3.

In some embodiments, the HCDR1 sequence includes amino acid sequence shown in SEQ ID any one of NOs:1-4 Column.In some embodiments, the HCDR2 sequence includes amino acid sequence shown in SEQ ID any one of NOs:5-8.? In some embodiments, the HCDR3 sequence includes amino acid sequence shown in SEQ ID any one of NOs:9-12.Optional Embodiment in, above-mentioned antigen-binding portion is selected from Fab segment, Fab ' segment, F (ab ')₂Segment, Fv segment, scFv piece Section, Fd segment or single domain antibody.

In some embodiments, it is described specific binding OX40 antibody or its antigen-binding portion thereof also include light chain can Become area, wherein the light chain variable region includes LCDR1, LCDR2 and/or LCDR3 sequence.

In certain embodiments, the LCDR1 sequence includes amino acid shown in SEQ ID any one of NOs:13-16 Sequence.In certain embodiments, the LCDR2 sequence includes amino acid sequence shown in SEQ ID any one of NOs:17-19 Column.In certain embodiments, the LCDR3 sequence includes amino acid sequence shown in SEQ ID any one of NOs:20-23.

In some embodiments, the heavy chain variable region of the antibody of the specific binding OX40 or its antigen-binding portion thereof Have comprising the amino acid sequence shown in the SEQ ID any one of NOs:24-30 or with above-mentioned sequence at least 80% homologous The amino acid sequence of property；Preferably, the heavy chain variable region includes amino acid sequence shown in SEQ ID NO:24 or 29.

In some embodiments, the light chain variable region of the antibody of the specific binding OX40 or its antigen-binding portion thereof Have comprising the amino acid sequence shown in the SEQ ID any one of NOs:31-37 or with above-mentioned sequence at least 80% homologous The amino acid sequence of property；Preferably, the light chain variable region includes to be selected from amino acid sequence shown in SEQ ID NO:31 or 36.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof are specifically bound to Primate OX40.

In some embodiments, the antibody of the specific binding OX40 is monoclonal antibody.

In some embodiments, the antibody of the specific binding OX40 is source of mouse antibody.

In some specific embodiments, the heavy chain variable amino acid sequence of the antibody of the specific binding OX40 As shown in SEQ ID NO:24 or 29 and it is described specific binding OX40 antibody chain variable region amino acid sequence such as Shown in SEQ ID NO:31 or 36.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof are with 1 × 10^-8To 1 ×10^-7The KD of M specifically binds to OX40 molecule.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof have OX40 excitement Agent function.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof can stimulate T thin The activation and proliferation of born of the same parents.

Second aspect encodes specific binding OX40 described in first aspect this application provides nucleic acid molecule Antibody or its antigen-binding portion thereof.

The third aspect, this application provides expression vectors, and it includes the nucleic acid molecules described in second aspect.

Fourth aspect, this application provides host cell, it includes described in second aspect nucleic acid molecule or third party Expression vector described in face.

5th aspect, this application provides pharmaceutical compositions, and it includes the specific binding OX40's described in first aspect Antibody or its antigen-binding portion thereof and pharmaceutically acceptable carrier.

In some embodiments, described pharmaceutical composition also includes one or more other active components.In some realities It applies in scheme, the active constituent is chemotherapeutics, PD-1 binding antagonists etc..

6th aspect, this application provides vaccine, it includes the antibody of the specific binding OX40 described in first aspect or Its antigen-binding portion thereof and optional immunologic adjuvant.

In some embodiments, vaccine described in pharmaceutical composition or the 6th aspect described in the 5th aspect is for treating The relevant disease of OX40, such as tumour.

7th aspect, this application provides the antibody or its antigen-binding portion of the specific binding OX40 described in first aspect Point or the 5th aspect described in pharmaceutical composition preparation for inhibit Treg function, kill expression OX40 cell, cause The function of reaction, raising effector T cell that T cell mediates improves the function of memory T cell, and/or effectively inhibits tumour growth Drug in purposes.

Eighth aspect, this application provides the antibody or its antigen-binding portion of the specific binding OX40 described in first aspect Point or the 5th aspect described in pharmaceutical composition preparation for preventing and/or treating the relevant disease of OX40, such as tumour Drug in purposes.

In some embodiments, the tumour is selected from colon cancer, melanoma, cortical tumor, clear-cell carcinoma, lymph Tumor, advanced solid tumor and above-mentioned metastatic tumor.

9th aspect, this application provides detection reagent or kits, and it includes the specificity knots described in first aspect Close the antibody or its antigen-binding portion thereof of OX40.

In other respects, this application provides preventing and/or the relevant disease for the treatment of OX40, such as the method for tumour, The antibody or its antigen-binding portion thereof or the 5th of OX40 are specifically bound described in individual first aspect in need including giving Vaccine described in pharmaceutical composition described in aspect or the 6th aspect.Optionally, other the method also includes being administered in combination Therapeutic agent, such as chemotherapeutics, PD-1 binding antagonists etc..

The antibody of the specific binding OX40 of the application or its antigen-binding portion thereof can be specific in conjunction with OX40, have One or more effects below: having the function of OX40 agonist, stimulates the Proliferative Activated of T cell, and induction OX40 is mediated anti- Tumor immune response, and/or inhibit tumour growth etc..

The brief description of accompanying drawing

Fig. 1 shows the result that the functional activity of candidate authentic monoclonal antibody is verified by NF- κ B signal access: its Middle Figure 1A be 30 monoclonal antibodies are carried out primary dcreening operation as a result, Figure 1B is the knot for 7 candidate monoclonal antibodies that screening obtains Fruit.

Fig. 2 shows the result that the functional activity of candidate authentic monoclonal antibody is verified by NFAT signal path.

Fig. 3 shows that Fortebio detects the KD value of anti-OX40 monoclonal antibody: wherein Fig. 3 A to Fig. 3 G is respectively corresponded anti- The KD value of OX40 monoclonal antibody 18B10,4F2,6H12,6A6,4B9,6C1 and 13F7.

Fig. 4 shows experimental result of the anti-OX40 monoclonal antibody in conjunction with people OX40 and monkey OX40.

Fig. 5 shows blocking effect of the anti-OX40 monoclonal antibody to people OX40 in conjunction with its ligand OX40L.

Fig. 6 shows the EC50 value functional activity testing result of anti-OX40 monoclonal antibody.

Fig. 7 shows anti-OX40 monoclonal antibody stimulation people CD4⁺The result of T cell proliferation and IL-2 and IFN-γ secretion: Wherein Fig. 7 A is CFSE flow cytometer detection CD4⁺T cell proliferation as a result, Fig. 7 B be ELISA detect IL-2 secretion as a result, Fig. 7 C The result of IFN-γ secretion is detected for ELISA.

Fig. 8 shows internal drug effect and safety of the anti-OX40 monoclonal antibody in the MC38 mice with tumor of humanization OX40 Research: wherein Fig. 8 A is the tumor volume change tendency chart of mouse, and Fig. 8 B is the changes of weight tendency chart of mouse, and Fig. 8 C is ALT Testing result figure, Fig. 8 D are AST testing result figure, and Fig. 8 E is the photo of the tumour obtained after dissecting, and Fig. 8 F accounts for for tumor weight The percentage of mouse total weight.

Specific embodiment

This application provides the novel anti-OX40 antibody or its antigen-binding portion thereof that specifically bind to OX40.Preferred real It applies in scheme, the antibody of the application or its antigen-binding portion thereof are incorporated into the OX40 molecule of target cell surface and have OX40 excitement Agent function.Present invention also provides encode the nucleic acid molecule of the antibody or its antigen-binding fragment, divide comprising the nucleotide Son expression vector, the host cell comprising the nucleic acid molecule or expression vector, prepare and purify the method for the antibody with And the medicine and biological applications of the antibody or its antigen-binding fragment, such as prevention or treatment OX40 related disease or disease Disease.The application also cover using the antibody or its antigen-binding fragment detect OX40 and adjust the active method of OX40 and Coherent detection reagent or kit.

To will be readily understood that the application, certain terms used herein are defined first.

As used herein, term " antibody " refers to comprising four polypeptide chains, i.e., two heavy chains (H) interconnected by cystine linkage And the immunoglobulin molecules and its polymer (such as IgM) of two light chains (L).Each heavy chain includes heavy chain variable region (abbreviation For VH) and heavy chain constant region (being abbreviated as CH).Heavy chain constant region includes three domains, i.e. CH1, CH2 and CH3.Each light chain includes light Chain variable region (being abbreviated as VL) and constant region of light chain (being abbreviated as CL).Constant region of light chain includes a domain (CL1).The area VH and VL can It is further subdivided into the hypervariable region of referred to as complementary determining region (CDR), wherein being interspersed with the conserved region of referred to as framework region (FR).One In a little embodiments, from the end N- to the end C-, light chain and heavy-chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

As used herein, " antigen-binding portion thereof " of term antibody, which refers to, is responsible for the complete antibody molecule in conjunction with antigen A part or section.Antigen binding domain may include heavy chain variable region (VH), light chain variable region (VL) or both.Antibody Any suitable standard technique can be used to prepare from complete antibody molecule for antigen-binding fragment, and the standard technique includes albumen water Solution digestion or genetic recombination engineering technology etc..The non-limiting example of antigen-binding portion thereof includes: Fab segment, F (ab')₂Piece Section, Fd segment, Fv segment, scFv (scFv) molecule, single domain antibody, dAb segment and the amino acid by analog antibody hypervariable region The minimum recognition unit (such as isolated CDR) of residue composition.Term " antigen-binding portion thereof " also includes point of other engineering Son, such as double antibody, three antibody, four antibody and miniantibody.For example, Fd segment described herein refers to by VH and CH1 structural domain The antibody fragment of composition；Fv segment is made of VL and VH structural domain in the single armed of antibody；DAb segment (Ward et al., Nature 1989；341:544-546) it is made of VH structural domain.

Well known to those skilled in the art, complementary determining region (CDR usually has CDR1, CDR2 and CDR3) is right in variable region The maximum region of affinity and specific effect of antibody.There are two types of common definition modes for the CDR sequence of VH or VL, i.e., Kabat definition and Chothia definition, for example, see Kabat et al., " Sequences of Proteins of Immunological Interest ", National Institutes of Health, Bethesda, MD. (1991)；Al- Lazikani et al., J Mol Biol 273:927-948 (1997)；And Martin et al., Proc.Natl.Acad.Sci.USA 86:9268-9272(1989).It, can basis for giving the variable region sequences of antibody Kabat definition or Chothia definition are to determine CDR region sequence in VH and VL sequence.In the embodiment of the application, utilize Kabat defines CDR sequence.Herein, CDR1, CDR2 and CDR3 of heavy chain variable region be briefly referred to as HCDR1, HCDR2 and HCDR3；CDR1, CDR2 and CDR3 of light chain variable region are briefly referred to as LCDR1, LCDR2 and LCDR3.

For giving the variable region sequences of antibody, CDR region sequence in variable region sequences, example can be analyzed in several ways It such as can use online software Abysis and determine (http://www.abysis.org/).

As used herein, term " specific binding " refers to the nonrandom association reaction between two molecules, such as anti- Body is to the combination of epitope, such as antibody is to combine the compatibility of the big at least twice of the compatibility of heterogenetic antigen than it In the ability of specific antigen.It is to be appreciated, however, that can to specifically bind to two or more relevant to its sequence for antibody Antigen.For example, antibody of the invention can specifically bind to the mankind and non-human (such as mouse or non-human primate) OX40。

As used herein, term " monoclonal antibody " refers to by the antibody of the antibody population acquisition of basic homogeneity, that is, in addition to It may be identical there are each antibody for other than abiogenous mutation, forming group in a small amount of individual.List described herein Clonal antibody particularly including " chimeric " antibody, wherein a part of heavy chain and/or light chain with from specific species or belong to tool Corresponding sequence in the antibody of body antibody class or subclass is identical or homologous, and the remaining part of heavy chain and/or light chain with derive from Another species or the corresponding sequence belonged in the antibody of another antibody class or subclass are identical or homologous, and further include such anti- The segment of body, as long as they can show desired biological activity (referring to U.S. Patent number 4,816,567；With Morrison et al.,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。

As used herein, term " source of mouse antibody " refers to that wherein all constant domain sequences are mouse sequence Any antibody.Such antibody can be generated by hybridoma.

As used herein, term " chimeric antibody " refers to anti-comprising the section from two or more different antibodies Body.In some embodiments, one or more CDR are derived from the anti-OX40 antibody of mouse.In other embodiments, own CDR is derived from the anti-OX40 antibody of mouse.In some embodiments, combination is anti-from more than one mouse in chimeric antibody The CDR of OX40 antibody.For example, chimeric antibody may include in the anti-OX40 antibody of the first mouse the CDR1 of light chain, from the The CDR2 of light chain, the CDR3 with the light chain in the anti-OX40 antibody of the third mouse in the anti-OX40 antibody of two kinds of mouse, and come One or more other anti-OX40 antibody can be derived from from the CDR of heavy chain.In addition, framework region may be from identical anti-OX40 antibody or From one or more different individuals.

Can additionally serve as the suitable technology in antibody method includes that affinity purification, native gel based on OX40 are pure Change, HPLC or RP-HPLC, molecular exclusion, any combination of purifying or these technologies on albumin A column.ELISA can be used to survey Determine method measurement OX40 antibody isotype, such as the anti-human Ig surveyor Ig of mouse Ig absorption can be used.

It can be generated by any one of the protein purification of multiple standards known in the art or recombination and expression techniques Suitable for generating the OX40 of antibody.The form of OX40 suitable for generating immune response includes OX40 subsequence (such as immunogenic fragments Section).The form of other OX40 includes that OX40 expression cell, the product containing OX40 or cell extract or fraction, part are pure The OX40 of change.

As used herein, term " homology " is defined as by sequence alignment and after introducing vacancy, amino acid or core The percentage of identical residue in nucleotide sequence variant, if it is desired, reach the homology of largest percentage.Side for comparison Method and computer program are well known in the art." at least 80% homology " as described herein refer to homology be 80% to 100% any value, such as 85%, 90%, 95%, 99% etc..

As used herein, term " OX40 related disease " includes disease relevant to OX40 signal path and/or symptom. Exemplary OX40 related disease or illness include tumour, such as colon cancer, melanoma, cortical tumor, clear-cell carcinoma, lymph Tumor, advanced solid tumor and above-mentioned metastatic tumor.

As used herein, term " EC50 " refers to half maximal effect concentration (concentration for50%of Maximal effect, EC50), refer to the concentration that can cause 50% ceiling effect.

In a first aspect, it includes heavy chains this application provides the antibody or its antigen-binding portion thereof of specific binding OX40 HCDR1, HCDR2 and/or HCDR3 of variable region.It is exemplary in the following table 1-4 to list suitable for antibody disclosed in the present application CDR, heavy chain variable region and chain variable region amino acid sequence.In certain embodiments, anti-OX40 antibody or its antigen binding Part includes HCDR1, HCDR2 and/or HCDR3 sequence, independently selected from HCDR1, HCDR2 shown in table 1 and/or Any one in HCDR3 sequence.In certain embodiments, the anti-OX40 antibody of the application can further include LCDR1, LCDR2 And/or LCDR3, independently selected from any one in LCDR1, LCDR2 and/or LCDR3 sequence shown in table 2.For example, The anti-OX40 antibody of the application may include any one of heavy chain variable region shown in table 3, optionally with it is light shown in table 4 Any one combination or pairing of chain variable region.

Table 1: the sequence number of the heavy chain CDR amino acid sequence of exemplary anti-OX40 antibody

Table 2: the sequence number of the light chain CDR amino acid sequence of exemplary anti-OX40 antibody

Table 3: the sequence number of the heavy chain variable amino acid sequence of exemplary anti-OX40 antibody

Table 4: the sequence number of the chain variable region amino acid sequence of exemplary anti-OX40 antibody

In some embodiments, the amino acid sequence of antibody disclosed herein or the HCDR1 of its antigen-binding portion thereof is such as Shown in SEQ ID any one of NOs:1-4.In some embodiments, the amino acid sequence of HCDR2 such as SEQ ID NOs:5-8 appoints Shown in one.In some embodiments, the amino acid sequence of HCDR3 is as shown in SEQ ID any one of NOs:9-12.

In alternative embodiments, the antigen-binding portion is selected from Fab segment, Fab ' segment, F (ab ')₂Segment, Fv segment, scFv segment, Fd segment or single domain antibody.

Antibody disclosed herein or its antigen-binding portion thereof can also be wrapped further on the basis of comprising heavy chain variable region Containing light chain variable region.

In some embodiments, the amino acid sequence of antibody disclosed herein or the LCDR1 of its antigen-binding portion thereof is such as Shown in SEQ ID any one of NOs:13-16.In some embodiments, the amino acid sequence of LCDR2 such as SEQ ID NOs:17- Shown in 19 any one.In some embodiments, the amino acid sequence of LCDR3 is as shown in SEQ ID any one of NOs:20-23.

In some specific embodiments, the heavy chain variable region and choosing of antibody disclosed herein or its antigen-binding portion thereof There is at least 80% homology from the amino acid sequence of SEQ ID NOs:24-30, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology.In some specific embodiments, above-mentioned antibody weight Chain variable region is made of the amino acid sequence selected from any one of SEQ ID NOs:24-30.Scheme is put in some more specific implementations In, the amino acid sequence of above-mentioned antibody heavy chain variable region is as shown in SEQ ID NO:24 or 29.

In some specific embodiments, the light chain variable region and choosing of antibody disclosed herein or its antigen-binding portion thereof There is at least 80% homology from the sequence of SEQ ID NOs:31-37, for example, with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology.In some specific embodiments, the antibody light chain can Become area to be made of the amino acid sequence selected from any one of SEQ ID NOs:31-37.In some more particular embodiments, on The amino acid sequence of antibody's light chain variable region is stated as shown in SEQ ID NO:31 or 36.

In some embodiments, the heavy chain variable region or light chain variable region of antibody disclosed herein can be above-mentioned listed The change for replacing, missing or adding at least one amino acid on the basis of the corresponding specific amino acid sequence lifted, and obtaining Body still keeps the activity in conjunction with OX40.

In certain embodiments, the number of above-mentioned amino acid substitutions, deletions, or additions is between 1-30 or 1-30 Any value, preferably 1-20, more preferably 1-10.In preferred embodiments, sequence variants and original acid Sequence differs the substitution of about 1,2,3,4,5,6,7,8,9 or 10 amino acid, deletion and/or addition.In preferred embodiment party In case, sequence variants differ replacing, missing or adding for about 1,2,3,4 or 5 amino acid with original acid sequence.Specific In embodiment, the amino acid substitution is conservative replaces.

In preferred embodiments, antibody disclosed herein is antibody 6A6 or 4F2, the wherein weight chain variable of antibody 6A6 Region amino acid sequence is as shown in SEQ ID NO:29, and chain variable region amino acid sequence is as shown in SEQ ID NO:36；Antibody 4F2 Heavy chain variable amino acid sequence as shown in SEQ ID NO:24, chain variable region amino acid sequence such as SEQ ID NO:31 institute Show.

Antibody disclosed herein or its antigen-binding portion thereof can specifically bind OX40.In some embodiments, OX40 antibody shows species and molecular selection.In some specific embodiments, the antibody or its antigen-binding portion thereof Specifically bind primate OX40, or the OX40 of the species with primate OX40 with high homology.Some specific In embodiment, the antibody or its antigen-binding portion thereof specifically bind people OX40 and monkey OX40.In some more specific realities It applies in scheme, anti-OX40 antibody is in conjunction with people, macaque or rhesus macaque OX40.In some other embodiments, anti-OX40 is anti- Body is not in conjunction with mouse, rat, dog or rabbit OX40.

As used herein, term " KD " equilibrium dissociation constant to be referred to is to obtain from ratio (that is, kd/ka) of the kd to ka It obtains and is indicated with molar concentration (M).The KD value of method measurement antibody sufficiently established in the industry can be used.Measure the excellent of the KD of antibody Choosing method system is by using surface plasma resonance, it is preferable to use bio-sensor system (such as SPR system) or fluidic cell Art and Scatchard analysis.

As used herein, term means that antibody has 10 for target antigen to " high-affinity " of IgG antibody^-8M or more It is small, preferably 10^-9M or smaller, and more preferable 10^-10M or smaller KD.However, for other antibody morphisms, " high-affinity " knot Conjunction may be variation.For example, antibody, which has 10, to be meant for the " high-affinity " combination of IgM homotype^-7M or smaller, it is excellent Select 10^-8M or smaller KD.

As used herein, the antibody of term " inhibiting OX40-L in conjunction with OX40 " refers to the knot for inhibiting OX40-L and OX40 Close, for example, using HEK::OX40 100B5 cell binding assay in inhibit OX40-L and OX40 combination antibody, this It, can be part or all of under certain antibody concentration in the generally acknowledged method (such as binding assay described herein based on FACS) in field Block the combination of the OX40-L and OX40 of 0.5 μ g.

As used herein, term " inhibition " or " blocking " are (for example, the combination of OX40-L and OX40 on referring to cell When inhibition/blocking) it is used interchangeably, and cover part and complete inhibition/blocking.In certain embodiments, anti-OX40 Antibody by the combination of OX40-L and OX40 inhibit at least about 50%, for example, about 60%, 70%, 80%, 90%, 95%, 99% or 100%.In certain embodiments, anti-OX40 antibody inhibits the combination of OX40-L and OX40 to be no more than 50%, for example, about 40%, 30%, 20%, 10%, 5% or 1%.

As used herein, term " nucleic acid molecule " is intended to include DNA molecular and RNA molecule.Nucleic acid molecule can be It is single-stranded or double-stranded, and can be cDNA.

In some more particular embodiments, antibody disclosed herein is anti-human OX40 monoclonal antibody.OX40 antibody Type and hypotype can be determined by any mode known in the art.In general, Antibody types and hypotype, which can be used, is specific to specific resist The antibody of body type and hypotype determines.

As used herein, term " agonist antibody " refer to when by the antibody add to expression OX40 cell, tissue or When in organism, one or more OX40 activity can be made to improve at least about 20%.In certain embodiments, there is agonist function The antibody of energy makes OX40 activity improve at least 40%, 50% or 60%.Activated form antibody can expand or replace OX40L to OX40's Effect.In some embodiments, activated form antibody is substantially the analogies of OX40L, and with OX40L competitive binding OX40. Herein, term " agonist antibody ", " agonistic antibody " and " activated form antibody " may be used interchangeably.

In some embodiments, anti-OX40 activated form monoclonal antibody can pass through following at least one mechanism inhibition or root Except tumour: enhancing activated tumor specific C D4⁺With CD8⁺Lymphocyte inhibits Treg cell in tumor, and enhances tumoricidal Memory cell etc..In some embodiments, other immune strengthening effects that other anti-tumor activities can be transmitted by OX40 signal (such as generating chemotactic factor (CF) and cell factor, and reinforce CTL and NK lysis activity etc.) is mediated, and is withered by inducing cell Die or stimulate the humoral response for being directed to ADCC and directly tumors destroyed etc..

In some embodiments, antibody disclosed herein is anti-OX40 agonistic antibody, with existing Genentech The IgG1 hypotype OX40 antibody (Genentech company's numbering: MOXR0916) of company compares, in cellular functional activity and stimulation People CD4⁺T cell proliferation aspect has certain advantage, and can be living with the proliferation of rapid stimulation specific T-cells in Mice Body Change, and inhibits the growth of tumour cell.

In some specific embodiments, antibody disclosed herein can block people OX40 albumen and its ligand OX40L Combination, and have a certain amount imitate relationship.

In some embodiments, antibody disclosed herein can stimulate CD4⁺T cell is proliferated and increases IL-2 and IFN- The secretion of γ.For example, antibody disclosed herein can effectively facilitate CD4 in some specific embodiments⁺T cell proliferation And the secretion of cell factor, including but not limited to IL-2, IFN-γ etc..

In some embodiments, antibody disclosed herein can effectively activate tumoricidal T cell (such as to activate Specific T-cells in hOX40 KI mouse body), and inhibit Treg cell in tumor.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof can kill expression The cell of OX40, such as the cell of expression high level OX40.

In some embodiments, it is thin can to cause T for the antibody of the specific binding OX40 or its antigen-binding portion thereof The reaction that born of the same parents mediate.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof can be improved effect The function of T cell.

In some embodiments, the antibody of the specific binding OX40 or its antigen-binding portion thereof can be improved memory The function of T cell.

In some embodiments, antibody disclosed herein can inhibit the growth of tumour.For example, in some specific realities It applies in scheme, antibody disclosed herein can inhibit the growth of the MC38 subcutaneous transplantation tumor of hOX40 KI mouse.Some specific In embodiment, above-mentioned antibody inhibiting tumor growth at least up to 80%, 85% or 90%.In some embodiments, in lotus knurl Individual can be detected the situation for inhibiting tumour growth for 14 days after receiving antibody processing disclosed herein.In other embodiments In, it can be detected within 6 days the situation for inhibiting tumour growth after receiving antibody processing for the first time.

Present invention also provides encode the nucleic acid molecule of antibody disclosed herein or its antigen-binding portion thereof, include described The expression vector of nucleic acid molecule, the host cell comprising the nucleic acid molecule or expression vector and preparation and purification should The method of antibody.

In some embodiments, the nucleic acid molecule of encoding said antibody or its antigen-binding portion thereof is operably connected To regulating and controlling sequence, regulating and controlling sequence can be identified with the transformed host cell of the expression vector.

In some embodiments, any suitable expression vector may be used to the application.For example, the expression vector It can be any one of pQK1, pTT5, pUC57, pDR1, pcDNA3.1 (+), pDHFF and pCHO1.0.It can in expression vector To include the fusion dna sequence for being connected with suitable transcription and translation and adjusting sequence.

In some embodiments, available host cell is the cell containing above-mentioned expression vector, and it is thin to can be eukaryon Born of the same parents, such as mammal or insect host cell culture systems are used equally for the antibody of the application or the table of its antigen-binding portion thereof It reaches.For example, 293 cell of HEK, COS cell, Chinese hamster ovary celI, NS0 cell, sf9 cell and sf21 cell etc. may be applicable to this hair It is bright.The host cell may be the prokaryotic cell containing above-mentioned expression vector, for example, can for DH5 α, BL21 (DE3) or TG1 etc..

In some embodiments, the preparation method of anti-OX40 monoclonal antibody disclosed herein includes: in expression condition Under, host cell is cultivated, to express anti-OX40 monoclonal antibody；The anti-OX40 monoclonal antibody of separation and purifying expression.Benefit In aforementioned manners, it can be substantially uniform substance by recombinant protein purification, such as be single band on SDS-PAGE electrophoresis.

In some embodiments, the method that can use affinity chromatography separates anti-OX40 antibody disclosed herein Purifying can be used the methods of conventional method such as high-salt buffer, change pH and wash according to the characteristic of the affinity column utilized It is de- to combine the anti-OX40 antibody on affinity column.

In some embodiments, after animal inoculation pvaccination OX40 antigen, antibody can be obtained from animal body and/or generates antibody Cell.The immortalized cell line for generating antibody can be prepared by the cell isolated in immunized animal body.After immunity inoculation, Animal is killed, takes lymph node and/or splenic B cells to carry out immortalization processing, at tumorigenic compound and mutagenesis compound Reason, merges with immortalized cells (such as myeloma cell), removes the activity of tumor suppressor gene.If using myeloma cell into When row fusion, which does not secrete immunoglobulin polypeptides (non-secretory cell line) preferably.Use OX40, its part Or the cell screening immortalized cells of expression OX40.In preferred embodiments, first screening is to utilize ELISA Method (ELISA) carries out.The cell such as hybridoma for choosing the anti-OX40 antibody of generation is cloned, and required spy is further screened Property, including well-grown, antibody production are high and have required antibody characteristic etc..Screening, clone and the amplification method of hybridoma It is well known within the skill of those ordinarily skilled.In some embodiments, the animal through immunity inoculation is non-human animal, wherein Splenic B cells with from being merged with the myeloma cell line of non-human animal's same species.In some embodiments, the warp The animal of immunity inoculation is Balb/c mouse and myeloma cell line is non-secreting mouse myeloma cell SP2/0.

This application provides pharmaceutical compositions, it includes antibody disclosed herein or its antigen-binding portion thereof and pharmaceutically Acceptable carrier.Above-mentioned anti-OX40 antibody (such as anti-human OX40 monoclonal antibody) disclosed herein can with can pharmaceutically connect The carrier received is configured to pharmaceutical preparation together, to more stably play curative effect.In some embodiments, these preparations can be with Guarantee the Conformational Integrity of the amino acid core sequence of anti-OX40 antibody disclosed herein (such as anti-human OX40 monoclonal antibody), The polyfunctional group for going back protected protein matter simultaneously prevents its degradation (including but not limited to cohesion, deamidation or oxidation).In some realities It applies in scheme, for liquid preparation, at least a year preservation can usually stablize under the conditions of 2 DEG C -8 DEG C.In some embodiments In, for lyophilized preparation, holding at least six months is stablized at 30 DEG C.In some embodiments, described pharmaceutical composition is also Include one or more other active components.In some embodiments, the active constituent is anti-tumor drug.

This application provides vaccines, and it includes the antibody of the specific binding OX40 described in first aspect or its antigen bindings Part and optional immunologic adjuvant.The immunologic adjuvant includes but is not limited to: 1) biological adjuvant, such as bacterium or its production Object (such as mycobacteria (tubercle bacillus, BCG vaccine), corynebacterium, Bordetella pertussis, gram negative bacilli endotoxin), grain Granulocytemacrophage colony stimulating factor, interleukins-l, interleukin 2, interferon-γ etc.；2) inorganic adjuvant, example Such as aluminium hydroxide, alum, aluminum phosphate；3) artificial synthesized adjuvant, such as double-strand poly, cytidine monophosphate, double-strand poly gland Thuja acid；4) finish, such as adjuvant 65, mineral oil, vegetable oil, lanolin etc.；5) Freund's adjuvant, such as Freund are endless Full adjuvant and Freund's complete adjuvant.

Present invention also provides prevent and/or treat the relevant disease of OX40, such as the side of tumour in tumour immunity field Method comprising give the anti-OX40 antibody of individual or the drug comprising anti-OX40 antibody (such as anti-human OX40 monoclonal antibody) Composition or vaccine.In some embodiments, after to animal administration including people, antitumous effect is obvious.Specifically Ground says that anti-OX40 antibody disclosed herein can effectively prevent and/or treat tumour, can be used as anti-tumor drug use.

In some embodiments, can by the anti-OX40 antibody of the application or comprising anti-OX40 antibody it is (such as anti-human OX40 monoclonal antibody) pharmaceutical composition or vaccine and other therapeutic agents (such as chemotherapeutics, PD-1 binding antagonists etc.) It is administered in combination.

Present invention also provides anti-OX40 antibody or its antigen-binding portion thereofs, or comprising the anti-OX40 antibody or it is anti- The pharmaceutical composition of former bound fraction in preparation for preventing and/or treat the relevant disease of OX40, such as in the drug of tumour Purposes.

In some embodiments, the tumour be colon cancer, melanoma, cortical tumor, clear-cell carcinoma, lymthoma, Advanced solid tumor or its metastatic tumor etc..

Anti-human OX40 antibody disclosed herein and pharmaceutical composition comprising the anti-human OX40 antibody are to including that people exists When interior animal is administered, dosage is different because of the age of individual and weight, disease traits and seriousness and administration route, can With the result and comprehensive condition with reference to zoopery, total dosage is no more than a certain range.

The administration dosage and frequency of antibody or the pharmaceutical composition comprising the antibody can according to disease carry out prevention or It treats and changes.In prophylactic use, applies to the patient for being not yet in morbid state containing the antibody of the application or it is mixed The composition of object is closed to enhance patient's resistance, this amount is defined as " preventative effective dose ".In this purposes, specific dosage Again depending on patient health status and general immunity.Usually with relatively infrequent interval apply relatively low dosage it is longer when Between.In therapeutic application, it is sometimes desirable to apply relatively high dose up to progression of disease slows down or whole with relatively short interval Until only, and until showing that disease symptoms partially or completely improve preferably of up to patient.Hereafter, preventative side can be applied to patient Case.Those of ordinary skill in the art can easily grasp specific dosage and frequency according to actual needs.

Present invention also provides detection reagents or kit comprising antibody disclosed herein or its antigen-binding portion thereof.

As used herein, term " immune response " refers to the biological respinse that external agent is directed in vertebrate, this is anti- The disease that organism should be protected to resist such agent and be induced by it.Immune response system is by the cell of immune system (for example, T Lymphocyte, bone-marrow-derived lymphocyte, natural kill (NK) cell, macrophage, eosinophil, mast cell, dendritic cells or Neutrophil cell) and by any one of this class cell or by liver generate soluble large molecule (including antibody, cell because Son and complement) effect mediate, cause selectively targeting, combination, damage, destructions and/or from vertebrate body eliminate invade Enter pathogen, the cell or tissue through pathogenic infection, carcinous or other abnormal cells or in autoimmunity or Inflammation Normal human cells or tissue under situation.Immune response includes T cell (such as effector T cell) or Th cell (such as CD4⁺Or CD8⁺T cell) activation or inhibition or Treg cell inhibition.

As used herein, term " T cell mediate reaction " means that (including effector T cell is (for example, CD8 by T cell⁺ Cell) and T helper cell (for example, CD4⁺Cell)) mediate reaction.T cell mediate reaction include T cell cytotoxicity and Proliferation.

As used herein, term " cancer " refers to the major class disease with the characteristics of the uncontrolled growth of internal abnormal cell. The cell division of imbalance can form invasion adjacent tissue and can be transferred to the distal portion of body via lymphatic system or blood flow Malignant tumour or cell.

As used herein, term " growth for inhibiting tumour " includes any measurable reduction of tumour growth, such as Tumor growth inhibition at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% or 100%.

As used herein, term " treatment " refers to any kind of intervention implement to subject or method or applies to it Activating agent, wherein purpose is reverse, mitigates, improves, inhibits or alleviate or prevent symptom, complication, the patient's condition or related to disease Biochemical biomarker progress, development, severity or recurrence.Prevent to refer to subject's application to not suffering from the disease, to prevent Disease occurs or it is made to influence (if present) minimum.

In the specification and claims, word "include", "comprise" and " containing " mean " including but not limited to ", and It is not intended to exclude other parts, additive, component or step.

It should be understood that the feature described in the particular aspects, embodiment or embodiment of the application, characteristic, component or Step is applicable to any other aspect, embodiment or embodiment described herein, unless contradiction therewith.

Above disclosure describes the application on the whole, and following embodiment is further described to the application, It should not be construed as the limitation to the application.Embodiment does not include detailed descriptions of conventional methods, as those are used for carrier construction With the method for plasmid, the gene for encoding albumen is inserted into the method for carrier and plasmid or plasmid is introduced to the side of host cell Method.Such method is well-known for those of ordinary skill in the art, and in many publications It is all described, for example, see Sambrook, J., Fritsch, EF.and Maniais, T. (1989) Molecular Cloning:A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

The invention discloses specific binding mammal (people, primate etc.) OX40 antibody, and the antibody or Antibody moiety is used to adjust for receptor active, stimulates activation and proliferation of T cell etc..The present invention provides this proteinoid and is controlling The application of treat, screen and detect etc., purposes such as in cancer treatment.Those skilled in the art can use for reference in this paper Hold, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art For be it will be apparent that they are considered as being included within the scope of the invention.Those skilled in the art can not depart from this Method described herein and application are modified or appropriate changes and combinations in summary of the invention, spirit and scope, realizing and Using the present invention.

In order to make those skilled in the art more fully understand technical solution of the present invention, below with reference to embodiment to this hair It is bright to be described in further detail.

Embodiment

Embodiment 1. generates the preparation of the hybridoma of anti-OX40 antibody

1. take 8-10 weeks big Balb/c mouse, it is intraperitoneally inoculated with OX40 albumen (10 μ g/ agent/only).People OX40 (TNFRSF4) albumen, which is ordered from justice, sticks up Divine Land, article No. No.10481-H08H, DNA sequence dna NP_003318.1, protein sequence For extracellular region Met1-Ala 216, C-terminal contains His label, verifies its molecular weight in 40-45kDa by SDS-PAGE.In 3- In 8 weeks, repeat this dosage 5-7 times.Merge first 4 days, injects last one OX40 albumen for mouse.

2. merge first 1 day, macrophage in common Balb/c mouse peritoneal is taken to be inoculated in 96 orifice plates as trophoderm In.

3. fetching thin from the spleen of the mouse through immunity inoculation and the lymphocyte and non-secretory myeloma SP2/0 of lymph node Born of the same parents are fusion, and cell is added to and is covered in trophoblastic 96 orifice plate in advance, carry out HAT selection (Galfre to the cell of fusion and Milstein,Methods Enzymol 1981；73:3-46).

4. the hybridoma that anti-OX40 specific antibody is secreted in one group of recycling.First screening is to utilize enzyme linked immunological point Analysis method (ELISA) determines the titre of the anti-OX40 antibody of hybridoma secretion.

Embodiment 2. detects antibody activity by NF- κ B signal access

1. 1 bottle of (T75) HEK::OX40 100B5 cell of collection is transferred in 15mL centrifuge tube, contain 10% with 5mL The DMEM culture medium (Gibco) of FBS (Gemini) is washed 1 time, and 1000rpm is centrifuged 5min；Supernatant is abandoned, 5mL is added containing 2%FBS's Cell count is resuspended in PBS.

2. cell is diluted to 2 × 10⁵A cell/mL is added 96 orifice plates according to 100 holes μ L/, is put into 5%CO₂37 DEG C Cell incubator is incubated for 30min.

3. by each monoclonal antibody to be detected and control antibodies, (antibody MOXR0916, IgG1 hypotype are being ground by Roche Holding Ag Humanized antibody), the 4-1BB ligand (h-OX40L, hundred Pu Saisi) of people, same to subclass antibodies (1 antibody of mouse IgG (mIgG1), people IgG1 antibody (hIgG1), Biolegend), it is diluted respectively according to following concentration: 4 μ g/mL, 0.4 μ g/mL and 0.04 μ g/ ML is added in 96 orifice plates according to 50 holes μ L/, multiple holes is arranged, so that the final concentration of reaction system monoclonal antibody is respectively 1 μ g/ ML, 0.1 μ g/mL and 0.01 μ g/mL.The DMEM culture medium group that antibody is not added completely, single plus secondary antibody anti-mIgG-Fc are added simultaneously Group is compareed as background value.

4. anti-mouse IgG Fc (Biolegend) and anti-human igg Fc (Biolegend) are added as secondary antibody, analogue body is played The effect of interior Fc receptor crosslinking, secondary antibody final concentration is 3 times of primary antibody, is prepared according to the system in 50 holes μ L/, and 96 orifice plates are added In.

5. 96 orifice plates are put into 5%CO₂37 DEG C of cell incubators in be incubated for 20h；Then by 96 orifice plate room temperature to be measured 1000rpm is centrifuged 5min, takes the every hole of 96 new orifice plates that 160 μ L are added in advance in the Quanti Blue of 37 DEG C of incubation 30min (Invivo Gen), after take 40 μ L cell conditioned mediums be added, be incubated for 1h in 37 DEG C.

6. detecting OD value at 620nm using microplate reader, and select GraphPad Prism mapping analysis data.

Partial results are as shown in Figure 1A, carry out primary dcreening operation to 30 monoclonal antibodies altogether；Further selective advantage clone verifying, As shown in Figure 1B, 7 candidate monoclonal antibodies are obtained, 4F2,4B9,6C1,6H12,13F7,6A6 and 18B10 are respectively labeled as.

Embodiment 3. detects antibody activity by NFAT signal path

1. 1 bottle of (T75) Jurkat OX40 cell of collection is transferred in 15mL centrifuge tube, with 5mL containing 10%FBS's 1640 culture mediums (Gibco) are washed 1 time, and 850rpm is centrifuged 5min, abandon supernatant, and RPMI 1640 culture medium of the 5mL containing 1%FBS is added Cell is resuspended and counts.

2. cell is diluted to 1.0 × 10⁶A cell/mL is added 96 orifice plates according to 25 holes μ L/, is put into 5%CO₂37 DEG C Cell incubator is incubated for 30min.

3. the control group with embodiment 2 designs, the addition same subclass antibodies of control antibodies MOXR0916 (mIgG1 and hIgG1, Biolegend), each antibody to be detected is diluted according to following concentration respectively: 3 μ g/ml, 0.3 μ g/ml and 0.03 μ g/ Ml is added in 96 orifice plates according to 25 holes μ L/, and multiple holes are arranged.The 1640 culture medium group of RPMI that antibody is not added completely is added simultaneously It (is marked in figure are as follows: 1640), compareed as background value.

4. anti-mouse IgG Fc (Biolegend) and anti-human igg Fc (Biolegend) are the 3 of primary antibody as secondary antibody final concentration Times, prepare in 96 orifice plates of addition according to the system in 25 holes μ L/.

5. 96 orifice plates are put into 5%CO₂37 DEG C of cell incubators be incubated for 6h.

6. weighing apparatus of making even is added in 96 orifice plate according to 75 holes μ L/ to the Bio-Glo (Promega) of room temperature, carries out the inspection that shines It surveys.

As a result as shown in Fig. 2, obtaining 7 candidate monoclonal antibodies, be respectively labeled as 4F2,4B9,6C1,6H12,13F7, 6A6 and 18B10.

The Determination of Kinetic Parameters of 4. antibody of embodiment

1. using Fortebio interaction of molecules instrument measurement people OX40 and anti-OX40 monoclonal antibody (4F2,4B9,6C1, 6H12,13F7,6A6 and 18B10) combine kinetic parameter.

2. with the fixed each monoclonal antibody of ProA sensor capture, after being balanced in PBS, in conjunction with antigen people OX40.

3. people OX40 is diluted to 2 concentration, respectively 400nM and 200nM with PBS, dissociated in PBS.

The results are shown in Table 1 for parameter detecting, and dynamics combines dissociation to scheme the affinity as shown in figure 3, all candidate antibodies It is horizontal to be in sub- nM.

Kinetic parameter of the 1. people OX40 of table in conjunction with anti-OX40 monoclonal antibody

The Binding experiment of embodiment 5. candidate anti-OX40 monoclonal antibody and people OX40 and monkey OX40

1. transiently transfecting people OX40 and monkey OX40 film expression plasmid in advance in 293T cell.

2. selecting the 293T cell of untransfected as control cell, the 293T cell and expression monkey OX40 of expression people OX40 are taken 293T cell as experimental group, the PBS containing 2%FBS after three kinds of cell dissociations, will be taken to clean twice with pancreatin, 850rpm from Heart 5min abandons supernatant, the PBS containing 2%FBS is then added, cell is resuspended and counts.

3. it is 1.0 × 10 that cell, which is diluted to concentration,⁷A cell/mL is added in streaming pipe according to 50 holes μ L/.

4. PBS of each monoclonal antibody (4F2,4B9,6C1,6H12,13F7,6A6 and 18B10) containing 2%FBS is diluted To 2 μ g/mL, it is added according to 50 holes μ L/ in the streaming pipe of existing 50 μ L cells.

It is incubated for 30min at 5.4 DEG C, the PBS with 3mL containing 2%FBS is washed 2 times, and 1000rpm is centrifuged 5min.

6. abandoning supernatant, cell is resuspended in 1 × PBS with 100 μ L containing 2%FBS, and the anti-mouse IgG Fc (Biolegend) of APC is added As secondary antibody.

It is incubated for 30min at 7.4 DEG C, the PBS with 3mL containing 2%FBS is washed 1 time, and 1000rpm is centrifuged 5min.

8. abandoning supernatant, cell is resuspended in the PBS with 100 μ L containing 2%FBS, and streaming machine testing obtains positive rate.

As a result as shown in figure 4, candidate anti-OX40 monoclonal antibody 18B10,4F2,6C1,6H12 and 6A6 and people OX40 and monkey OX40 all has relatively higher affinity combination, and 4B9 and 13F7 show weaker affinity to monkey OX40, it is contemplated that convenient Subsequent pharmacological gives up the two candidate antibodies of 4B9 and 13F7 in the intracorporal safety evaluatio of monkey.

Blocking experiment of the anti-OX40 monoclonal antibody of embodiment 6. for the combination of human OX 40 L (hFc) and people OX40

1. 1 bottle of (T75) HEK::OX40 100B5 cell of collection is transferred in 15mL centrifuge tube, contain 2%FBS with 8mL PBS wash 1 time, 1000rpm be centrifuged 5min；Supernatant is abandoned, PBS of the 1mL containing 2%FBS is added, cell is resuspended and counts.

2. experiment sample group is arranged: blank group, 0 μ g people OX40 L (Speed biosystems) group, 0.5 μ g people OX40 L Four concentration gradient groups of group and each monoclonal antibody (4F2,6C1,6H12,6A6 and 18B10).

3. cell is diluted to 1 × 10⁷A cell/mL is added in streaming pipe according to 10 holes μ L/, monoclonal antibody is used PBS dilution containing 2%FBS, the final concentration of monoclonal antibody is respectively 0.01 μ g/mL, 0.1 μ g/mL, 1 μ g/mL and 10 μ g/mL.

4. each 100 μ L of dilution antibody of corresponding four concentration gradients is added in monoclonal antibody sample group, corresponding Dan Ke Grand amount of antibody is respectively 1ng, 10ng, 100ng and 1000ng.100 PBSs of the μ L containing 2%FBS are added in remaining set, guarantee all Reaction system is 110 μ L.

5.4 DEG C combine 30min, the PBS with 3mL containing 2%FBS to wash 2 times, and 1000rpm is centrifuged 5min.

6. abandoning supernatant, in addition to blank group and 0 μ g people's OX40 L group, the people that concentration is 5 μ g/mL is added in remaining sample tube OX40 L 100μL。

7.4 DEG C combine 30min, the PBS with 3mL containing 2%FBS to wash 2 times, and 1000rpm is centrifuged 5min.

8. cell is resuspended in 1 × PBS with 100 μ L containing 2%FBS, in addition to blank group, APC anti-human igg is added in remaining each group Fc (Biolegend) is used as secondary antibody.

9.4 DEG C combine 30min, are washed 1 time with the PBS of the 2%FBS of 3mL, and 1000rpm is centrifuged 5min.

10. abandoning supernatant, cell is resuspended in 1 × PBS with 100 μ L containing 2%FBS, and streaming machine testing obtains positive rate.

As a result as shown in figure 5, candidate antibodies dosage can partially or completely block 0.5 μ g in 100ng and concentrations above The combination of OX40L and OX40 albumen.Wherein candidate antibodies 18B10 and 6C1 barrier effect are slightly worse.

The EC50 value of the anti-OX40 monoclonal antibody of embodiment 7. detects

EC50 value test experience detects antibody activity using NF- κ B signal access

1. 1 bottle of (T75) HEK::OX40 100B5 cell of collection is transferred in 15mL centrifuge tube, contain 10% with 5mL The DMEM culture medium (Gibco) of FBS (Gemini) is washed 1 time, and 1000rpm is centrifuged 5min；Supernatant is abandoned, 5mL is added containing 2%FBS's PBS is resuspended cell and counts.

2. cell is diluted to 2 × 10⁵A cell/mL is added 96 orifice plates according to 100 holes μ L/, is put into 5%CO₂37 DEG C Cell incubator is incubated for 30min.

3. candidate 5 monoclonal antibodies (4F2,6C1,6H12,6A6 and 18B10) and control antibodies MOXR0916's matches Concentration processed reduces dilution according to initial concentration 200nM, 3 times of gradients, totally 10 concentration points.It is added in 96 orifice plates according to 50 holes μ L/, That is the initial concentration of the final concentration of 50nM of monoclonal antibody, 3 times of gradients reduce, and multiple holes are arranged.

4. anti-mouse IgG Fc (Biolegend) is as 3 times that the final concentration of secondary antibody is primary antibody, according to the system in 50 holes μ L/ It is prepared, is added in 96 orifice plates.

5. 96 orifice plates are put into 5%CO₂37 DEG C of cell incubators be incubated for 20h.

6. 96 orifice plate room temperature 1000rpm to be measured is centrifuged 5min, take the every hole of 96 new orifice plates that 160 μ L are added in advance in 37 DEG C of temperature Case be incubated for 30min Quanti Blue (Invivo Gen), after take 40 μ L cell conditioned mediums be added, be incubated for 1h in 37 DEG C.

7. detecting OD value at 620nm using microplate reader, and select GraphPad Prism mapping analysis data.

The corresponding EC50 value of anti-OX40 monoclonal antibody is as shown in table 2, and EC50 value fitted figure is as shown in Figure 6.Early period streaming The binding force of antibody and cellular level is detected, the functional activity that antibody plays a role is investigated in this experiment.The results show that candidate antibodies 6A6 and 4F2 has the functional activity better than control antibodies；6C1 is relatively poor, therefore gives up the antibody in subsequent experimental.Further Selected candidate monoclonal antibody is 4F2,6H12,6A6 and 18B10.

The EC50 value bioactivity summary sheet of the candidate anti-OX40 monoclonal antibody of table 2.

The anti-OX40 monoclonal antibody of embodiment 8. stimulates people CD4⁺The experiment of T cell proliferation

1.CD3 is coated with 96 orifice plates: the CD3 (OKT3) of 100 holes μ L/, 0.05 μ g/mL or 0.01 μ g/mL, 4 DEG C of overnight incubations.

2. sorting cell: using CD4⁺The magnetic bead sorting kit (Miltenyi Biotec) of yin choosing is obtained from PBMC CD4⁺Cell is simultaneously counted.

3.CFSE (MCE) label: appropriate cell, remaining cell (10 are left and taken⁷It is a) it is resuspended in the PBS containing 5%FBS of 1mL In, CFSE is added to final concentration of 2 μM of progress CFSE label, is incubated at room temperature 10min, the PBS with 5mL containing 5%FBS is washed 3 times Dyeing is terminated, is resuspended with 1640 culture mediums containing 10%FBS.

4. the 96 orifice plate bed boards that selection is coated with different CD3 concentration: 100 holes μ L/, 3 × 10⁵The CD4 of a cells/well⁺T cell, 2 multiple holes.

5. adding monoclonal antibody: 100 holes μ L/, 5 μ g/mL (final concentration of 2.5 μ g/mL) antibody (4F2,6H12,6A6, 18B10 and control antibodies MOXR0916), negative antibody control (mIgG1 and hIgG1, Biolegend) and blank well pair are set According to 37 DEG C of incubations.

6. being incubated for 20h or so, 60 μ L supernatant of centrifuging and taking carries out the ELISA detection of IL-2, as a result as shown in Figure 7 B.

7. 96 orifice plates are continued to be incubated for 5-7 days, 100 μ L supernatants are taken to carry out the ELISA detection of IFN-γ, as a result such as Fig. 7 C institute Show.

8. collecting the CFSE flow cytometer detection that cell carries out CD4, as a result as shown in Figure 7 A.

The results show that the proliferation of 4 equal effective stimulus people CD4+T cells of candidate monoclonal antibody, and effectively increase IL-2 And the secretion of IFN-γ, under the experiment condition, candidate antibodies are superior to control antibodies.It is special in conjunction with first few items experimental result Not Guan Zhu the detection of EC50 value bioactivity, comprehensively consider, select 6A6 and 4F2 and carry out drug effect inhibiting tumor assay research in Mice Body.

The anti-human OX40 monoclonal antibody of embodiment 9. is in the intracorporal inhibiting tumor assay of OX40 humanization mouse

1. cell culture: by mouse colorectal cancer MC38 cell in 37 DEG C, 5%CO₂Incubator in cultivate, culture medium at It is divided into the DMEM culture medium containing 10%FBS, cell passed on 1 time every 2 days sub-bottles.

2. mouse inoculation cell: the MC38 tumour cell that PBS is resuspended is with 5 × 10⁶The concentration of a cell/mL, 0.1mL/ The right side side of body rib portion that volume is inoculated in OX40 humanization mouse (hundred Olympic Competition figure Genetic Biotechnologies Co., Ltd of Beijing) is subcutaneous, Corotation connects 26 mouse.When mean tumour volume reaches about 100mm³When, the moderate mouse of picking individual gross tumor volume enters group, Animal is assigned randomly in 4 groups by gross tumor volume, including control group (PBS group) and three (MOXR0916 pairs of experimental group According to antibody group, 6A6 group and 4F2 group), every group 6, the grouping same day starts to be administered, and specific dosage regimen is shown in Table 3:

3. inhibiting tumor assay dosage regimen of table

Note:^aRefer to every 3 days and be administered once, is administered 4 times altogether.

3. continuing to observe to experimental animal weight and tumour growth state after administration.Measure weekly gross tumor volume twice and The weight of animals, and record measured value.The measurement result of gross tumor volume is as shown in Figure 8 A, wherein MOXR0916 control antibodies group with When control is compared,^*P < 0.05,^**P<0.01；6A6 group compared with the control when,^#P < 0.05,^##P<0.01；4F2 group is compared with the control When,^&P<0.05.The measurement result of the weight of animals is as shown in Figure 8 B.

4. being observed and recorded 14 days after administration, mouse is dissected, serum is taken to carry out ALT and AST biochemistry detection, testing number According to respectively as shown in 8C and 8D, the results show that its data in the normal range, has no obvious hepatotoxicity wind agitation.Dissect tumour, liver And spleen tissue, it carries out weighing and goes forward side by side line number according to statistics, tumour internal anatomy is as illustrated in fig. 8e.It calculates dissection knurl weight and accounts for mouse totality The percentage of weight, has more intuitively investigated group difference, data statistics result is as shown in Figure 8 F, experimental group and control group phase Than, statistically difference highly significant,^***P<0.001。

Synchronization Analysis tumor control rate 5. (TGI) data, as a result as shown in Table 4.After administration, candidate antibodies (6A6 and 4F2) Show with control antibodies (MOXR0916) comparable tumor killing effect, and clone 6A6 be slightly better than 4F2.

4. experimental group TGI of table statistics

It is appreciated that although the application describes related invention with above-mentioned concrete form, these inventions not office It is limited to the specific content of these concrete forms description.It should be apparent to those skilled in the art that without departing from the application Under the premise of described spirit, also various equivalent changes can be carried out to the technical characteristic for including is invented involved in it Change, these variations all should belong within the scope of the invention.

Sequence table

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<213>artificial sequence (Artificial Sequence)

<400> 13

Lys Ala Ser Gln Asp Val Ser Ser Ala Val Ala

1 5 10

<210> 14

<211> 11

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 15

<211> 11

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser

1 5 10

<210> 16

<211> 11

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 17

<211> 7

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 17

Trp Ala Ser Ile Arg His Thr

1 5

<210> 18

<211> 7

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 18

Ala Ala Ser Thr Leu Asp Ser

1 5

<210> 19

<211> 7

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 19

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 20

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 20

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210> 21

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 21

Gln Pro His Tyr Ser Thr Pro Trp Thr

1 5

<210> 22

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 22

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr

1 5

<210> 23

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 23

Gln Gln Leu Asn Thr Leu Pro Trp Thr

1 5

<210> 24

<211> 140

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg Gln Gly Ser Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 25

<211> 140

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 26

<211> 141

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 26

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr

50 55 60

Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn

65 70 75 80

Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn

85 90 95

Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr

100 105 110

Tyr Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 27

<211> 140

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

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

20 25 30

Phe Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

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

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 28

<211> 140

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 29

<211> 138

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 29

Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly

1 5 10 15

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

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

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

50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn

65 70 75 80

Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Leu Pro Tyr Tyr Asp Val Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 30

<211> 134

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 30

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Asn Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr Asn

65 70 75 80

Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val

100 105 110

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

115 120 125

Thr Leu Thr Val Ser Ser

130

<210> 31

<211> 131

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 31

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

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

100 105 110

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

115 120 125

Glu Ile Lys

130

<210> 32

<211> 131

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 32

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

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

100 105 110

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu

115 120 125

Glu Leu Lys

130

<210> 33

<211> 131

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 33

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

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

100 105 110

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

115 120 125

Glu Ile Lys

130

<210> 34

<211> 127

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 34

Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe Leu Trp Leu Ser

1 5 10 15

Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser

20 25 30

Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Pro His Tyr

100 105 110

Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 35

<211> 131

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 35

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

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

100 105 110

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

115 120 125

Glu Ile Lys

130

<210> 36

<211> 129

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 36

Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

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

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu His Gln Lys Pro Asp Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu

115 120 125

Lys

<210> 37

<211> 127

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 37

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu Ser

20 25 30

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

35 40 45

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

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

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

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu Asn

100 105 110

Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Claims (16)

1. specifically bind the antibody or its antigen-binding portion thereof of OX40, it includes heavy chain variable region HCDR1, HCDR2 and/or HCDR3, wherein the HCDR1 sequence include amino acid sequence SGYNWN (SEQ ID NO:1), SGYYWN (SEQ ID NO:2), GYYMN (SEQ ID NO:3) or NSWMN (SEQ ID NO:4)；The HCDR2 sequence includes amino acid sequence YISYDGSNNYNPSLKN(SEQ ID NO:5)、YISYDGSDNYNPSLKN(SEQ ID NO:6)、EINPSTGGTTYNQKFKA (SEQ ID NO:7) or RIYPGDENTNYNGKFKG (SEQ ID NO:8)；And/or the HCDR3 sequence includes amino acid sequence Arrange QGSSGHFYYAMDY (SEQ ID NO:9), EGRSGHFYYAMDY (SEQ ID NO:10), SGLPYYDVDY (SEQ ID NO:11),GLYSVQ(SEQ ID NO:12)；Preferably, the antigen-binding portion is selected from: Fab segment, Fab ' segment, F (ab’)₂Segment, Fv segment, scFv segment, Fd segment or single domain antibody.

2. antibody as described in claim 1 or its antigen-binding portion thereof, wherein the heavy chain variable region includes to be selected from SEQ ID Amino acid sequence shown in any one of NOs:24-30 or the amino acid sequence with above-mentioned sequence at least 80% homology； Preferably, the heavy chain variable region includes amino acid sequence shown in SEQ ID NO:24 or 29.

3. antibody as claimed in claim 1 or 2 or its antigen-binding portion thereof, also include light chain variable region, the light chain can Becoming area includes LCDR1, LCDR2 and/or LCDR3 sequence, wherein the LCDR1 sequence includes amino acid sequence KASQDVSSAVA (SEQ ID NO:13), KASQDVNTAVA (SEQ ID NO:14), RASQEISGYLS (SEQ ID NO:15) or RASQDISNYLN(SEQ ID NO:16)；The LCDR2 sequence include amino acid sequence WASIRHT (SEQ ID NO:17), AASTLDS (SEQ ID NO:18) or YTSRLHS (SEQ ID NO:19)；And/or the LCDR3 sequence includes amino acid sequence QQHYSTPWT (SEQ ID NO:20), QPHYSTPWT (SEQ ID NO:21), LQYASYPLT (SEQ ID NO:22) or QQLNTLPWT(SEQ ID NO:23)。

4. antibody as claimed in claim 3 or its antigen-binding portion thereof, wherein the light chain variable region includes to be selected from SEQ ID Amino acid sequence shown in any one of NOs:31-37 or the amino acid sequence with above-mentioned sequence at least 80% homology； Preferably, the light chain variable region includes to be selected from amino acid sequence shown in SEQ ID NO:31 or 36.

5. such as the described in any item antibody of preceding claims or its antigen-binding portion thereof, wherein the antibody or its antigen binding Part specifically binds to primate OX40；Preferably, the primate OX40 is selected from people OX40 or monkey OX40.

6. such as the described in any item antibody of preceding claims or its antigen-binding portion thereof, wherein the antibody is anti-for source of mouseization Body；Preferably, the heavy chain variable amino acid sequence of the antibody is as shown in SEQ ID NO:24 or 29 and/or the antibody Chain variable region amino acid sequence as shown in SEQ ID NO:31 or 36.

7. such as the described in any item antibody of preceding claims or its antigen-binding portion thereof, wherein the antibody or its antigen binding Part is with 1 × 10^-8To 1 × 10^-7The KD of M specifically binds to OX40 molecule；Preferably, the antibody has OX40 agonist Function stimulates the activation and proliferation of T cell.

8. pharmaceutical composition, it includes antibody of any of claims 1-7 or its antigen-binding portion thereofs and pharmacy Upper acceptable carrier；

Optionally, described pharmaceutical composition also includes one or more other active components, such as chemotherapeutics, PD-1 binding antagonists Deng.

9. vaccine, it includes antibody of any of claims 1-7 or its antigen-binding portion thereofs, and it is optional immune Adjuvant.

10. nucleic acid molecule encodes antibody of any of claims 1-7 or its antigen-binding portion thereof.

11. expression vector, it includes nucleic acid molecules described in any one of claim 10.

12. host cell, it includes expression vectors described in nucleic acid molecule described in any one of claim 10 or claim 11.

13. antibody of any of claims 1-7 or its antigen-binding portion thereof or drug according to any one of claims 8 Composition is in preparation for inhibiting Treg function, the cell for killing expression OX40, the reaction for causing T cell mediation, improving effect T Purposes in the function of cell, the function of improving memory T cell, and/or the drug for effectively inhibiting tumour growth.

14. antibody of any of claims 1-7 or its antigen-binding portion thereof or drug according to any one of claims 8 Composition is preparing the purposes in the drug for preventing and/or treating the relevant disease of OX40.

15. purposes as claimed in claim 14, wherein the disease is tumour；

Preferably, the tumour is selected from one of following or a variety of: colon cancer, melanoma, cortical tumor, clear-cell carcinoma, Lymthoma, advanced solid tumor and above-mentioned metastatic tumor.

16. detection reagent or kit, it includes antibody of any of claims 1-7 or its antigen-binding portion thereofs.