CN107847570A

CN107847570A - Target construct of HPV16 HPV-16 E7s/MHC complexs and application thereof

Info

Publication number: CN107847570A
Application number: CN201680039553.5A
Authority: CN
Inventors: 刘诚; 刘宏; P·王; V·W-F·陈
Original assignee: Yurico Biotech Corp
Current assignee: Yurico Biotech Corp; Eureka Therapeutics Inc
Priority date: 2015-05-08
Filing date: 2016-05-06
Publication date: 2018-03-27
Also published as: WO2016182957A1; SG11201708674XA; RU2017142716A; CA2988397A1; EP3294328A1; MX2017014056A; JP2018516879A; AU2016261356A1; KR20170141256A; TW201713695A; IL255228A0

Abstract

Present application provides the construct of the antibody moiety comprising complex of the specific binding comprising HPV16 HPV-16 E7s and MHC I proteinoid.Manufacture and the method using these constructs are also provided.

Description

Target construct of HPV16-E7 peptides/MHC complexs and application thereof

The cross reference of related application

This application claims U.S. Provisional Application No. filed in 3 days April in 2015 62/158,735, Shen on May 8th, 2015 U.S. Provisional Application No. 62/304,915 filed in U.S. Provisional Application No. please 62/197,480 and 27 days July in 2015 Priority, its all be incorporated in entirety by reference hereby.

Technical field

The present invention relates to specific binding and the antibody construct of the compound MHC molecule of HPV16-E7 peptides, and application thereof, bag Include treatment and diagnose the illness.

Sequence table is submitted in the form of ASCII text documents

It is incorporated herein in entirety by reference with the herein below that ASCII text documents are submitted：Computer-readable shape Sequence table (the file name of formula (CRF)：750042000240SEQLIST.txt record date：On May 6th, 2016, size： 125KB)。

Background technology

HPV (HPV) causes wart or benign papillomatous small no coating when being and being infected in epithelial cell DNA virus.Persistent infection high-risk HPV can cause more serious cytological abnormal or lesion, may development if untreated To cancer.In the U.S. (according to CDC), the annual morbidity of HPV associated cancers>26,000, in the world>600,000 (Forman D. et al., Vaccine, 30:Suppl 5:F12-23,2012) the different types of HPV of are to cause to cause almost institute The reason for some cases of cervical cancer, global female cancer associated death the third-largest reason (Doorbar J., Clin.Sci., 110:525–541,2006).In addition to cervical carcinoma, HPV also with anogenital cancer disease (anus, penis, vagina and vulva) and head Neck cancer disease (pars oralis pharyngis：Throat back, including the root of the tongue and amygdaline root) it is relevant.To 5,046 from 60 research In the meta-analysis of neck squamous cell cancer (HNSCC) cancer sample, it is 25.9% to find HPV total incidences.Positive in HPV In HNSCC, in 86.7% HPV positive oropharyngeal squamous cell cancers, 68.2% OSCC, 69.2% larynx squamous HPV-16 is found in cell cancer.(Kreimer A.R. et al., Cancer Epidemiol Biomarkers Prev.14:467- 475,2005).(The Cancer Genome Atlas Network, Nature, 517 in a newer research:576– 582,2015) HPV E6 and E7 albumen, is passed through>The RNA sequencings (RNA-Seq) of 1,000 matchings are read long by HPV states point Class, it is the HPV positives to find 36 (12.9%) in 279.

The cancer illness of excessive risk HPV hypotype persistent infections is mainly due to the table of two kinds of potential oncogenes, E6 and E7 Reach, it has been demonstrated the p53 and Rb that degraded in a manner of proteasome dependence, so as to promote genomic instability and cell transformation (Doorbar, see on).HPV E6 and E7 cancer proteins continuous expression in lesion, and tumour is only in initial cell immortality thing The several years occurs after part.In fact, E6 and E7 continuous expression for maintain conversion phenotype and prevent cell growth arrest and/ Or apoptosis is required (McLaughlin-Drubin M.E.＆M ü nger K., Virology, 384:335-344,2009).By L1 is not expressed in fixed lesion, so even if can use the pre- of the approval of the L1 albumen for the hypotype of HPV-6,11,16 and 18 Anti- property vaccine, for HPV⁺The treatment of cancer still suffers from unsatisfied demand.

The protein expressed by such as HPV oncogenic virus is represented and is used for due to expression is not present in normal cell The excellent target of immunization therapy.E6 and E7 is using low molecular weight compound or for the anti-of cell surface protein not by traditional The intracellular protein that the drug discovery effort of body method is targetted.The positive advanced vulva intraepithelial neoplasia sample diseases of nearest HPV16 The research of change is it has been proved that having T cell and facing in 50% patient treated with the methods of vaccination using E6/E7 peptides Bed reaction (Kenter G.G. et al., N.Engl.J.Med.361:1838–1847,2009；Welters M.J.et al., N.Engl.J.Med.361:1838–1847,2010；Daayana S.et al.,Br.J.Cancer 102:1129–1136, 2010).The combination HLA-A*02 of the different prediction at 13 kinds:In 01 HPV-16 E7, find only have E7_11–19(YMLDLQPET) by day The HLA-A*02 that so processing and HPV-16 in vitro are converted:01 expression cell fasten displaying (Riemer A.B. et al., J.Biol.Chem.285(38):29608-29622,2010).In HPV databases, this peptide is in 17 HPV-16 variants It is conservative (Zhang G.L. et al., Database, 1-12,2014) to have 16.

Limit of the selection for next arrogant antibody repertoire system is allowed to using phage display to produce mAb latest developments Determining epitope has the medicament of fine specificity.It is many this to entity tumor antigen tool in the case of HLA-A01 and HLA-A02 There is specific mAb to be successfully selected from phage display library (Noy et al., Expert Rev.Anticancer Ther.5 (3):523-536,2005；Chames et al., Proc.Natl.Acad.Sci.USA97:7969-7974,2000；Held et al., Eur.J.Immunol.34:2919-2929,2004；Lev et al., Cancer Res.62:3184-3194,2002； Klechevsky et al., Cancer Res.68 (15):6360-6367,2008).Recently, shown to mankind WT1/HLA-A02 There is complex specific people mAb (t cell epitope fully described) to be surveyed via the effector cell function that Fc is mediated in cell Suppress a variety of cancerous cell line and primary cancer cells (Dao et al., Sci.Transl.Med.5 in fixed and In vivo model:176ra33, 2013；Veomett et al., Clin.Cancer Res.doi:10.1158/1078-0432,2014).

The disclosure of all publications, patent, patent application and the patent application of announcement involved by this paper is hereby with full The mode of reference is incorporated herein.

The content of the invention

In one aspect, the application, which provides, is bound to the complex comprising HPV16-E7 peptides and MHC I proteinoid (at this Referred to herein as " HPV16-E7/MHC I classes complex " or " E7MC ") construct (such as through separate construct).In some implementations In scheme, construct (" anti-E7MC constructs ") includes specific binding answering comprising HPV16-E7 peptides and MHC I proteinoid Fit antibody moiety (being referred to herein as " anti-E7MC antibody moieties ").

Therefore, in some embodiments, there is provided include HPV16-E7 peptides and MHC I proteinoid comprising specific binding Complex antibody moiety anti-E7MC constructs (as separation anti-E7MC constructs).In some embodiments, HPV16-E7/MHC I class complexs are present on cell surface.In some embodiments, HPV16-E7/MHC I classes are compound Body is present on cancer cell surfaces.

In some embodiments, anti-E7MC constructs include specific binding and include HPV16-E7 peptides and MHC I class eggs The antibody moiety of the complex of white matter, wherein MHC I proteinoid are HLA-A.In some embodiments, MHC I albuminoids Matter is HLA-A02.In some embodiments, MHCI proteinoid is the HLA-A*02 of HLA-A02 allele:01 hypotype.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein antibody moiety (have different with MHC I proteinoid from comprising HPV16-E7 peptides and the 2nd MHC I proteinoid HLA allele) complex cross reaction.In some embodiments, antibody moiety with comprising being taken containing an amino acid The variant of HPV16-E7 peptides in generation (such as conserved amino acid substitution) and the complex cross reaction of MHC I proteinoid.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein HPV16-E7 peptides length are about 8 to about 12 (any one of such as from about 8,9,10,11 or 12) individual amino acid.One In a little embodiments, HPV16-E7 peptides, which have, is selected from SEQ ID NO:3-14 amino acid sequence.In some embodiments, HPV16-E7 peptides have amino acid sequence YMLDLQPET (SEQ ID NO:4).

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein antibody moiety are full length antibody, Fab, Fab', (Fab') 2, Fv or scFv (scFv).In some embodiments In, antibody moiety is whole mankind's antibody moiety, semi-synthetic antibody moiety or the humanized antibody portion with human antibody framework region Point.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein antibody moiety with about 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, Any scope between any one of 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values) equilibrium solution From constant (K_d) it is bound to HPV16-E7/MHC I class complexs.In some embodiments, the anti-E7MC constructs of separation with About 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, Any scope between any one of 100nM or 500nM, including these values) K_dHPV16-E7/MHC I classes are bound to answer It is fit.

In some embodiments, anti-E7MC constructs include specific binding and include HPV16-E7 peptides and MHC I class eggs The antibody moiety of the complex of white matter, wherein antibody moiety include：I) weight chain variable district, it includes complementary determining region of heavy chain (HC- CDR) 1, the HC-CDR1 include amino acid sequence G-F/G/Y-S/T-F-S/T-S-Y-A/G (SEQ ID NO:183), or it is wrapped Variant containing at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, the HC-CDR2 include amino acid Sequence I-N/I-P-X-X-G-G/T/I-T/A/P or I-S-X-S/D-G/N-G/S-N-T/I/K (SEQ ID NO:184 or 185), Or it includes the variant of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 bags A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D containing amino acid sequence, A-R-G-X-X-X-Y-Y/G/S or A-R-G-X-X-Y-Q/ W-W-S-X-D-D(SEQ ID NO:Any in 186-188), or it includes at most about 3 (any one of such as from about 1,2 or 3) The variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes complementary determining region of light chain (LC-CDR) 1, the LC-CDR1 bags N-I-G-S-N/K containing amino acid sequence or L-R-S/N-X-Y (SEQ ID NO:189 or 190), or it includes at most about 3 (such as from about 1st, any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include amino acid sequence A/Q/N-S/ A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V(SEQ ID NO:191), or it includes at most about 3 (in such as from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, wherein X can be any amino acid.

In some embodiments, anti-E7MC constructs include specific binding and include HPV16-E7 peptides and MHC I class eggs The antibody moiety of the complex of white matter, wherein antibody moiety include：I) weight chain variable district, it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:Any one of 57-77 amino acid sequence or its include at most about 5 (in such as from about 1,2,3,4 or 5 appoint One) variant (and being made from it in some embodiments) of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence or its include at most about 5 (any one of such as from about 1,2,3,4 or 5) it is individual The variant (and being made from it in some embodiments) of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 or its include at most about 5 (in such as from about 1,2,3,4 or 5 appoint One) variant (and being made from it in some embodiments) of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence or its include at most about 5 The variant (and being made from it in some embodiments) of (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, LC- CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence or its include at most about 3 (such as from about 1st, any one of 2 or 3) variant (and being made from it in some embodiments) of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, should LC-CDR3 includes SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence or its include at most about 5 (such as Any one of about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant (and being made from it in some embodiments).

In some embodiments, anti-E7MC constructs include specific binding and include HPV16-E7 peptides and MHC I class eggs The antibody moiety of the complex of white matter, wherein antibody moiety include：I) weight chain variable district, it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence (and being made from it in some embodiments)；And HC-CDR3, the HC-CDR3 are included SEQ ID NO:The amino acid sequence (and being made from it in some embodiments) of any one of 99-119,244 and 245； Or it includes the variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at most about 5 (any one of such as from about 1,2,3,4 or 5) individual HC-CDR areas；And ii) Light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino Acid sequence (and being made from it in some embodiments)；LC-CDR2, the LC-CDR2 include SEQ ID NO:In 141-161 The amino acid sequence (and being made from it in some embodiments) of any one；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence (and being made from it in some embodiments)；Or its bag Variant containing the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at most about 5 (any one of such as from about 1,2,3,4 or 5) individual LC-CDR areas.

In some embodiments, anti-E7MC constructs include specific binding and include HPV16-E7 peptides and MHC I class eggs The antibody moiety of the complex of white matter, wherein antibody moiety include a) weight chain variable district, and it includes SEQ ID NO:15-35 and Any one of 233-237 amino acid sequence or itself and SEQ ID NO:Any one of 15-35 and 233-237 have at least The variant of about 95% (as any one of at least about 95%, 96%, 97%, 98% or 99%) sequence identity is (and at some It is made from it in embodiment)；And b) light chain variable district, it includes SEQ ID NO:Any one of 36-56's and 238-243 Amino acid sequence or itself and SEQ ID NO:Any one of 36-56 and 238-243 have at least about 95% (as at least about 95%th, any one of 96%, 97%, 98% or 99%) variant of sequence identity is (and in some embodiments by its group Into).In some embodiments, antibody moiety includes weight chain variable district, and it includes SEQ ID NO:In 15-35 and 233-237 Any one amino acid sequence (and being made from it in some embodiments), and light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence (and being made from it in some embodiments).

In some embodiments, anti-E7MC constructs include with according to any one of antibody moiety described above Secondary antibody partial competition is bound to the first antibody part of target HPV16-E7/MHC I class complexs.In some embodiments In, first antibody part is bound to identical or substantially the same epitope with secondary antibody part.In some embodiments, The combination of one antibody moiety and target HPV16-E7/MHC I class complexs suppresses secondary antibody part and target HPV16-E7/MHC I The combination of class complex at least about 70% is (such as any at least about 75%, 80%, 85%, 90%, 95%, 98% or 99% ), or vice versa it is as the same.In some embodiments, first antibody part and secondary antibody partial intersection competition binding are to target HPV16-E7/MHC I class complexs, i.e., each of first and second antibody moiety, which contends with one other, is bound to target HPV16- E7/MHC I class complexs.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, the anti-E7MC constructs of separation are full length antibody.In some embodiments, the anti-E7MC constructs of separation are Dan Te The opposite sex.In some embodiments, the anti-E7MC constructs of separation are polyspecific.In some embodiments, separate Anti- E7MC constructs for bispecific.In some embodiments, the anti-E7MC molecules of separation are series connection scFv, difunctional Antibody (Db), Single-chain bifunctional antibody (scDb), double affinity target again (DART) antibody, Double variable regions (DVD) antibody, pestle- Mortar (KiH) antibody, depressed place lock (dock and lock) (DNL) antibody, chemical crosslinking antibody, heteromultimeric antibody or different conjugate Antibody.In some embodiments, the anti-E7MC constructs of separation are to include two scFv by peptide linker connection series connection scFv.In some embodiments, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments).

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein the anti-E7MC constructs separated further include the second antigen-binding portion thereof of the second antigen of specific binding. In some embodiments, the second antigen-binding portion is divided into antibody moiety.In some embodiments, the second antigen is T cell table Antigen on face.In some embodiments, T cell is selected from the group consisted of：Cytotoxic T cell, helper cell and Natural killer T cell.In some embodiments, the second antigen is selected from the group consisted of：CD3γ、CD3δ、CD3ε、CD3 ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.In some embodiments, the second antigen is CD3 ε, and point From anti-E7MC constructs be comprising to HPV16-E7/MHC I classes complexs with specific N-terminal scFv and to CD3 ε with Specific C-terminal scFv series connection scFv.In some embodiments, the second antigen be constant killer cell, neutrophil leucocyte, Antigen on monocyte, macrophage or surface of dendritic cells.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein the anti-E7MC constructs separated are Chimeric antigen receptor (CAR).In some embodiments, Chimeric antigen receptor Include the extracellular containing antibody moiety, membrane-spanning domain and intracellular signal transduction domain.In some embodiments, intracellular signal transduction Domain includes CD3 ζ intracellular signal transductions sequences and costimulatory signal conduction sequence.In some embodiments, costimulatory signal passes It is CD28 intracellular signal transduction sequences to lead sequence.

In some embodiments, according in anti-E7MC constructs (such as the anti-E7MC constructs of separation) described above Any one, anti-E7MC constructs include the antibody of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Part, wherein the anti-E7MC constructs separated are the immunoconjugates comprising antibody moiety and effector molecule.In some embodiment party In case, effector molecule is the therapeutic agent selected from the group consisted of：Medicine, toxin, radio isotope, protein, peptide and Nucleic acid.In some embodiments, therapeutic agent is medicine or toxin.In some embodiments, effector molecule is mark.

In other embodiments, there is provided include the anti-E7MC constructs according to any one of embodiment as described above The pharmaceutical composition of (such as the anti-E7MC constructs of separation).In some embodiments, pharmaceutical composition is further included with resisting The related cell (such as effector cell) of E7MC constructs.In some embodiments, there is provided express anti-E7MC constructs or it is more Peptide composition or relative host cell.In some embodiments, there is provided encode anti-E7MC constructs or its polypeptide fractions Nucleic acid.In some embodiments, there is provided the carrier comprising nucleic acid.In some embodiments, there is provided express anti-E7MC structures Build body or relative effector cell.In some embodiments, effector cell is T cell.

In some embodiments, there is provided detection presents comprising HPV16-E7 peptides and MHC I proteinoid on the surface The method of the cell of complex, it includes real cell and the anti-E7MC constructs according to any one of embodiment as described above (such as the anti-E7MC constructs of separation) contact, the anti-E7MC constructs include a) specific binding and include HPV16-E7 peptides and MHC The antibody moiety of the complex of I proteinoid, and b) mark, and the presence of the mark on detection cell.

In some embodiments, there is provided individual method of the treatment with HPV16-E7 positive diseases, it includes item Body applies anti-E7MC constructs (such as anti-E7MC of separation included according to any one of embodiment as described above of effective dose Construct) pharmaceutical composition.In some embodiments, pharmaceutical composition further includes the anti-E7MC constructs with separating Related cell (such as effector cell).In some embodiments, there is provided treatment is individual with HPV16-E7 positive diseases Method, it includes the effector cell for expressing any one of anti-E7MC CAR described above that item individual applies effective dose. In some embodiments, effector cell is T cell.In some embodiments, HPV16-E7 positive diseases are cancer.At some In embodiment, cancer is squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharynx Cancer.In some embodiments, cancer is HPV16-E7 positive squamous cells cancer (SCC).

In some embodiments, there is provided individual method of the diagnosis with HPV16-E7 positive diseases, it is included：A) to Individual applies the anti-E7MC constructs of the separation comprising mark according to any one of embodiment as described above of effective dose； And b) measure individual in labelled content, wherein the level marked higher than threshold level instruction individual suffer from HPV16-E7 positive diseases Disease.In some embodiments, there is provided individual method of the diagnosis with HPV16-E7 positive diseases, it is included：A) derivative is made Sample and the anti-E7MC constructs of the separation comprising mark according to any one of embodiment as described above from individual Contact；And the quantity of cell that b) measure is combined with the anti-E7MC constructs of the separation in sample, wherein with the anti-E7MC that separates The quantitative value for the cell that construct combines suffers from HPV16-E7 positive diseases higher than threshold level instruction individual.In some embodiment party In case, HPV16-E7 positive diseases are cancer.In some embodiments, cancer be squamous cell carcinoma, cervical carcinoma, cancer of anus, Carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is that HPV16-E7 positive squamous is thin Born of the same parents' cancer (SCC).

The method for manufacturing any one of construct as described herein, the product for being suitable for approach described herein are also provided And kit.

Brief description of the drawings

Fig. 1 is shown by HPV16-E7 11-19 peptides/HLA-A*02 after ultrafiltration concentration:The size of 01 complex is exclusive Chromatography (SEC) tomographic map.The peptide appropriately folded /MHC complex monomers：212mL；The aggregation of false folding：111mL；Trip From β 2M：267mL.

Fig. 2 is shown on biotin labeling HPV16-E7 11-19 peptides/HLA-A*02:01 relative to C3 pairs of biotin labeling According to peptide/HLA-A*02:The phage clone ELISA results of 01 specific binding.

Fig. 3 shows the bacteriophage for loading T2 cells relative to C3 control peptides on HPV16-E7 11-19 peptides load T2 cells Clone FACS binding analysis results.1：Only cell negative control；2：Only 2 ° of antibody controls；3：HPV16-E7 11-19 peptides/HLA- A*02:01 specific antibody phage clone.

Fig. 4 shows the T2 cells being loaded with 1,5 or 8 HPV16-E7 11-19 peptide with single alanine replacement The result of phage clone #11FACS binding analysis：1：Only cell negative control；2：Only 2 ° of antibody controls；3：HPV16-E7 11-19 peptides/HLA-A*02:01 specific antibody phage clone #11.

Fig. 5 shows the T2 cells of HPV16-E7 11-19 peptides load relative to the endogenous egg for being loaded with being derived from normal expression The result of the phage clone FACS binding analysis of the combination of the T2 cells of white matter.It is past from a left side for each phage clone The peptide of right load is：HPV16-E7 11-19、A2E1、A2E2、A2E3、A2E4、A2E5、A2E6、A2E7、A2E8、A2E9、 A2E11 and A2E17.

Fig. 6 shows the SDS-PAGE analyses of the purity for determining anti-HPV16-E7 11-19/MHC bispecific antibodies.

Fig. 7 shows the anti-HPV16-E7 11- by being prepared from 1 μ g/ml and the different bacteriophages of 0.2 μ g/ml concentration clone 19/HLA-A*02:The T cell killing of the HPV16-E7 11-19 peptides load T2 cells of 01 Mediated by Bi-specific Antibodies.It is negative right According to including being loaded with the T2 cells of AFP158 peptides and for AFP158/HLA-A*02:01 bispecific antibody.For each Phage clone, the peptide loaded from left to right are：1μg/ml HPV16-E7 11-19、1μg/ml AFP158、0.2μg/ml HPV16-E711-19 and 0.2 μ g/ml AFP158.NC peptides：AFP15 peptides；NC antibody：Anti- AFP158/HLA-A*02:01 pair special Property antibody.

Fig. 8 A show the two kinds of cancerous cell lines mediated by anti-HPV16-E7 11-19/MHC bispecific antibodies (BsAb) The T cell killing of (CaSki and MS-751).

Fig. 8 B are shown in what is mediated under various concentrations by anti-HPV16-E7 11-19/MHC bispecific antibodies (BsAb) The dose-dependant of the T cell killing of two kinds of cancerous cell lines (CaSki and MS-751).

Fig. 9 shows the schematic diagram of Chimeric antigen receptor construct.

Figure 10 A and 10B show the HPV16-E7 11-19 for being loaded with having WT sequences or single alanine replacement in 1-9 positions The result of the BsAb FACS combination mensurations of the T2 cells of peptide.Figure 10 A show the BsAb based on clone US-7,7-1,7-3 and 7-6 Result.Figure 10 B show the result of the BsAb based on clone 7-7 and 7-8.

The T that Figure 11 A-11C displays are transduceed using different CAR or the parent scFv with anti-HPV16-E7 affinity maturations is thin The flow cytometry of born of the same parents；Cell HPV16-E7 11-19 peptides/HLA-A*02:01 tetramer staining and use anti-CD 4 antibodies Dyed altogether with anti-CD8 antibody.Figure 11 A show US-7 4-1BB, 7-1 4-1BB, 7-3 4-1BB, 7-6 4-1BB, 7-7 4-1BB With the flow cytometry of 7-8 4-1BB CAR-T cells.Figure 11 B show 7-9 4-1BB, US-7CD28,7-1CD28,7- The flow cytometry of 3CD28,7-6CD28 and 7-7CD28CAR-T cell.Figure 11 C show 7-8CD28 and 7-9CD28CAR- The flow cytometry of T cell and the T cell of simulation transduction.

Figure 12 is shown has the anti-HPV 16-E7/HLA-A*02 of affinity maturation by expression:01CAR's or parent scFv T cell expression mediation for HLA-A*02:01 is positive and is positive for HPV16-E7 or negative cancerous cell line Killing.Tmock- transducer cells including simulation transduction are as control.

Detailed description of the invention

The application is provided comprising complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid (herein In be referred to as " HPV16-E7/MHC I classes complex " or " E7MC ") antibody moiety (be referred to herein as " anti-E7MC antibody portion Point ") through separate construct (being referred to herein as " anti-E7MC constructs ").Anti- E7MC constructs specific recognition HPV16- E7/MHC I class complexs, the HPV16-E7 peptides that such as MHC on expression HPV16-E7 cell surface is presented.Anti- E7MC structures Body can specifically bind N-terminal portion, C-terminal part or the center section of the HPV16-E7 peptides of the complex, and/or with comprising At least one complex cross reaction of HPV16-E7 peptides and the different subtype of MHC I proteinoid is (for example, anti-E7MC constructs With reference to HPV16-E7 peptides/HLA-A*02:01 complex and HPV16-E7 peptides/HLA-A*02:02 complex).Anti- E7MC constructs Allow selectively targeted E7MC in delivery cell (that is, in its surface present be bound to MHC molecule HPV16-E7 peptides it is thin Born of the same parents), such as expression HPV16-E7 disease cells.This strategy, which provides, to be better than using the notable of the antibody for HPV16-E7 protein Technical advantage, using such antibody can not selectively targeted E7MC be in delivery cell.In addition, when being fused to detectable part, resist E7MC antibody moieties allow with for E7MC in delivery cell number and changes in distribution (than circulation the potential more phase of HPV16-E7 contents The progression of disease of pass is measured) high sensitivity HPV16-E7 positive diseases or illness are diagnosed and prognosis.

Using display technique of bacteriophage, we, which generate, a variety of is directed to HPV16-E7 11-19 peptides/HLA-A*02:01 is compound The monoclonal antibody of body specificity and high-affinity.Flow cytometry and T cell mediation cytotoxicity analysis displaying antibody with HPV16-E7 and HLA-A*02:The mode of 01 limitation identifies the T2 cells of HPV16-E7 peptides impact.Resist when with AntiCD3 McAb bispecific When body is equiped with arms, antibody redirects human T cells to kill the HPV16-E7 positives and HLA-A*02:01 positive target cell.Herein The data display of presentation can be that (such as entity swells cancer indication for the antibody of HPV16-E7 peptides in the case of HLA complexs Knurl indication) effective therapeutic agent.

Therefore the application is provided comprising the anti-of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid The construct of body portion (such as through separating construct).Construct may be, for example, anti-E7MC points of the anti-E7MC antibody of total length, polyspecific Sub (the anti-E7MC antibody of such as bispecific), anti-E7MC Chimeric antigen receptors (" CAR ") or anti-E7MC immunoconjugates.

In another aspect, there is provided encode the anti-E7MC antibody moieties part (anti-of anti-E7MC constructs or construct E7MC antibody moiety portion) nucleic acid.

In another aspect, there is provided include the composition of anti-E7MC constructs, the construct includes comprising specific binding The antibody moiety of HPV16-E7 peptides and the complex of MHC I proteinoid.Composition can be to include anti-E7MC constructs or expression Anti- E7MC constructs or the pharmaceutical composition of relative effector cell (such as expressing anti-E7MC CAR T cell).

Also provide for treatment or diagnostic purpose manufacture and using anti-E7MC constructs (the anti-E7MC constructs of expression or with Its related cell) method, and kit and product suitable for such method.

Definition

As used herein, " treatment (treatment/treating) " is (including to face for obtaining beneficial or desired result Bed result) method.For purposes of the present invention, it is beneficial or it is expected clinical effectiveness include but is not limited to it is following in one or It is multinomial：Alleviate one or more result from the symptom of disease, reduce disease degree, make stable disease (such as prevention or delay Disease progression), prevention or delay disease's spread (such as transfer), prevention or delay palindromia, postpone or slow down progression of disease, Improve disease state, remission (partially or completely) is provided, reduces one or more other drugs needed for treatment disease Dosage, delay progression of disease, increase or quality of making the life better, put on weight growth and/or extend survival period." treatment " is also covered The pathological consequences (such as gross tumor volume) of cancer reduce.The method of the present invention covers any one in these aspects for the treatment of It is or multinomial.

Term " recurrence (recurrence/relapse/relapsed) " refers to cancer or disease to be commented in the clinic of disappearance of disease Recovery after estimating.Distal end metastasis of cancer or the diagnosis of local recurrence can be considered recurrence.

Term " intractable " or " tolerance " refer to not in response to the cancer or disease for the treatment of.

Such as refer to fully stimulation in " activation " used herein on T cell and induce the T of detectable cell propagation thin Born of the same parents' state.Activation can also produce with the cytohormone of induction and to can detect effector function associated.

Term " antibody moiety " includes full length antibody and its antigen-binding fragment.Full length antibody includes two heavy chains and two Light chain.The variable region of light chain and heavy chain is responsible for antigen binding.Variable region in two kinds of chains is generally referred to as complementation containing three (light chain (LC) CDR includes LC-CDR1, LC-CDR2 and LC-CDR3, heavy chain (HC) CDR bags to the alterable height ring of decision area (CDR) Include HC-CDR1, HC-CDR2 and HC-CDR3).Antibody disclosed herein and the CDR borders of antigen-binding fragment can be by Kabat, Chothia or Al-Lazikani pact (Al-Lazikani 1997；Chothia 1985；Chothia 1987； Chothia 1989；Kabat 1987；Kabat 1991) limit or differentiate.Three CDR of heavy chain or light chain are inserted in referred to as frame Between the side joint elongated portion in frame area (FR), the side joint elongated portion is more highly conserved than CDR and forms the framework of support hypervariable loop. The constant region of heavy chain and light chain is not involved in antigen binding, but shows various effector functions.Constant region ammonia of the antibody based on its heavy chain Base acid sequence is distributed to various classifications.The antibody of five kinds of primary categories or homotype is IgA, IgD, IgE, IgG and IgM, its feature To be respectively present α, δ, ε, γ and μ heavy chain.Some main antibody classifications are divided into subclass, such as IgG1 (heavy chains of γ 1), the IgG2 (weights of γ 2 Chain), IgG3 (heavy chains of γ 3), IgG4 (heavy chains of γ 4), IgA1 (heavy chains of α 1) or IgA2 (heavy chains of α 2).

Term " antigen-binding fragment " as used herein refers to antibody fragment, including for example bifunctional antibody, Fab, Fab', F (ab') 2, Fv fragments, disulfide bond stability Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv'), two sulphur (divalence is difunctional anti-for key stability bifunctional antibody (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers Body), the multi-specificity antibody that is formed by a part for the antibody comprising one or more CDR, the single domain antibody of camel, nanometer resist Body, domain antibodies, divalence domain antibodies are bound to antigen but any other antibody fragment not comprising complete antibody structure.Antigen knot Conjunction fragment can be bound to combines identical antigen with parental antibody or parental antibody fragment (such as parent scFv).At some In embodiment, antigen-binding fragment can be included from one of specific human antibodies kind or a variety of CDR, and it is grafted to from one kind Or the framework region of a variety of different human antibodies.

Term " epitope " as used herein refers to specific atoms or amino on the antigen that antibody or antibody moiety are combined Sour group.If two antibody or antibody moiety show the competitive binding for antigen, it can combine the same epitope in antigen.

As used herein, when first antibody part suppresses secondary antibody in the presence of the first antibody part of equimolar concentration Partial target E7MC combine at least about 50% (such as at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%th, any one of 98% or 99%) when, first antibody part and secondary antibody part " competition " target E7MC is bound to, or Vice versa.The high throughput method of its " vanning " is described in by PCT Publication WO 03/48731 based on the cross competition of antibody In number.

As used herein, term " specific binding " or " right ... to have specificity " refer to measurable and reproducible phase Interaction, such as the combination between target and antibody or antibody moiety, it exists in heterogeneous molecular (including biomolecule) colony Make decision the presence of target.For example, specifically bind target (it can be epitope) antibody or antibody moiety for compared to The bigger affinity of other targets, affinity are bound to, be easier and/or the anti-of this target is combined with longer duration in it Body or antibody moiety.In some embodiments, the antibody of molecule of the antigen binding or antibody moiety are to be at least it for it The binding affinity that about 10 times of the binding affinity of his target and antigen (such as HPV16-E7 peptides/MHC I proteinoid is compound Body) one or more antigenic determinants (antigenic determinant) reaction.

As used herein " through separation " anti-E7MC constructs refer to (1) not with the albumen qualitative correlation found in nature, (2) Without other protein from identical source, (3) are expressed by the cell from different plant species, or (4) are not present in nature In anti-E7MC constructs.

Term as used herein is intended to mean genome, cDNA or synthesis source or some combinations " through seperated nuclear acid " Nucleic acid, by means of its source, " through seperated nuclear acid " (1) is not with being found in all in nature or one " through seperated nuclear acid " Polynucleotides are divided to be associated, (2) are operably connected in nature not connected polynucleotides, or (3) in nature In not as larger sequence part exist.

As used herein, term " CDR " or " complementary determining region " are intended to mean the variable region internal memory of heavy chain and light chain polypeptide Non-adjacent antigen combination site.These specific regions are by Kabat et al., J.Biol.Chem.252:6609-6616 (1977)；Kabat et al., U.S.'s health and Human Services (U.S.Dept.of Health and Human Services), “Sequences of proteins of immunological interest”(1991)；Chothia et al., J.Mol.Biol.196:901-917(1987)；And MacCallum et al., J.Mol.Biol.262:732-745 (1996) is retouched State, overlapping or subgroup when amino acid residue compares relative to each other is included defined in it.Nevertheless, come using any definition Refer to that the CDR of antibody or grafted antibodies or its variant is intended to belong to the category of the term as defined herein and used.Forgive with CDR amino acid residue is set forth in table 1 thing as a comparison as follows defined in each piece in upper cited bibliography.

Table 1：CDR is defined

¹Residue numbering follows the nomenclature of Kabat et al. (foregoing)

²Residue numbering follows the nomenclature of Chothia et al. (foregoing)

³Residue numbering follows the nomenclature of MacCallum et al. (foregoing)

Term " chimeric antibody " refers to following antibody：Wherein a part for heavy chain and/or light chain with derived from particular species or The corresponding sequence belonged in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain is another with being derived from One species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass are identical or homologous；And the piece of this antibody-like Section, as long as it shows the bioactivity of the present invention (referring to U.S. Patent No. 4,816,567；And Morrison et al., Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。

Mean that antibody or antibody moiety have one or more natural on the term of antibody or antibody moiety " semi-synthetic " Existing sequence and one or more non-naturally occurring (that is, synthesis) sequences.

" Fv " is the minimum antibody fragment containing intact antigen identification and binding site.This fragment is formed by close, non-covalent The heavy chain variable domain closed and the dimer composition of a light chain variable region.The folding in two domains sends six height since then Become ring (3 rings respectively from heavy chain and light chain), it promotes the antigen binding of amino acid residue and assigns antigen binding to antibody Specificity.However, even if single variable region (or half only comprising three specificity for the CDR of antigen Fv) can identify And with reference to antigen, but affinity is lower than whole binding site.

" scFv " (be also abbreviated as " sFv " or " scFv ") be comprising VH the and VL antibody domains being connected in Single polypeptide chain Antibody fragment.In some embodiments, scFv polypeptides further include polypeptide linker between VH and VL domains, and it causes ScFv can form the required structure for antigen binding.On scFv summary, referring to Pluckthun, The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore compile Springer- Verlag, New York, the 269-315 pages (1994).

Term " bifunctional antibody " refers to small antibody fragment, and it generally has short circuit head by being constructed between VH and VL domains The scFv fragments (referring to aforementioned paragraphs) of (such as from about 5 to about 10 residues) so that realize in the interchain rather than chain in V domains and match, production Raw bivalent fragment, i.e., fragment with two antigen binding sites and prepare.Bispecific diabodies are two " intersections " The heterodimer of scFv fragments, VH the and VL domains of two of which antibody are present on different polypeptide chains.Bifunctional antibody is more abundant It is described in such as EP404,097；WO 93/11161；And Hollinger et al., Proc.Natl.Acad.Sci.USA, 90: In 6444-6448 (1993).

" humanization " form of non-human (such as rodent) antibody is to contain the minmal sequence from non-human antibody Chimeric antibody.Largely, humanized antibody is human immunoglobulin (recipient's antibody), wherein from recipient The residue of hypervariable region (HVR) pass through from mouse, rat, the rabbit or non-such as with required specificity, affinity and ability The residue substitutions of the hypervariable region of non-human species' (donor antibody) of human primate.In some cases, human immunity Framework region (FR) residue substitutions of globulin are corresponding non-human residues.In addition, humanized antibody can be included in recipient's antibody Or undiscovered residue in donor antibody.These modifications are carried out further to improve antibody efficiency.In general, humanized antibody Generally whole at least one and usual two variable regions will be included, wherein whole or generally whole hypervariable loops are exempted from non-human Those regions of epidemic disease globulin are corresponding and all or those areas that generally whole FR are human immunoglobulin sequence.People source Changing antibody optionally will also include at least a portion of constant region for immunoglobulin (Fc), generally, the perseverance of human immunoglobulin Determine at least a portion in area.On other details, referring to Jones et al., Nature 321:522-525(1986)； Riechmann et al., Nature 332:323-329(1988)；And Presta, Curr.Op.Struct.Biol.2:593-596 (1992)。

On the " amino acid sequence identity percentage (%) " or " homologous of the polypeptide that differentiates herein and antibody sequence Property " be defined as after sequence alignment, in the case where considering any conservative replacement as a part for sequence identity, Hou Xuanxu In row with compared with polypeptide the consistent amino acid residue of amino acid residue percentage.For measure amino acid sequence identity Comparing for percentage purpose can be reached in a manner of various in the range of the technical ability in technique, such as using obtained by disclosure Computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE softwares.Those skilled in the art can It is determined that for measuring the suitable parameter compared, including high specific for reaching in the total length of comparative sequences is to required any Algorithm.However, for the purposes herein, compare computer program MUSCLE using sequence and produce amino acid sequence identity % values (Edgar,R.C.,Nucleic Acids Research 32(5):1792-1797,2004；Edgar,R.C.,BMC Bioinformatics 5(1):113,2004)。

Term " Fc acceptors " or " FcR " are used for the acceptor for describing to be bound to the Fc areas of antibody.In some embodiments, originally The FcR of invention be with reference to IgG antibody (γ acceptors) and including Fc γ RI, Fc γ RII and Fc γ RIII subclasses acceptor (including this The allele variant and Alternate splice forms of a little acceptors) FcR.Fc γ RII acceptors include Fc γ RIIA (" activated receptor ") And Fc γ RIIB (" suppression acceptor "), both have similar amino acid sequence mainly different in terms of its cytoplasmic domain.Activation Acceptor Fc γ RIIA contain the activation motifs (ITAM) based on immunity receptor tyrosine in its cytoplasmic domain.Suppress acceptor Fc γ RIIB in its cytoplasmic domain containing based on immunity receptor tyrosine suppression motif (ITIM) (referring to summary M., Annu.Rev.Immunol.15:203-234(1997)).The term includes allograft, such as Fc γ RIIIA allografts：Fc γ RIIIA-Phe158, Fc γ RIIIA-Val158, Fc γ RIIA-R131 and/or Fc γ RIIA-H131.FcR summarize in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92(1991)；Capel et al., Immunomethods 4:25- 34(1994)；And de Haas et al., J.Lab.Clin.Med.126:In 330-41 (1995).Other FcR include differentiating in the future FcR, by this paper terms " FcR " covers.The term also includes neonatal receptor FcRn, and it is responsible for conveying Maternal immunoglobulin G to fetus (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249(1994)).

Term " FcRn " refers to neonatal Fc receptor (FcRn).FcRn is similar in construction to major histocompatibility complex (MHC) and by the α chains of Non-covalent binding B2M form.Neonatal Fc receptor FcRn multiple functions comment in In Ghetie and Ward (2000) Annu.Rev.Immunol.18,739-766.FcRn exempts from passively being transmitted from parent to the young Worked in epidemic disease globulin IgG and serum IgG content regulation.FcRn may act as succouring acceptor, its in the cell with across cell With reference to and conveying complete form saved from default degradative pathway through pinocytosis IgG, and by it.

" CH1 domains " (" C1 " that is also known as " H1 " domain) in IgG Fc areas generally extends to about ammonia from about amino acid/11 18 Base acid 215 (EU numbering systems).

" hinge area " be normally defined from the Glu216 of IgG 1 extend to Pro230 (Burton, Molec.Immunol.22:161-206(1985)).The hinge area of other IgG homotypes can be by will form S -- S between heavy chain First and last cysteine residues be seated in same position and with IgG1 sequence alignments.

" CH2 domains " (" C2 " that is also known as " H2 " domain) in IgG Fc areas generally extends to about ammonia from about amino acid 231 Base acid 340.CH2 domains are unique in that it is not matched closely with another domain.In fact, two N- connection branched chain carbon hydrates Thing chain is inserted between two CH2 domains of intact native l: gG molecule.Speculate that carbohydrate can be provided and replaced for domain-domain pairing For product and CH2 domains are helped to stabilize.Burton,Molec Immunol.22:161-206(1985).

" CH3 domains " (be also known as " C2 " or " H3 " domain) includes residue C-terminal in Fc areas to CH2 domains (that is, antibody sequence About amino acid residue 341 is to C-terminal, generally at IgG amino acid residue 446 or 447) tract (stretch).

" feature Fc fragments " has " effector function " in native sequences Fc areas.Exemplary " effector function " is tied including C1q Close；Complement-dependent cytotoxicity (CDC)；Fc acceptors combine；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis Effect；Downward cell surface receptor (such as B-cell receptor；BCR) etc..Such effector function generally requires Fc areas and binding domain (such as antibody variable region) combines and various analysis and evaluations known in the art can be used.

The antibody of variant IgG Fc with " through changing " FcR binding affinities or ADCC activity is compared to parental polypeptide Or include the polypeptide in native sequences Fc areas, have enhancing or weaken FcR binding activity (such as Fc γ R or FcRn) and/or The antibody of ADCC activity.The variant Fc of " showing " and FcR " increase combines " is with compared to parental polypeptide or native sequences IgG Higher Fc affinity (such as relatively low apparent K_dOr IC50 values) combine at least one FcR.According to some embodiments, compared to The combination of parental polypeptide rise to about 3 times (such as from about 5,10,25,50,60,100,150,200 or at most any one of 500 times, Or about 25% to 1000%) combine and improve.The polypeptide variants of " showing " and FcR " reduce and combine " with compared to parental polypeptide compared with Low affinity (such as higher apparent K_dOr higher IC 50 is worth) combine at least one FcR.Combination compared to parental polypeptide subtracts The combination that can be about 40% or more than 40% less is reduced.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refers to some form of cytotoxicity, wherein secreted Ig be bound to and be present in some cytotoxic cells (such as natural killer (NK) cell, neutrophils and macrophage) On Fc acceptors (FcR) so that these cytotoxic effect cells can be specifically bound to carry antigen target cell and with Afterwards target cell is killed with cytotoxin.Antibody " arms " cytotoxic cell and killed for such as absolute demand.For Mediation ADCC primary cell NK cells only express Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Expression of the FcR on hematopoietic cell is summarized in Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 In the table 3 of page (1991) the 464th.In order to assess the ADCC activity of correlation molecule, external ADCC analyses can be carried out, such as U.S. is special Analysis described in profit No. 5,500,362 or No. 5,821,337.Include periphery blood suitable for the effector cell of this alanysis Monocyte (PBMC) and natural killer (NK) cell.Besides or furthermore, the ADCC activity of molecule of interest can be commented in vivo Estimate, such as be such as disclosed in Clynes et al. PNAS (USA) 95:In animal model in 652-656 (1998).

Comprising " show increased ADCC " or in the presence of mankind effector cell compare with wild type IgG Fc polypeptide or Effectively the polypeptide of the variant Fc regions of the cytotoxicity (ADCC) of mediate antibody dependent cell mediation is to analyze to parental polypeptide In the polypeptide with variant Fc regions it is substantially the same with the amount of the polypeptide (or parental polypeptide) with wild type Fc areas when it is external or Substantially more effective regulation ADCC polypeptide in vivo.In general, such variant will be using known in the art any external ADCC analysis and identifications, such as it is used for analysis or the method for determining ADCC activity, for example, it is medium in animal model.In some embodiments In, variant effectively adjusts ADCC than wild type Fc (or parental polypeptide) about 5 times to about 100 times (e.g., from about 25 to about 50 times).

" complement-dependent cytotoxicity " or " CDC " refers to target cell and cracked in the presence of complement.The work of classical complement pathway Change and be bound to the starting of (appropriate subclass) antibody by the first component of complement system (C1q), the antibody binding is homologous to its Antigen.In order to assess complement activation, CDC analyses, such as such as Gazzano-Santoro et al. can be carried out, J.Immunol.Methods 202:Described in 163 (1996).Fc region amino acid sequences with change and increase or decrease The polypeptide variants of C1q binding abilities are described in U.S. Patent No. 6,194,551B1 and WO99/51642.Those patents disclose The content of case is specifically incorporated to herein by reference.Also referring to Idusogie et al. J.Immunol.164:4178-4184 (2000)。

Unless specified otherwise herein, it is mutual degeneracy form and volume that otherwise " nucleotide sequence of encoding amino acid sequence ", which includes, All nucleotide sequences of the code-phase with amino acid sequence.Phrase encoding proteins matter or RNA nucleotide sequence also may include to include Son, its reach the nucleotide sequence of encoding proteins matter can in some patterns the degree containing introne.

Term " being operably connected " refers to the function key between regulatory sequence and heterologous nucleic acid sequence, and it causes the table of the latter Reach.For example, when the first nucleotide sequence and second nucleotide sequence are in functional relationship, the first nucleotide sequence is operationally Connect second nucleotide sequence.For example, if promoter influences the transcription or expression of coded sequence, promoter operationally connects It is connected to coded sequence.In general, the DNA sequence dna being operably connected is continuous, and compiled when two protein must be engaged During code area, in identical reading frame.

" homologous " refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules.When two When a position in comparative sequences is occupied by identical base or amino acid monomer subunit, if such as every in two DNA moleculars A position in one is occupied by adenine, then the molecule is homologous in the opening position.Homology % between two sequences The matching shared for two sequences or the quantity of homologous position divided by the quantity of institute's comparison position are multiplied by 100 function.Citing and Speech, if 6/10 location matches or homologous in two sequences, two sequences are 60% homologous.For example, DNA sequence dna ATTGCC and TATGGC shares 50% homology.In general, when two sequences it is aligned with produce largest percentage it is homologous when It is compared.

Anti- E7MC constructs or " effective dose " of composition as disclosed herein is to be sufficient for specific statement purpose Amount." effective dose " can be determined by rule of thumb and by the known method related to the purpose.

Term " therapeutically effective amount " refers to resisting as disclosed herein of disease in effective " treatment " individual or illness The amount of E7MC constructs or composition.In the case of cancer, anti-E7MC constructs or composition or group as herein disclosed The therapeutically effective amount of compound can reduce cancer cell count；Reduce tumor size or weight；Suppress (that is, slow down to a certain extent And preferably terminate) cancer cell infiltrated into peripheral organs；Suppress (that is, slow down to a certain extent or terminate) metastases； Suppress tumour growth to a certain extent；And/or one or more symptoms related to cancer are reduced to a certain extent.Such as originally Anti- E7MC constructs or composition disclosed in text can prevent to grow and/or kill in the degree of existing cancer cell, and it can be Cell growth inhibition and/or cytotoxicity.In some embodiments, therapeutically effective amount is growth amount of suppression.In some realities Apply in scheme, therapeutically effective amount is to extend the amount of patient's survival period.In some embodiments, therapeutically effective amount is improvement patient The progresson free survival phase amount.

As used herein, " pharmaceutically acceptable " or " pharmacologically compatible " means not as biologically or its other party The unfavorable material in face, that is, the material may be incorporated into any significantly improper without causing in the pharmaceutical composition using patient Biological action is interacted with harmful way and any other component containing its composition.Pharmaceutically acceptable Supporting agent or excipient preferably meet the required standard of toxicology and manufacture test and/or are included in Food and Drug Administration Non-active ingredient guiding (the Inactive Ingredient that (U.S.Food and Drug administration) is formulated Guide in).

Term " mark " refers to as used herein can directly or indirectly be bound to the detectable of anti-E7MC antibody moieties Compound or composition.Mark itself can detect (such as labelled with radioisotope or fluorescent labelling), or in enzyme mark In the case of, the chemical modification of substrate compounds or composition can be catalyzed, this changes into detectable.

It will be appreciated that invention as described herein embodiment includes " by " embodiment " composition " and/or " substantially by " Embodiment " composition ".

Include (and description) reference is made to " about " and be directed to the change of the value or parameter in itself.For example, " about X " is referred to Description include the description of " X ".

As used herein, refer to " not for " value or parameter typically mean and describe " an except " value or parameter " in addition to ".Lift For example, method be not used in treatment X-type cancer mean method be used for treat type in addition to X cancer.

Unless the context clearly, otherwise as used in herein and appended claim, odd number shape Formula " one (a/an) " and " be somebody's turn to do " include a plurality of indicants.

Anti- E7MC constructs

In one aspect, the present invention provides HPV16-E7/MHC I class complexs specific construct (anti-E7MC structures Body), it includes complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, and (" HPV16-E7/MHC I classes are answered It is fit " or " E7MC ") antibody moiety.Anti- E7MC antibody of the specificity of anti-E7MC constructs from specific binding E7MC Part, such as full length antibody or its antigen-binding fragment.In some embodiments, refer to that specific binding includes HPV16-E7 peptides And the part (such as antibody moiety) of the complex of MHCI proteinoid means that the part is bound to E7MC in the case where there：A) it is Its for total length HPV16-E7, free HPV16-E7 peptides, be not bound to the MHCI proteinoid of peptide and be bound to non-HPV16-E7 The binding affinity of each in the MHC I proteinoid of peptide at least about 10 (including for example, at least about 10,20,30,40, 50th, any of the above item in 75,100,200,300,400,500,750,1000 or 1000) times affinity；Or b) it is no more than It is bound to total length HPV16-E7, free HPV16-E7 peptides, is not bound to the MHC I proteinoid of peptide and is bound to non-HPV16- The K of each in the MHC I proteinoid of HPV-16 E7_dAbout 1/10 (such as no more than about 1/10,1/20,1/30,1/40,1/50,1/ 75th, any one of 1/100,1/200,1/300,1/400,1/500,1/750,1/1000 or less than 1/1000) K again_d.Knot Closing affinity can be by methods known in the art, such as ELIS fluorescence activated cell sorts (FACS) analysis or radioimmunoprecipitation Analyze (RIA) measure.K_dThe surface plasma resonance of such as Biacore instruments can be such as utilized by methods known in the art (SPR) analyze, or determined using the dynamics Exclusion analysis (KinExA) of such as Sapidyne instruments.

Expected anti-E7MC constructs include the anti-E7MC antibody of such as total length, polyspecific (such as bispecific) anti-E7MC point Sub, anti-E7MC Chimeric antigen receptors (CAR) and anti-E7MC immunoconjugates.

For example, in some embodiments, there is provided anti-E7MC constructs (such as the anti-E7MC constructs of separation), it is wrapped The anti-E7MC antibody moieties of the complex comprising HPV16-E7 peptides and MHC I proteinoid containing specific binding.In some implementations In scheme, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid For HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01 (Genbank accession number： AAO20853).In some embodiments, anti-E7MC constructs are non-naturally occurring.In some embodiments, anti-E7MC Construct is full length antibody.In some embodiments, anti-E7MC constructs are polyspecific (such as bispecific) molecule.One In a little embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments, anti-E7MC constructs are sewed to be immune Compound.In some embodiments, anti-E7MC constructs combination E7MC, with reference to K_dAbout 0.1pM between about 500nM (such as About any one of 0.1pM, 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including Any scope between these values).In some embodiments, anti-E7MC constructs with it is at least one (such as at least 2,3,4,5 or Any one of 6) include MHC I proteinoid and the HPV16- with a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution) The complex cross reaction of the variant of HPV-16 E7.In some embodiments, anti-E7MC constructs with it is at least one (such as at least 2,3, Any one of 4 or 5) the complex cross reaction of the different subtype comprising HPV16-E7 peptides and MHC I proteinoid.

In some embodiments, there is provided anti-E7MC constructs, it includes specific binding and includes HPV16-E7 11-19 Peptide (SEQ ID NO:And HLA-A*02 4):The anti-E7MC antibody moieties of 01 complex.In some embodiments, anti-E7MC Construct is non-naturally occurring.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments In, anti-E7MC constructs are polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are chimeric Antigen receptor.In some embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, anti-E7MC structures Build body combination E7MC, with reference to K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, Any scope between any one of 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values).In some realities Apply in scheme, anti-E7MC constructs include MHC I albuminoids with least one (such as any one of at least 2,3,4,5 or 6) The complex cross reaction of the variant of matter and HPV16-E7 peptides with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution). In some embodiments, anti-E7MC constructs include HPV16-E7 with least one (such as any one of at least 2,3,4 or 5) The complex cross reaction of peptide and the different subtype of MHC I proteinoid.

In some embodiments, there is provided include following anti-E7MC constructs：Specific binding includes HPV16-E7 peptides And the anti-E7MC antibody moieties of the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC-CDR1, should HC-CDR1 includes SEQ ID NO:183 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) The variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, or its bag Variant containing at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 are included SEQ ID NO:Any one of 186-188 amino acid sequence, or it includes at most about 3 (any in e.g., from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:189 Or 190 amino acid sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, And LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.In some embodiments, anti-E7MC constructs are non-naturally occurring. In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments In, anti-E7MC constructs are immunoconjugates.In some embodiments, anti-E7MC constructs combination E7MC, with reference to K_d About 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, Any scope between any one of 100nM or 500nM, including these values).In some embodiments, anti-E7MC structures Body takes with least one (such as any one of at least 2,3,4,5 or 6) comprising MHC I proteinoid and with an amino acid The complex cross reaction of the variant of the HPV16-E7 peptides in generation (such as conserved amino acid substitution).In some embodiments, resist E7MC constructs include HPV16-E7 peptides and MHC I proteinoid with least one (such as any one of at least 2,3,4 or 5) Different subtype complex cross reaction.

In some embodiments, there is provided include following anti-E7MC constructs：Specific binding includes HPV16-E7 peptides And the anti-E7MC antibody moieties of the complex of MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variable district sequence Row, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence is (and at some It is made from it in embodiment)；Or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence (and some implementation It is made from it in scheme)；Or it includes the change of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Body；And HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 99-119,244 and 245 amino acid sequence (and It is made from it in some embodiments)；Or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual amino acid Substituted variant；And ii) light-chain variable sequence, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:120-140 and Any one of 246 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 5 (e.g., from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；LC-CDR2, the LC-CDR2 include SEQ ID NO:141- Any one of 161 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 3 (e.g., from about 1st, any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.In some embodiments, anti-E7MC constructs To be non-naturally occurring.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, resist E7MC constructs are polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are chimeric antigen Acceptor.In some embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, anti-E7MC constructs With reference to E7MC, with reference to K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, Any scope between any one of 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values).In some realities Apply in scheme, anti-E7MC constructs include MHC I albuminoids with least one (such as any one of at least 2,3,4,5 or 6) The complex cross reaction of the variant of matter and HPV16-E7 peptides with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution). In some embodiments, anti-E7MC constructs include HPV16-E7 with least one (such as any one of at least 2,3,4 or 5) The complex cross reaction of peptide and the different subtype of MHC I proteinoid.

In some embodiments, there is provided include following anti-E7MC constructs：Specific binding includes HPV16-E7 peptides And the anti-E7MC antibody moieties of the complex of MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variable district sequence Row, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence is (and at some It is made from it in embodiment)；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence Arrange (and being made from it in some embodiments)；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119,244 and Any one of 245 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 5 (e.g., from about 1st, any one of 2,3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, its SEQ ID NO are included comprising LC-CDR1, the LC-CDR1:Any one of 120-140 and 246 amino acid sequence is (and one It is made from it in a little embodiments)；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino Acid sequence (and being made from it in some embodiments)；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and Any one of 247-250 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 5 (examples Any one of such as from about 1,2,3,4 or 5) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences variant.In some embodiments, resist E7MC constructs are non-naturally occurring.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiment party In case, anti-E7MC constructs are polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are embedding Close antigen receptor.In some embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, anti-E7MC Construct combination E7MC, with reference to K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10pM, 50pM, Any scope between any one of 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values). In some embodiments, anti-E7MC constructs include MHC I with least one (such as any one of at least 2,3,4,5 or 6) The complex of the variant of proteinoid and HPV16-E7 peptides with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution) intersects Reaction.In some embodiments, anti-E7MC constructs include with least one (such as any one of at least 2,3,4 or 5) The complex cross reaction of HPV16-E7 peptides and the different subtype of MHC I proteinoid.

In some embodiments, there is provided include following anti-E7MC constructs：Specific binding includes HPV16-E7 peptides And the anti-E7MC antibody moieties of the complex of MHC I proteinoid, its moderate resistance E7MC antibody moieties include：Weight chain variable district, its Include SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence is (and in some embodiments by its group Into), or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity Variant, and light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence is (and one It is made from it in a little embodiments), or it had at least about 95% (appointing in for example, at least about 96%, 97%, 98% or 99% One) variant of sequence identity.In some embodiments, anti-E7MC constructs are non-naturally occurring.In some implementations In scheme, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are polyspecific (such as double spies The opposite sex) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments, anti-E7MC Construct is immunoconjugates.In some embodiments, anti-E7MC constructs combination E7MC, with reference to K_dIn about 0.1pM extremely (such as from about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM between about 500nM Any one of, including any scope between these values).In some embodiments, anti-E7MC constructs with it is at least one (such as any one of at least 2,3,4,5 or 6) is comprising MHC I proteinoid and with a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conservative ammonia Base acid substitution) HPV16-E7 peptides variant complex cross reaction.In some embodiments, anti-E7MC constructs with extremely Few one different subtype of (such as any one of at least 2,3,4 or 5) comprising HPV16-E7 peptides and MHC I proteinoid is answered Fit cross reaction.

In some embodiments, there is provided anti-E7MC constructs, its include with according to anti-E7MC antibody portion as described herein Point any one of the second anti-E7MC antibody moieties competition binding it is first anti-to target HPV16-E7/MHC I class complexs E7MC antibody moieties.In some embodiments, the first anti-E7MC antibody moieties are bound to phase with the second anti-E7MC antibody moieties Together, or substantially the same epitope.In some embodiments, the first anti-E7MC antibody moieties and target HPV16-E7/MHC I classes The combination of complex suppresses the combination at least about 70% of the second anti-E7MC antibody moieties and target HPV16-E7/MHC I class complexs (as any one of at least about 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa it is as the same.In some implementations In scheme, the first anti-E7MC antibody moieties and the second anti-E7MC antibody moieties cross competition are bound to target HPV16-E7/MHC I classes Each in complex, i.e. first and second antibody moiety, which contends with one other, is bound to target HPV16-E7/MHC I class complexs.

For example, in some embodiments, there is provided anti-E7MC constructs, it is included and antibody moiety competition binding to target The anti-E7MC antibody moieties of HPV16-E7/MHC I class complexs, wherein antibody moiety include：I) weight chain variabl area sequence, it is wrapped SEQ ID NO are included containing HC-CDR1, the HC-CDR1:183 amino acid sequence, or include at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, should HC-CDR3 includes SEQ ID NO:Any one of 186-188 amino acid；Or comprising at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant；And ii) light-chain variable sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or include at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or comprising at most about 3 (e.g., from about 1st, any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, there is provided anti-E7MC constructs, it is included and antibody moiety competition binding to target HPV16- The anti-E7MC antibody moieties of E7/MHC I class complexs, wherein antibody moiety include：I) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence (and in some embodiments by Composition), or the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors is included, HC-CDR2 should HC-CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence (and being made from it in some embodiments)； , should or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3 HC-CDR3 includes SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 is (and in some embodiments It is made from it)；Or include the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light Chain variable region sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 ammonia Base acid sequence (and being made from it in some embodiments), or include at most about 5 (any in e.g., from about 1,2,3,4 or 5 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino Acid sequence (and being made from it in some embodiments), or include at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182's and 247-250 Amino acid sequence (and being made from it in some embodiments), or include at most about 5 (any in e.g., from about 1,2,3,4 or 5 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, there is provided anti-E7MC constructs, it is included and antibody moiety competition binding to target HPV16- The anti-E7MC antibody moieties of E7/MHC I class complexs, wherein antibody moiety include：I) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence (and in some embodiments by It is formed)；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence is (and in some realities Apply in scheme and be made from it)；And HC-CDR3, the HC-CDR3 include SEQ ID NO:Any in 99-119,244 and 245 The amino acid sequence (and being made from it in some embodiments) of item；Or comprising at most about 5 (in e.g., from about 1,2,3,4 or 5 Any one) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences variant；And ii) light-chain variable sequence, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence (and in some embodiments by It is formed)；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence is (and at some It is made from it in embodiment)；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any in 162-182 and 247-250 The amino acid sequence (and being made from it in some embodiments) of item, or comprising at most about 5 (in e.g., from about 1,2,3,4 or 5 Any one) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences variant.

In some embodiments, there is provided anti-E7MC constructs, it is included and antibody moiety competition binding to target HPV16- The anti-E7MC antibody moieties of E7/MHC I class complexs, wherein antibody moiety include：I) weight chain variable district weight chain variable district, it is wrapped The NO of ID containing SEQ:Any one of 15-35 and 233-237 amino acid sequence (and being made from it in some embodiments), Or it has the variant of at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity, And light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence is (and in some realities Apply in scheme and be made from it), or it has at least about 95% (any in for example, at least about 96%, 97%, 98% or 99% ) variant of sequence identity.

Different aspect is discussed in more detail in hereafter each several part.

Anti- E7MC antibody moieties

Anti- E7MC constructs include the anti-of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid E7MC antibody moieties.

In some embodiments, the E7MC that anti-E7MC antibody moieties specific binding is present on cell surface.One In a little embodiments, the cell is cancer cell.In some embodiments, cancerous cell line is in entity tumor.In some implementations In scheme, cancer cell is metastatic carcinoma cell.

In some embodiments, HPV16-E7 peptides are that MHC I classes limit peptide.In some embodiments, HPV16-E7 The length of peptide is about 8 to about 12 (any one of such as from about 8,9,10,11 or 12) individual amino acid.

In some embodiments, HPV16-E7 peptides include the sequence of the following (and in some embodiments by it Composition)：HPV16-E7 amino acid 7-15 (TLHEYMLDL, SEQ ID NO:3), HPV16-E7 amino acid/11 1-19 (YMLDLQPET, SEQ ID NO:4, herein be also known as " HPV16-E7 11-19 "), HPV16-E7 amino acid/11 6-25 (QPETTDLYCY, SEQ ID NO:5), HPV16-E7 amino acid 44-52 (QAEPDRAHY, SEQ ID NO:6)、HPV16- E7 amino acid 46-55 (EPDRAHYNIV, SEQ ID NO:7), HPV16-E7 amino acid 49-57 (RAHYNIVTF, SEQ ID NO:8), HPV16-E7 amino acid 82-90 (LLMGTLGIV, SEQ ID NO:9), or HPV16-E7 amino acid 86-93 (TLGIVCPI, SEQ ID NO:10)

In some embodiments, MHC I proteinoid is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. In some embodiments, MHC I proteinoid is HLA-A.In some embodiments, HLA-A HLA-A02.At some In embodiment, HLA-A02 HLA-A*02:01.

In some embodiments, anti-E7MC antibody moieties are full length antibody.In some embodiments, anti-E7MC antibody Part is antigen-binding fragment, is selected from the antigen-binding fragment of group consisted of：Fab, Fab', F (ab') 2, Fv pieces Section, disulfide bond stability Fv fragments (dsFv) and single-chain antibody molecules (scFv).In some embodiments, anti-E7MC antibody portion It is divided into scFv.In some embodiments, anti-E7MC antibody moieties are the mankind, humanization or semi-synthetic.

In some embodiments, the N-terminal portion of the HPV16-E7 peptides in anti-E7MC antibody moieties specific binding complex Point.In some embodiments, the C-terminal part of the HPV16-E7 peptides in anti-E7MC antibody moieties specific binding complex. In some embodiments, the center section of the HPV16-E7 peptides in anti-E7MC antibody moieties specific binding complex.

In some embodiments, anti-E7MC antibody moieties (or anti-E7MC constructs comprising anti-E7MC antibody moieties) knot It is bonded to the complex comprising HPV16-E7 peptides and MHC I proteinoid, binding affinity is it for total length HPV16-E7, free HPV16-E7 peptides, the MHC I proteinoid for not being bound to peptide and it is bound to every in the MHC I proteinoid of non-HPV16-E7 peptides A kind of binding affinity at least about 10 (including for example, at least about 10,20,30,40,50,75,100,200,300,400, 500th, any of the above item in 750,1000 or 1000) times.In some embodiments, anti-E7MC antibody moieties are (or comprising anti- The anti-E7MC constructs of E7MC antibody moieties) it is bound to the complex comprising HPV16-E7 peptides and MHC I proteinoid, K_dDo not surpass It is crossed to be bound to total length HPV16-E7, free HPV16-E7 peptides, be not bound to the MHC I proteinoid of peptide and be bound to non- The K of each in the MHC I proteinoid of HPV16-E7 peptides_dAbout 1/10 (such as no more than about 1/10,1/20,1/30,1/ 40th, any in 1/50,1/75,1/100,1/200,1/300,1/400,1/500,1/750,1/1000 or less than 1/1000 ) times.

In some embodiments, anti-E7MC antibody moieties (or anti-E7MC constructs comprising anti-E7MC antibody moieties) knot It is bonded to the complex comprising HPV16-E7 peptides and MHC I proteinoid, K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, Any one of 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values it Between any scope).In some embodiments, anti-E7MC antibody moieties (or anti-E7MC structures comprising anti-E7MC antibody moieties Build body) it is bound to the complex comprising HPV16-E7 peptides and MHC I proteinoid, K_dAbout 1pM between about 250pM (such as from about 1st, any scope between any one of 10,25,50,75,100,150,200 or 250pM, including these values).In some realities Apply in scheme, anti-E7MC antibody moieties (or anti-E7MC constructs comprising anti-E7MC antibody moieties) are bound to comprising HPV16-E7 The complex of peptide and MHC I proteinoid, K_dAbout 1nM between about 500nM (such as from about 1,10,25,50,75,100,150, 200th, any scope between any one of 250,300,350,400,450 or 500nM, including these values).

In some embodiments, anti-E7MC antibody moieties specific binding includes HPV16-E7 peptides and MHC I albuminoids The complex of matter, its moderate resistance E7MC antibody moieties and at least one equipotential base comprising HPV16-E7 peptides and MHC I proteinoid Because of the complex cross reaction of variant.In some embodiments, when compared to MHC I proteinoid, allele variant With at most about 10 (any one of such as from about 1,2,3,4,5,6,7,8,9 or 10) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.In some embodiments In, allele variant is and MHC I proteinoid identical serotypes.In some embodiments, allele variant is The serotype different from MHC I proteinoid.In some embodiments, anti-E7MC antibody moieties are not with including HPV16-E7 peptides And the complex cross reaction of any allele variant of MHC I proteinoid.In some embodiments, anti-E7MC antibody Part and at least one difference of (such as any one of at least 2,3,4 or 5) comprising HPV16-E7 peptides and MHC I proteinoid The complex cross reaction of hypotype.

In some embodiments, anti-E7MC antibody moieties specific binding includes HPV16-E7 peptides and MHC I albuminoids The complex of matter, its moderate resistance E7MC antibody moieties comprising MHC I proteinoid and have a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor with least one The complex cross reaction of the variant of the HPV16-E7 peptides of (such as conserved amino acid substitution).In some embodiments, anti-E7MC The antibody moiety not complex cross reaction with the variant comprising MHC I proteinoid and HPV16-E7 peptides.

For example, in some embodiments, anti-E7MC antibody moieties specific binding includes HPV16-E711-19 (SEQ ID NO:And MHC I proteinoid (such as HLA-A02, such as HLA-A*02 4):01) complex.In some embodiments, Anti- E7MC antibody moieties are in addition combined with least one in the following (including any case at least about 2 or 3)：Comprising SEQ ID NO:The HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02 of 12 alanine substitution: 01) complex；Include SEQ ID NO:The HPV16-E7 peptides and MHC I proteinoid (such as HLA- of 13 alanine substitution A02, such as HLA-A*02:01) complex；And include SEQ ID NO:14 alanine substitution HPV16-E7 peptides and MHC I Proteinoid (such as HLA-A02, such as HLA-A*02:01) complex.

In some embodiments, anti-E7MC antibody moieties specific binding：Include SEQ ID NO:4 HPV16-E7 peptides And MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex；Include SEQ ID NO:12 alanine takes The HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02 in generation:01) complex；And include SEQ ID NO:The HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02 of 13 alanine substitution:01) compound Body.

In some embodiments, anti-E7MC antibody moieties specific binding：Include SEQ ID NO:4 HPV16-E7 peptides And MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex；Include SEQ ID NO:12 alanine takes The HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02 in generation:01) complex；And include SEQ ID NO:The HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02 of 14 alanine substitution:01) compound Body.

In some embodiments, anti-E7MC antibody moieties specific binding：Include SEQ ID NO:4 HPV16-E7 peptides And MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex；And include SEQ ID NO:12 alanine Substituted HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex.

In some embodiments, anti-E7MC antibody moieties specific binding：Include SEQ ID NO:4 HPV16-E7 peptides And MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex；And include SEQ ID NO:14 alanine Substituted HPV16-E7 peptides and MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex.

In some embodiments, anti-E7MC antibody moieties specific binding includes HPV16-E7 11-19 (SEQ ID NO:And HLA-A*02 4):01 complex.In some embodiments, anti-E7MC antibody moieties and at least one in the following (including any case at least about 2,3,4,5 or 6) cross reaction：Include HPV16-E7 11-19 (SEQ ID NO:4) And HLA-A*02:02 (GenBank accession number：AFL91480 complex), HPV16-E7 11-19 (SEQ ID NO are included:4) And HLA-A*02:03 (GenBank accession number：AAA03604 complex), HPV16-E7 11-19 (SEQ ID NO are included:4) And HLA-A*02:05 (GenBank accession number：AAA03603 complex), HPV16-E7 11-19 (SEQ ID NO are included:4) And HLA-A*02:06 (GenBank accession number：CCB78868 complex), HPV16-E7 11-19 (SEQ ID NO are included:4) And HLA-A*02:07 (GenBank accession number：ACR55712 complex), and include HPV16-E7 11-19 (SEQ ID NO: And HLA-A*02 4):11 (GenBank accession number：CAB56609 complex).

In some embodiments, anti-E7MC antibody moieties are specifically bound to：Include HPV16-E711-19 (SEQ ID NO:4) and MHC I proteinoid complex (such as HLA-A02, such as HLA-A*02:01), and comprising with YMLDVQPET's HPV16-E7 11-19 variants (the SEQ ID NO of amino acid sequence:11) and MHC I proteinoid (such as HLA-A02, such as HLA-A*02:01) complex.

In some embodiments, anti-E7MC antibody moieties are half comprising whole mankind's sequence and one or more synthesis zones Synthetic antibody part.In some embodiments, anti-E7MC antibody moieties are comprising whole mankind's light chain variable district and semi-synthetic heavy The semi-synthetic antibody moiety of chain variable region, the semi-synthetic weight chain variable district include whole mankind FR1, HC-CDR1, FR2, HC-CDR2, FR3 and FR4 areas and synthesis HC-CDR3.In some embodiments, semi-synthetic weight chain variable district includes fully synthetic HC-CDR3, its With length be about 5 to about 25 (such as from about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23, Any one of 24 or 25) sequence of individual amino acid.In some embodiments, semi-synthetic weight chain variable district or synthesis HC- CDR3 is obtained from semi-synthetic storehouse (such as semi-synthetic people's class libraries), its include have length be about 5 to about 25 (such as from about 5,6,7,8,9,10, 11st, any one of 12,13,14,15,16,17,18,19,20,21,22,23,24 or 25) the full conjunction of the sequence of individual amino acid Into HC-CDR3, each amino acid wherein in sequence is selected from standard human's amino acid, subtracts cysteine at random.In some implementations In scheme, the length for synthesizing HC-CDR3 is that about 7 to about 15 (any one of such as from about 7,8,9,10,11,12,13,14 or 15) are individual Amino acid.

In some embodiments, anti-E7MC antibody moieties include particular sequence or some variants of such sequence.One In a little embodiments, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in series of variation does not weaken anti-E7MC antibody moieties combination E7MC ability generally. For example, the change of E7MC binding affinities can not generally be weakened.Also cover and generally improve E7MC with reference to affine Power influences some other characteristics, such as specificity and/or the change with the cross reactivity of E7MC related variants.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or or its include at most about 3 (e.g., from about 1,2 or Any one of 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR3, and the LC-CDR3 includes SEQ ID NO:191 amino acid sequence, or it includes the change of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Body.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence；And ii) light chain variable district, it includes LC- CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:183 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, or its include extremely The variant of more about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 Amino acid sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and LC- CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:183 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, or its include extremely The variant of more about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 is included SEQ ID NO:Any one of 189 or 190 amino acid sequence, or it includes at most about 3 (any in e.g., from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 ammonia Base acid sequence, and HC-CDR3, the HC-CDR3 include NO:Any one of 186-188 amino acid sequence；Or it includes HC- The variant of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors in CDR sequence；And ii) light chain variable district, its Comprising LC-CDR1, the LC-CDR1 includes 189 or 190 amino acid sequence, and LC-CDR3, and the LC-CDR3 includes SEQ ID NO:191 amino acid sequence；Or it includes at most about 3 (any one of such as from about 1,2 or 3) individual amino in LC-CDR sequences The variant of acid substitution.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 ammonia Base acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino acid sequence；And ii) Light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, and LC- CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence.The sequence of CDR mentioned by this paper is provided in table 2 below In.

Table 2

HC-CDR1 consensus sequences	SEQ ID NO:183	G-F/G/Y-S/T-F-S/T-S-Y-A/G
			HC-CDR2 consensus sequences 1	SEQ ID NO:184	I-N/I-P-X-X-G-G/T/I-T/A/P
HC-CDR2 consensus sequences 2	SEQ ID NO:185	I-S-X-S/D-G/N-G/S-N-T/I/K
			HC-CDR3 consensus sequences 1	SEQ ID NO:186	A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D
HC-CDR3 consensus sequences 2	SEQ ID NO:187	A-R-G-X-X-X-Y-Y/G/S
			HC-CDR3 consensus sequences 3	SEQ ID NO:188	A-R-G-X-X-Y-Q/W-W-S-X-D-D
LC-CDR1 consensus sequences 1	SEQ ID NO:189	N-I-G-S-N/K
			LC-CDR1 consensus sequences 2	SEQ ID NO:190	L-R-S/N-X-Y
LC-CDR3 consensus sequences	SEQ ID NO:191	A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR3, the HC- CDR3 includes SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR3, the LC- CDR3 includes SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR3, the HC- CDR3 includes any one of 99-119,244 and 245 amino acid sequence；And ii) light chain variable district, it includes LC-CD3, bag The NO of ID containing SEQ:Any one of 162-182 and 247-250 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 Amino acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245, or it includes The variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable district, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or its include at most about The variant of 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 include SEQ ID NO: Any one of 141-161 amino acid sequence, or it takes comprising at most about 3 (any one of such as from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid Sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variable district, and it includes HC-CDR1, the HC- CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 Amino acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light Chain variable region, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid Sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2 should LC-CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence, or comprising at most about 3 (in 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:In 162-182 and 247-250 Any one amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variabl area sequence, and it includes HC-CDR1, should HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119,244 and Any one of 245 amino acid sequence；Or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual HC-CDR sequences The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in row；And ii) light-chain variable sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:141-161 Any one of amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:In 162-182 and 247-250 The amino acid sequence of any one；Or it includes the amino in about 5 (any one of such as from about 1,2,3,4 or 5) individual LC-CDR sequences The variant of acid substitution.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variabl area sequence, and it includes HC-CDR1, should HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119,244 and Any one of 245 amino acid sequence；Or it takes comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid The variant in generation, wherein 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor are in HC-CDR1 or HC-CDR2；And ii) light-chain variable sequence, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC- CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；Or it includes at most about 5 (in such as from about 1,2,3,4 or 5 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, wherein 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is in HC-CDR1 or HC-CDR2.

In some embodiments, anti-E7MC antibody moieties include i) weight chain variabl area sequence, and it includes HC-CDR1, should HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119,244 and Any one of 245 amino acid sequence；And ii) light-chain variable sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:141- Any one of 161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:In 162-182 and 247-250 Any one amino acid sequence.HC-CDR mentioned in this article sequence is provided in table 3 below and LC- mentioned in this article CDR is provided in table 4 below.

Table 3

Table 4

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:15-35 and Any one of 233-237 amino acid sequence, or it has at least about 95% (including for example, at least about 96%, 97%, 98% Or any one of 99%) variant of sequence identity, and light chain variable district, it includes SEQ ID NO:36-56 and 238-243 Any one of amino acid sequence, or it has at least about 95% (including in for example, at least 96%, 97%, 98% or 99% Any one) sequence identity variant.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:15-35 and Any one of 233-237 amino acid sequence, and light chain variable district, it includes SEQ ID NO:In 36-56 and 238-243 The amino acid sequence of any one.

Heavy chain and light chain variable district can be combined to produce a variety of anti-E7MC antibody moieties with various pair-wise combinations.

For example, in some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, The HC-CDR1 includes SEQ ID NO:57 amino acid sequence, or its include at most about 5 (in e.g., from about 1,2,3,4 or 5 appoint One) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；HC-CDR2, the HC-CDR2 include SEQ ID NO:78 amino acid sequence, or its bag Variant containing at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And HC-CDR3, the HC-CDR3 bags The NO of ID containing SEQ:99 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual amino The variant of acid substitution；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:120 amino acid sequence, Or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC- CDR2 includes SEQ ID NO:141 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162 amino acid sequence, or its include at most The variant of about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:57 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:78 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:99 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:120 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:141 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:162 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:57 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:78 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:99 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:120 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:141 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:162 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:58 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:79 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:100 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:121 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:142 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:163 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:58 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:79 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:100 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:121 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:142 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:163 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:58 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:79 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:100 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:121 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:142 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:163 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:59 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:80 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:101 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:122 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:143 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:164 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:59 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:80 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:101 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:122 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:143 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:164 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:59 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:80 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:101 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:122 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:143 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:164 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:60 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:81 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:102 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:123 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:144 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:165 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:60 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:81 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:102 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:123 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:144 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:165 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:60 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:81 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:102 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:123 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:144 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:165 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:61 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:82 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:103 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:124 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:145 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:166 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:61 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:82 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:103 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:124 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:145 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:166 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:61 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:82 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:103 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:124 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:145 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:166 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:62 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:83 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:104 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:125 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:146 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:167 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:62 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:83 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:104 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:125 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:146 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:167 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:62 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:83 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:104 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:125 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:146 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:167 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:63 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:84 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:105 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:126 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:147 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:168 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:63 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:84 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:105 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:126 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:147 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:168 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:63 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:84 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:105 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:126 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:147 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:168 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:64 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:85 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:106 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:127 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:148 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:169 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:64 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:85 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:106 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:127 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:148 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:169 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:64 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:85 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:106 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:127 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:148 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:169 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:65 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:86 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:107 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:128 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:149 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:170 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:65 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:86 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:107 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:128 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:149 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:170 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:65 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:86 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:107 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:128 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:149 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:170 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:66 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:87 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:108 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:129 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:150 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:171 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:66 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:87 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:108 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:129 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:150 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:171 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:66 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:87 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:108 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:129 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:150 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:171 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:67 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:88 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:109 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:130 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:151 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:172 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:67 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:88 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:109 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:130 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:151 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:172 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:67 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:88 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:109 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:130 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:151 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:172 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:68 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:89 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:110 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:131 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:152 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:173 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:68 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:89 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:110 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:131 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:152 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:173 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:68 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:89 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:110 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:131 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:152 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:173 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:69 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:90 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:111 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:132 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:153 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:174 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:69 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:90 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:111 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:132 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:153 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:174 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:69 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:90 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:111 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:132 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:153 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:174 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:70 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:91 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:112 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:133 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:154 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:175 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:70 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:91 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:112 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:133 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:154 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:175 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:70 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:91 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:112 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:133 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:154 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:175 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:71 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:92 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:113 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:134 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:155 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:176 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:71 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:92 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:113 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:134 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:155 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:176 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:71 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:92 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:113 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:134 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:155 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:176 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:72 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:93 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:114 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:135 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:156 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:177 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:72 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:93 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:114 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:135 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:156 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:177 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:72 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:93 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:114 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:135 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:156 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:177 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:73 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:94 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:115 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:136 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:157 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:178 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:73 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:94 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:115 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:136 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:157 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:178 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:73 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:94 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:115 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:136 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:157 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:178 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:74 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:95 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:116 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:137 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:158 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:179 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:74 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:95 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:116 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:137 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:158 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:179 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:74 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:95 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:116 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:137 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:158 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:179 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:75 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:96 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:117 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:138 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:159 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:180 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:75 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:96 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:117 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:138 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:159 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:180 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:75 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:96 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:117 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:138 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:159 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:180 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:76 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:97 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:118 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:139 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:160 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:181 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:76 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:97 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:118 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:139 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:160 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:181 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:76 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:97 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:118 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:139 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:160 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:181 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:244 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:244 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:244 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:245 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:245 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:245 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:247 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:247 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:247 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:248 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:248 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:248 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:246 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:246 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:246 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:182 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:249 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:249 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:249 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual ammonia The variant of base acid substitution；HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, or its include at most about 5 The variant of (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And light chain variable district, include SEQ ID NO comprising LC-CDR1, the LC-CDR1:140 amino acid sequence, or it includes The variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 are included SEQ ID NO:161 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant；And LC-CDR3, the LC-CDR3 include SEQ ID NO:250 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence, or it includes at most about 5 (such as from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And light chain variable district, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:250 amino acid sequence, or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes HC-CDR1, the HC-CDR1 Include SEQ ID NO:77 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:98 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:119 amino acid sequence；And light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:140 amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:161 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:250 amino acid sequence.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 15 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 36, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 15 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 36.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 16 The amino acid sequence of elaboration, or its have at least about 95% (including in for example, at least about 96%, 97%, 98% or 99% appoint One) variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 37, or It has the change of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity Body.In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The ammonia illustrated in 16 Base acid sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 37.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 17 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 38, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 17 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 38.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 18 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 39, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 18 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 39.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 19 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 40, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 19 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 40.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 20 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 41, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 20 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 41.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 21 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 42, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 21 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 42.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 22 The amino acid sequence of elaboration, or its have at least about 95% (including in for example, at least about 96%, 97%, 98% or 99% appoint One) variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 43, or It has the change of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity Body.In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The ammonia illustrated in 22 Base acid sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 43.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 23 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 44, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 23 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 44.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 24 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 45, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 24 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 45.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 25 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 46, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 25 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 46.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 26 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 47, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 26 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 47.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 27 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 48, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 27 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 48.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 28 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 49, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 28 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 49.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 29 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 50, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 29 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 50.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 30 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 51, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 30 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 51.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 31 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 52, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 31 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 52.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 32 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 53, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 32 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 53.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 33 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 54, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 33 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 54.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 34 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 55, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 34 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 55.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 56, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 35 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 56.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 233 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 56, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 233 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 56.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 234 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 56, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 234 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 56.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 235 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 56, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 235 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 56.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 238, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 35 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 238.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 239, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 35 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 239.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 240, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 35 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 240.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 241, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 35 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 241.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 35 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 242, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 35 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 242.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 236 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 243, or its tool There is the variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity. In some embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid illustrated in 236 Sequence, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 243.

In some embodiments, anti-E7MC antibody moieties include weight chain variable district, and it includes SEQ ID NO:Institute in 237 The amino acid sequence of elaboration, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) The variant of sequence identity, and light chain variable district, it includes SEQ ID NO:The amino acid sequence illustrated in 56, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity.One In a little embodiments, anti-E7MC antibody moieties include weight chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 237 Row, and light chain variable district, include SEQ ID NO:The amino acid sequence illustrated in 56.

In some embodiments, anti-E7MC antibody moieties and any in anti-E7MC antibody moieties as described herein The second anti-E7MC antibody moieties competition binding to target HPV16-E7/MHC I class complexs.In some embodiments, resist E7MC antibody moieties are bound to identical or substantially the same epitope with the second anti-E7MC antibody moieties.In some embodiments In, the combination of anti-E7MC antibody moieties and target HPV16-E7/MHC I class complexs suppresses the second anti-E7MC antibody moieties and target The combination at least about 70% (such as at least about 75%, 80%, 85%, 90%, 95%, 98% of HPV16-E7/MHC I class complexs Or any one of 99%), or vice versa it is as the same.In some embodiments, anti-E7MC antibody moieties and the second anti-E7MC antibody Partial intersection competition binding to target HPV16-E7/MHC I class complexs, i.e. each in antibody moiety is competed with another one and tied It is bonded to target HPV16-E7/MHC I class complexs.

For example, in some embodiments, anti-E7MC antibody moieties and antibody moiety competition binding to target HPV16-E7/ MHC I class complexs, wherein antibody moiety include：I) weight chain variable district, it includes HC-CDR 1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, or it includes the change of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Body, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, or its include at most about 3 (such as from about 1, Any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and HC-CDR3, the HC-CDR3 include SEQ ID NO:In 186-188 Any one amino acid sequence, or it includes the variant of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or It includes the variant of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and LC-CDR3, the LC-CDR3 are included SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant.

In some embodiments, anti-E7MC antibody moieties and antibody moiety competition binding to target HPV16-E7/MHC I classes Complex, wherein antibody moiety include：I) weight chain variable district, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:57- Any one of 77 amino acid sequence or its include at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant (and being made from it in some embodiments), HC-CDR2, the HC-CDR2 include SEQ ID NO:Appointing in 78-98 The amino acid sequence of one or its include the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors (and being made from it in some embodiments), and HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119,244 and 245 Any one of amino acid sequence or its include at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant (and being made from it in some embodiments)；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 is included SEQ ID NO:Any one of 120-140 and 246 amino acid sequence or its include at most about 5 (in such as from about 1,2,3,4 or 5 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant (and being made from it in some embodiments), LC-CDR2, the LC-CDR2 are included SEQ ID NO:Any one of 141-161 amino acid sequence or its include at most about 3 (any one of such as from about 1,2 or 3) The variant (and being made from it in some embodiments) of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence or its include at most about 5 (in such as from about 1,2,3,4 or 5 appoint One) variant (and being made from it in some embodiments) of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

In some embodiments, anti-E7MC antibody moieties and antibody moiety competition binding to target HPV16-E7/MHC I classes Complex, wherein antibody moiety include：I) weight chain variable district, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:57- Any one of 77 amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino Acid sequence (and being made from it in some embodiments)；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119、 Any one of 244 and 245 amino acid sequence (and being made from it in some embodiments)；Or it includes at most about 5 (such as Any one of about 1,2,3,4 or 5) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR areas variant；And ii) light chain variable district, it is included LC-CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 2469 amino acid sequence is (and in some realities Apply in scheme and be made from it)；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence Arrange (and being made from it in some embodiments)；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and 247- Any one of 250 amino acid sequence (and being made from it in some embodiments)；Or its include at most about 5 (such as from about 1, 2nd, any one of 3,4 or 5) variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR areas.

In some embodiments, anti-E7MC antibody moieties and antibody moiety competition binding to target HPV16-E7/MHC I classes Complex, wherein antibody moiety include weight chain variable district, and it includes SEQ ID NO:Any one of 15-35's and 233-237 Amino acid sequence or itself and SEQ ID NO:Any one of 15-35 and 233-237 have at least about 95% (as at least about 95%th, any one of 96%, 97%, 98% or 99%) variant of sequence identity is (and in some embodiments by its group Into)；And light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence or its with SEQ ID NO:Any one of 36-56 and 238-243 have at least about 95% (such as at least about 95%, 96%, 97%, 98% Or any one of 99%) variant (and being made from it in some embodiments) of sequence identity.

The anti-E7MC antibody of total length

In some embodiments, anti-E7MC constructs are (herein for the full length antibody comprising anti-E7MC antibody moieties Also known as " the anti-E7MC antibody of total length ").In some embodiments, full length antibody is monoclonal antibody.

In some embodiments, the anti-E7MC antibody of total length includes and comes from immunoglobulin, such as IgA, IgD, IgE, IgG and IgM Fc sequences.In some embodiments, the anti-E7MC antibody of total length includes IgG, in IgG1, IgG2, IgG3 or IgG4 The Fc sequences of any one.In some embodiments, the anti-E7MC antibody of total length includes the Fc sequences of human immunoglobulin.One In a little embodiments, the anti-E7MC antibody of total length includes the Fc sequences of mouse immuning ball protein.In some embodiments, total length resists E7MC antibody includes altered or other change to make it have the antibody-dependent cytotoxicity of enhancing (ADCC) or complement The Fc sequences of dependent cellular cytotoxicity (CDC) effector function.

Therefore, for example, in some embodiments, there is provided include the following anti-E7MC antibody of total length：A) it is specific With reference to the anti-E7MC antibody moieties of the complex comprising HPV16-E7 peptides and MHC I proteinoid, and b) Fc areas.In some implementations In scheme, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid For HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01.In some embodiments, there is provided bag Containing the following anti-E7MC antibody of total length：A) specific binding includes HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A* 4) 02:The anti-E7MC antibody moieties of 01 complex, and b) Fc areas.In some embodiments, Fc areas include IgG1 Fc sequences. In some embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas include the Fc of mouse IgG 1 Sequence.In some embodiments, anti-E7MC antibody moieties are with comprising MHC I proteinoid and having a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor At least one (such as any one of at least 2,3,4,5 or 6) of the variant of the HPV16-E7 peptides of (such as conserved amino acid substitution) is again Fit cross reaction.In some embodiments, anti-E7MC antibody moieties with comprising HPV16-E7 peptides and MHC I proteinoid not With at least one (such as any one of at least 2,3,4 or 5) complex cross reaction of hypotype.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence；Or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or it takes comprising at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (examples Any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, and b) Fc areas.In some embodiments, Fc areas include IgG1 Fc sequences.In some embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas are wrapped Containing the Fc sequences of mouse IgG 1.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO: 184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NOs:Any one of 186-188 ammonia Base acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, and b) Fc areas.In some embodiments In, Fc areas include IgG1 Fc sequences.In some embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiment party In case, Fc areas include the Fc sequences of mouse IgG 1.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, comprising i) weight chain variable district, it includes HC-CDR1, should HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence, or its include at most about 5 (such as from about 1,2,3,4 Or any one of 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any in 78-98 Amino acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245, or it includes The variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable district, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or its include at most about The variant of 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 include SEQ ID NO: Any one of 141-161 amino acid sequence, or it takes comprising at most about 3 (any one of such as from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid Sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.In some embodiment party In case, Fc areas include IgG1 Fc sequences.In some embodiments, Fc areas include the Fc sequences of IgG 1.In some implementations In scheme, Fc areas include the Fc sequences of mouse IgG 1.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 are included SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99- 119th, any one of 244 and 245 amino acid sequence；Or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC-CDR1 Include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and Any one of 247-250 amino acid sequence；Or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual LC- The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in CDR sequence；And b) Fc areas.In some embodiments, Fc areas include IgG1 Fc sequences. In some embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas include the Fc sequences of mouse IgG 1 Row.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 are included SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99- 119th, any one of 244 and 245 amino acid sequence；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；And b) Fc areas.In some embodiments, Fc areas include IgG1 Fc sequences Row.In some embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas include mouse IgG1F c sequences.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) HPV16-E7 peptides are specifically bound And the anti-E7MC antibody moieties of the complex of MHC I proteinoid, it includes weight chain variable district, includes SEQ ID NO:15-35 With any one of 233-237 amino acid sequence, or its have at least about 95% (for example, at least about 96%, 97%, 98% or Any one of 99%) variant of sequence identity, and light chain variable district, SEQ ID NO are included:In 36-56 and 238-243 The amino acid sequence of any one, or it has at least about 95% (including in for example, at least about 96%, 97%, 98% or 99% Any one) sequence identity variant；And b) Fc areas.In some embodiments, Fc areas include IgG1Fc sequences.In some realities Apply in scheme, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas include the Fc sequences of mouse IgG 1.

In some embodiments, there is provided include the following anti-E7MC antibody of total length：A) specific binding includes HPV16- The anti-E7MC antibody moieties of HPV-16 E7 and the complex of MHC I proteinoid, it includes weight chain variable district, includes SEQ ID NO: Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, include SEQ ID NO:36-56 and 238- Any one of 243 amino acid sequence；And b) Fc areas.In some embodiments, Fc areas include IgG1 Fc sequences.One In a little embodiments, Fc areas include the Fc sequences of IgG 1.In some embodiments, Fc areas include the Fc sequences of mouse IgG 1 Row.

In some embodiments, the anti-E7MC antibody bindings of total length are to comprising HPV16-E7 peptides and MHC I proteinoid Complex, with reference to K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10PM, 50pM, 100pM, 500pM, Any scope between any one of 1nM, 10nM, 50nM, 100nM or 500nM, including these values).In some embodiments In, the anti-E7MC antibody bindings of total length to the complex comprising HPV16-E7 peptides and MHC I proteinoid, with reference to K_dIn about 1pM To (any one of such as from about 1,10,25,50,75,100,150,200 or 250pM, including between these values between about 250pM Any scope).

The anti-E7MC molecules of polyspecific

In some embodiments, anti-E7MC constructs include the anti-E7MC molecules of polyspecific, and it includes anti-E7MC antibody Part and the second bound fraction (such as the second antigen-binding portion thereof).In some embodiments, the anti-E7MC molecules bag of polyspecific Containing anti-E7MC antibody moieties and the second antigen-binding portion thereof.

Multispecific molecule is that synantigen or epitope do not have molecule (such as double spies of binding specificity at least two Heterogenetic antibody has binding specificity to two kinds of antigens or epitope).Also cover with more than two valencys and/or specific spy more Opposite molecule.For example, three-specific antibody can be prepared.Tutt et al. J.Immunol.147:60(1991).It will be appreciated that this The appropriate feature that indivedual multispecific molecules as described herein may be selected in art personnel forms the present invention to be combined with each other The anti-E7MC molecules of polyspecific.

Therefore, for example, in some embodiments, there is provided anti-comprising following polyspecific (such as bispecific) E7MC molecules：A) the anti-E7MC antibody moieties of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, and B) the second bound fraction (such as antigen-binding portion thereof).In some embodiments, the second bound fraction specific binding includes knot It is bonded to the complex of the different HPV16-E7 peptides of MHC I proteinoid.In some embodiments, the 2nd scFv is specifically bound Include the complex for the HPV16-E7 peptides for being bound to different MHC I proteinoid.In some embodiments, the second bound fraction Different epitopes on complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid.In some embodiments, Second bound fraction specifically binds not synantigen.In some embodiments, the second bound fraction specific binding cell, such as Antigen on cytotoxic cell surface.In some embodiments, the second bound fraction specific binding lymphocyte, such as T Antigen on cell, NK cells, neutrophil leucocyte, monocyte, macrophage or surface of dendritic cells.In some embodiment party In case, the second bound fraction specific binding effector T cell, as cytotoxic T cell (is also known as cytotoxic T lymphocyte (CTL) or T killer cell lines).

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, and b) specific binding CD3 the second antigen Bound fraction.In some embodiments, the second antigen-binding portion thereof specific binding CD3 ε.In some embodiments, Two antigen-binding portion thereofs specific binding CD3 ε exciting epitope (agonistic epitope).As used herein, term " swashs Dynamic epitope " means (a) when combining multispecific molecule, optionally when some multispecific molecules on combination same cell When, it is allowed to the multispecific molecule activates TCR signal transductions and the epitope of inducing T cell activation, and/or (b) only by CD3 it The amino acid residue of ε chains is formed and when being presented in T cell (that is, being surrounded by TCR, CD3 γ chains etc.) with natural situation, right In through multispecific molecule be combined into can and epitope, and/or (c) when combine multispecific molecule when, do not cause CD3 ε relative In the stabilized epitope in CD3 γ locus.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, and effector cell b) is specifically bound, including Such as the second antigen knot of CD3 γ, CD3 δ, CD3 ε, CD3 ζ, antigen on CD28, CD16a, CD56, CD68 and GDS2D surface Close part.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, and the b) group of specific binding complement system Point, such as C1q the second antigen-binding portion thereof.C1q is the subunit of the C1 combined enzyme agents of activated serum complement system.

In some embodiments, the second antigen-binding portion thereof specific binding Fc acceptors.In some embodiments, Two antigen-binding portion thereofs specific binding Fc γ acceptors (Fc γ R).Fc γ R can be to be present on natural killer (NK) cell surface Fc γ RIII or the Fc γ that are present on macrophage, monocyte, neutrophils and/or surface of dendritic cells One kind in RI, Fc γ RIIA, Fc γ RIIBI, Fc γ RIIB2 and Fc γ RIIIB.In some embodiments, the second antigen Bound fraction is Fc areas or its function fragment.It still is able to as used " function fragment " in this context refers to special enough Property and affinity be bound to FcR, in specific words Fc γ R to be to allow the effector cell for carrying Fc γ R, in specific words macrophage, Monocyte, neutrophil leucocyte and/or BMDC kill the antibody of target cell by cellular cytotoxicity cytolytic or phagocytosis Fc areas fragment.Feature Fc fragments can competitively suppress FcR initial, that total length Fc parts are with such as activating Fc γ RI knot Close.In some embodiments, feature Fc fragments keep at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% its affinity with activating Fc γ R.In some embodiments, Fc areas or its function fragment are enhanced Fc areas or its work( Can fragment.As used herein, term " enhanced Fc areas " refers to strengthens the receptor-mediated effector functions of Fc through modifying, in specific words Cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and the antibody-mediated phagocytosis of antibody dependent cellular mediation make Yong Fc areas.This can realize as known in the art, such as by cause activated receptor (such as to be expressed in natural killer (NK) Fc γ RIIIA (CD16A) on cell) increased affinity and/or with suppressing acceptor (such as Fc γ RIIB1/B2 (CD32B)) The mode of the combination of reduction changes Fc areas.In other embodiments, the second antigen-binding portion is divided into antibody or its antigen binding Fragment, it is with specificity enough and affinity specific binding FcR, especially in combination with Fc γ R to allow the effect for carrying Fc γ R thin Born of the same parents, especially, macrophage, monocyte, neutrophil leucocyte and/or BMDC make by cellular cytotoxicity cytolytic or phagocytosis With kill target cell.

In some embodiments, the anti-E7MC molecules of polyspecific allow kill E7MC presentation target cells and/or can be effective Ground redirects CTL and presents target cell to crack E7MC.In some embodiments, polyspecific of the invention is (such as double special Property) anti-E7MC molecules show external EC50 in the range of 10 to 500ng/ml, and can be about 1:1 to about 50:1 (such as from about 1:1 to about 15:1, or about 2:1 to about 10:1) resetting for about 50% target cell is induced under CTL and target cell ratio via CTL To cracking.

In some embodiments, the anti-E7MC molecules of polyspecific (such as bispecific) can be crosslinked through stimulating or not piercing Sharp CTL and target cell, cause target cell lysis in this way.It provides following advantage：Target-specific T cell clone is not produced Or it need not be presented by the common antigen of BMDC so that obtaining the anti-E7MC molecules of polyspecific plays activity needed for its. In some embodiments, the anti-E7MC molecules of polyspecific of the invention can redirect CTL with the absence of other activation signalses In the case of crack target cell.In some embodiments, the second antigen-binding portion thereof of the anti-E7MC molecules of polyspecific is special Property combination CD3 (such as specific binding CD3 ε), and CTL need not be redirected via CD28 and/or IL-2 signal transduction To crack target cell.

The anti-E7MC molecules of polyspecific are measured in combination with to two antigens (such as antigen on two kinds of different cells) The method of preference is in the normal capacity of those skilled in the art.For example, when the second bound fraction specifically binds CD3 When, the anti-E7MC molecules of polyspecific can be with CD3+/HPV16-E7^-Cell and CD3^-/HPV16-E7⁺The mixture contact of cell.It is more The quantity of the anti-E7MC molecules positive monocytes of specificity and it can be connect by the quantity of the cell of the anti-E7MC molecule cross-links of polyspecific By microscopy known in the art or fluorescence activated cell sorts (FACS) assessment.

For example, in some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specificity knot Close the anti-E7MC antibody moieties of the complex comprising HPV16-E7 peptides and MHC I proteinoid, and b) the second antigen-binding portion thereof. In some embodiments, HPV16-E7 peptides are HPV16-E711-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01.In some embodiments In, the specific binding of the second antigen-binding portion thereof includes the complex for the different HPV16-E7 peptides for being bound to MHC I proteinoid. In some embodiments, the specific binding of the second antigen-binding portion thereof, which includes, is bound to different MHC I proteinoid The complex of HPV16-E7 peptides.In some embodiments, the specific binding of the second antigen-binding portion thereof, which includes, is bound to MHC I Different epitopes on the complex of the HPV16-E7 peptides of proteinoid.In some embodiments, the second antigen-binding portion dtex The opposite sex combines another antigen.In some embodiments, the second antigen-binding portion thereof specific binding cell, as E7MC is presented carefully Antigen on cellular surface.In some embodiments, the thin of HPV16-E7 is not expressed in the second antigen-binding portion thereof specific binding Antigen on cellular surface.In some embodiments, on the second antigen-binding portion thereof specific binding cytotoxic cell surface Antigen.In some embodiments, the second antigen-binding portion thereof specific binding lymphocyte, as T cell, NK cells, in Antigen on property granulocyte, monocyte, macrophage or surface of dendritic cells.In some embodiments, the second antigen Bound fraction specifically binds effector T cell, such as the antigen on cytotoxic T cell surface.In some embodiments, second Antigen-binding portion thereof specifically bind effector cell, including such as CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD28, CD16a, CD56, Antigen on CD68 and GDS2D surfaces.In some embodiments, anti-E7MC antibody moieties are the mankind, humanization or semi-synthetic 's.In some embodiments, the second antigen-binding portion is divided into antibody moiety.In some embodiments, the second antigen binding Part is the mankind, humanization or semi-synthetic antibody moiety.In some embodiments, the anti-E7MC molecules of polyspecific further wrap Containing at least one (such as at least about 2,3,4,5 or 5 kind of any of the above item) additional antigens bound fraction.In some embodiments In, anti-E7MC antibody moieties and at least one (as any one of at least 2,3,4,5 or 6) comprising MHC I proteinoid and The complex cross reaction of the variant of HPV16-E7 peptides with a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution).At some In embodiment, anti-E7MC antibody moieties include HPV16-E7 peptides with least one (such as any one of at least 2,3,4 or 5) And the complex cross reaction of the different subtype of MHC I proteinoid.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):The anti-E7MC antibody moieties of 01 complex, and it is b) second anti- Former bound fraction.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or it takes comprising at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (examples Any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, and b) the second antigen-binding portion thereof.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequences, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 ammonia Base acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino Acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequences；Wherein X can be any amino acid, and B) the second antigen-binding portion thereof.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variable district, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:78-98 Any one of amino acid sequence, or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 Arrange, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable Area, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or It includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 bags The NO of ID containing SEQ:Any one of 141-161 amino acid sequence, or it includes at most about 3 (any in such as from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Appointing in 162-182 and 247-250 The amino acid sequence of one, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； And b) the second antigen-binding portion thereof.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 Include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO: The amino acid sequence of any one of 99-119,244 and 245；Or it includes at most about 5 (any in such as from about 1,2,3,4 or 5 ) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；Or it is individual comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in LC-CDR sequences；And b) the second antigen-binding portion thereof.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 Include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO: The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；And b) the second antigen-binding portion thereof.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) anti-E7MC antibody moieties, its Comprising weight chain variable district, SEQ ID NO are included:Any one of 15-35 and 233-237 amino acid sequence, or its have extremely The variant of few about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity, and light chain variable Area, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or its have at least about 95% (including For example, at least any one of about 96%, 97%, 98% or 99%) sequence identity variant；And b) the 2nd scFv.

In some embodiments, there is provided include the following anti-E7MC molecules of polyspecific：A) anti-E7MC antibody moieties, its Comprising weight chain variable district, SEQ ID NO are included:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable Area, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence；And b) the second antigen-binding portion thereof.

In some embodiments, the anti-E7MC molecules of polyspecific are that such as bifunctional antibody (Db), single-chain bifunctional resist Body (scDb), series connection scDb (Tandab), linear dimerization scDb (LD-scDb), circular dimerization scDb (CD-scDb), two-bis- work( Energy antibody, series connection scFv, two-scFv of series connection (such as bispecific T cell joint molecule), three-scFv of series connection, three functions resist Body, bispecific Fab2, two-miniantibody, four function antibodies, scFv-Fc-scFv fusions, double affinity target again (DART) antibody, Double variable regions (DVD) antibody, IgG-scFab, scFab-ds-scFv, Fv2-Fc, IgG-scFv fusion, depressed place Lock (dockand lock；DNL) antibody, pestle-mortar (knob-into-hole；KiH) antibody (is prepared double special by KiH technologies Different in nature IgG), DuoBody (the bispecific IgG prepared by Duobody technologies), heteromultimeric antibody or different conjugate resist Body.In some embodiments, the anti-E7MC molecules of polyspecific for series connection scFv (such as series connection two-scFv, such as bispecific T Cell joint molecule).

Connect scFv

In some embodiments, the anti-E7MC molecules of polyspecific are series connection scFv, and it contains anti-E7MC antibody moieties The first scFv and the 2nd scFv (be also known as herein " series connection scFv polyspecifics anti-E7MC antibody ").In some embodiment party In case, the anti-E7MC antibody of series connection scFv polyspecifics is further comprising at least one (in more than at least about 2,3,4,5 or 5 Any one) extra scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, and b) the 2nd scFv. In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01.In some embodiments In, the 2nd scFv specific bindings include the complex for the different HPV16-E7 peptides for being bound to MHC I proteinoid.In some realities Apply in scheme, the 2nd scFv specific bindings include the complex for the HPV16-E7 peptides for being bound to different MHC I proteinoid. In some embodiments, the 2nd scFv specific bindings include the complex for the HPV16-E7 peptides for being bound to MHC I proteinoid On different epitopes.In some embodiments, the 2nd scFv specifically binds another antigen.In some embodiments, Two scFv specifically bind cell, if E7MC is in the antigen on presenting cells.In some embodiments, the 2nd scFv is special Property combine and do not express antigen on HPV16-E7 cell surface.In some embodiments, the 2nd scFv specific bindings are thin Antigen on cellular toxicity cell surface.In some embodiments, the 2nd scFv specifically binds lymphocyte, such as T cell, NK Antigen on cell, neutrophil leucocyte, monocyte, macrophage or surface of dendritic cells.In some embodiments, Two scFv specifically bind effector T cell, such as the antigen on cytotoxic T cell surface.In some embodiments, second ScFv specifically bind effector cell, including such as CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD28, CD16a, CD56, CD68 and Antigen on GDS2D surfaces.In some embodiments, the first scFv is the mankind, humanization or semi-synthetic.In some implementations In scheme, the 2nd scFv is the mankind, humanization or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv are equal For the mankind, humanization or semi-synthetic.In some embodiments, the series connection anti-E7MC antibody of scFv polyspecifics further includes At least one scFv extra (such as at least about 2,3,4,5 or 5 any of the above items).In some embodiments, anti-E7MC Antibody moiety and the HPV16-E7 peptides comprising MHC I proteinoid and with a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution) Variant at least one (as any one of at least 2,3,4 or 5) complex cross reaction.In some embodiments, resist E7MC antibody moieties are with including at least one (such as at least 2,3, the 4 or 5 of HPV16-E7 peptides and MHC I proteinoid different subtypes Any one of) complex cross reaction.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) specific binding includes HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):The first of 01 complex ScFv, and b) the 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, includes i) weight chain variable Region sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 Or 185 amino acid sequences, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequences, or it includes at most about 3 (any in e.g., from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or its bag Variant containing at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and b) the 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, includes i) weight chain variable Region sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 Include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:In 186-188 Any one amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 Or 190 amino acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, and b) second scFv。

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, includes i) weight chain variable Area, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes The variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence, or its include at most about 5 (any one of such as from about 1,2,3,4 or 5) it is individual The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 99-119,244 and 245 Amino acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) Light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino Acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2 should LC-CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence, or it includes at most about 3 (such as from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and 247- Any one of 250 amino acid sequence, or it takes comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid The variant in generation；And b) the 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, includes i) weight chain variable Region sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC- CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 bags The NO of ID containing SEQ:The amino acid sequence of any one of 99-119,244 and 245；Or it includes at most about 5 (such as from about 1,2,3,4 Or any one of 5) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC- CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；Or it includes at most about 5 (in such as from about 1,2,3,4 or 5 Any one) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences variant；And b) the 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, includes i) weight chain variable Region sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC- CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 bags The NO of ID containing SEQ:The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it is included LC-CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, should LC-CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；And b) the 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid Sequence, or it has at least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity Variant, and light chain variable district, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or it has The variant of at least about 95% (including for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity；And b) 2nd scFv.

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid Sequence, and light chain variable district, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence；And b) second scFv。

In some embodiments, there is provided resist comprising following series connection scFv polyspecifics (such as bispecific) anti-E7MC Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, and b) the 2nd scFv, The anti-E7MC antibody of scFv polyspecifics wherein connect as three-scFv of two-scFv of series connection or series connection.In some embodiments, go here and there Join the anti-E7MC antibody of scFv polyspecifics for two-scFv of series connection.In some embodiments, connect the anti-E7MC of scFv polyspecifics Antibody is bispecific T cell joint molecule.

For example, in some embodiments, there is provided resist comprising the following anti-E7MC of-scFv bispecifics of series connection two Body：A) the first scFv of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid, and b) specifically bind 2nd scFv of the antigen on T cell surface.In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I albuminoids Matter is HLA-A*02:01.In some embodiments, the 2nd scFv specifically binds effector T cell, such as cytotoxic T cell Antigen on surface.In some embodiments, antigen of the 2nd scFv specific bindings selected from the group for example consisted of： CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.In some embodiments, The exciting epitope on antigen on 2nd scFv specific binding T cells surface, wherein the combination enhancing T of the 2nd scFv and antigen Cell activation.In some embodiments, the first scFv is the mankind, humanization or semi-synthetic.In some embodiments, Two scFv are the mankind, humanization or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv is the mankind, people Source is semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to including HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):First scFv of 01 complex, and it is b) special 2nd scFv of the antigen on property combination T cell surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it is wrapped SEQ ID NO are included containing HC-CDR1, the HC-CDR1:183 amino acid sequence, or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid Sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, should HC-CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or its include at most about 3 (e.g., from about 1,2 Or any one of 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 is included SEQ ID NO:189 or 190 amino acid sequences, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid Substituted variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and b) specifically bind antigen on T cell surface 2nd scFv.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it is wrapped SEQ ID NO are included containing HC-CDR1, the HC-CDR1:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include NOs:Any one of 186-188 amino acid Sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence Row, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, and b) specifically bind on T cell surface Antigen the 2nd scFv.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variable district, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as Any one of about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence, or it takes comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid The variant in generation, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 Arrange, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable Area, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or It includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 bags The NO of ID containing SEQ:Any one of 141-161 amino acid sequence, or it includes at most about 3 (any in such as from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Appointing in 162-182 and 247-250 The amino acid sequence of one, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； And b) specifically bind the 2nd scFv of the antigen on T cell surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it is wrapped SEQ ID NO are included containing HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it included at most about 5 (appointing in such as from about 1,2,3,4 or 5 One) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；Or it is individual comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in LC-CDR sequences, and b) the 2nd scFv of the antigen on specific binding T cell surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it is wrapped SEQ ID NO are included containing HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 are included SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162- Any one of 182 and 247-250 amino acid sequence；And b) specifically bind the 2nd scFv of the antigen on T cell surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) first ScFv, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or its Variant with least about 95% (for example, at least about 96%, 97%, 98%, any one of 99%) sequence identity, and gently Chain variable region, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or its have at least about The variant of 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity, and b) specifically bind 2nd scFv of the antigen on T cell surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) first ScFv, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and gently Chain variable region, include SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, and b) specific binding T is thin 2nd scFv of the antigen on cellular surface.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv of the complex comprising HPV16-E7 peptides and MHC I proteinoid, and b) specifically bind the second of CD3 ε scFv.In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments In, MHC I proteinoid is HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01.At some In embodiment, the first scFv is fused to the 2nd scFv via with the bonded of peptide linker.In some embodiments, peptide linker it Length about 5 to about 20 (any scope between any one of such as from about 5,10,15 or 20, including these values) individual amino acid it Between.In some embodiments, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments). In some embodiments, the first scFv is the mankind, humanization or semi-synthetic.In some embodiments, the 2nd scFv is people Class, humanization or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv is the mankind, humanization or hemizygous Into.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to including HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):First scFv of 01 complex, and it is b) special Property combination CD3 ε the 2nd scFv.In some embodiments, the first scFv is fused to second via with the bonded of peptide linker scFv.In some embodiments, the length of peptide linker about 5 to about 20 (any one of such as from about 5,10,15 or 20, including Any scope between these values) between individual amino acid.In some embodiments, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments).In some embodiments, the first scFv is the mankind, humanization or semi-synthetic 's.In some embodiments, the 2nd scFv is the mankind, humanization or semi-synthetic.In some embodiments, the first scFv And the 2nd scFv be the mankind, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence；Or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or it takes comprising at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (examples Any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, and b) specifically bind CD3 ε the 2nd scFv.In some realities Apply in scheme, the first scFv is fused to the 2nd scFv by using the link of too joint.In some embodiments, peptide linker Length about 5 to about 20 (any scope between any one of such as from about 5,10,15 or 20, including these values) individual amino acid it Between.In some embodiments, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments). In some embodiments, the first scFv is people, humanization or semi-synthetic.In some embodiments, the 2nd scFv is It is people, humanization or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv is to be people, humanization It is or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO: 184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino Acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, and b) specifically bind the of CD3 ε Two scFv.In some embodiments, the first scFv is fused to the 2nd scFv by using the link of too joint.In some implementations In scheme, the length of peptide linker is (any between any one of such as from about 5,10,15 or 20, including these values about 5 to about 20 Scope) between individual amino acid.In some embodiments, peptide linker includes amino acid sequence GGGGS (and in some embodiments In be made from it).In some embodiments, the first scFv is people, humanization or semi-synthetic.In some embodiments In, the 2nd scFv is people, humanization or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv are It is people, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 55-77 amino acid sequence, or its include at most about 5 (such as Any one of about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual amino acid Substituted variant, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid of any one of 99-119,244 and 245 Sequence；Or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain Variable region, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence Arrange, or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2 should LC-CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence, or its include at most about 3 (e.g., from about 1,2 Or any one of 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and Any one of 247-250 amino acid sequence, or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual amino The variant of acid substitution, and b) specific binding CD3 ε the 2nd scFv.In some embodiments, the first scFv is by using too The link of joint is fused to the 2nd scFv.In some embodiments, the length of peptide linker is about 5 to about 20 (such as from about 5,10,15 Or any scope between any one of 20, including these values) between individual amino acid.In some embodiments, peptide linker Include amino acid sequence GGGGS (and being made from it in some embodiments).In some embodiments, the first scFv is people , humanization or it is semi-synthetic.In some embodiments, the 2nd scFv is people, humanization or semi-synthetic.One In a little embodiments, the first scFv and the 2nd scFv are to be people, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 55-77 amino acid sequence；HC-CDR2, the HC-CDR2 are included SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99- 119th, any one of 244 and 245 amino acid sequence；Or it includes at most about 5 (any in e.g., from about 1,2,3,4 or 5 ) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light chain variable district, it includes LC-CDR1, the LC-CDR1 bags The NO of ID containing SEQ:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and Any one of 247-250 amino acid sequence；Or it includes at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual LC- The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in CDR sequence, and b) specific binding CD3 ε the 2nd scFv.In some embodiments, One scFv is fused to the 2nd scFv by using the link of too joint.In some embodiments, the length of peptide linker about 5 to Between about 20 (any scope between any one of such as from about 5,10,15 or 20, including these values) individual amino acid.In some realities Apply in scheme, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments).In some embodiments In, the first scFv is people, humanization or semi-synthetic.In some embodiments, the 2nd scFv is people, humanization It is or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv is to be people, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) it is specific With reference to the first scFv comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 55-77 amino acid sequence；HC-CDR2, the HC-CDR2 are included SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:99- 119th, any one of 244 and 245 amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, the LC-CDR1 bags The NO of ID containing SEQ:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 and Any one of 247-250 amino acid sequence；And b) specifically bind CD3 ε the 2nd scFv.In some embodiments, One scFv is fused to the 2nd scFv by using the link of too joint.In some embodiments, the length of peptide linker about 5 to Between about 20 (any scope between any one of such as from about 5,10,15 or 20, including these values) individual amino acid.In some realities Apply in scheme, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments).In some embodiments In, the first scFv is people, humanization or semi-synthetic.In some embodiments, the 2nd scFv is people, humanization It is or semi-synthetic.In some embodiments, the first scFv and the 2nd scFv is to be people, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) first ScFv, comprising weight chain variable district, it includes its SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or It has the change of at least about 95% (such as any one of at least about 95%, 96%, 97%, 98% or 99%) sequence identity Body, and light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or it has The variant of at least about 95% (such as any one of at least about 95%, 96%, 97%, 98% or 99%) sequence identity, and b) Specifically bind CD3 ε the 2nd scFv.In some embodiments, the first scFv is fused to by using the link of peptide linker 2nd scFv.In some embodiments, the length of peptide linker about 5 to about 20 (any one of such as from about 5,10,15 or 20, Including any scope between these values) between individual amino acid.In some embodiments, peptide linker includes amino acid sequence GGGGS (and being made from it in some embodiments).In some embodiments, the first scFv to be people, humanization or Semi-synthetic.In some embodiments, the 2nd scFv is people, humanization or semi-synthetic.In some embodiments, First scFv and the 2nd scFv is to be people, humanization or semi-synthetic.

In some embodiments, there is provided include the following anti-E7MC antibody of-scFv bispecifics of series connection two：A) first ScFv, comprising weight chain variable district, it includes its SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and Light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, and b) specificity knot Close CD3 ε the 2nd scFv.In some embodiments, the first scFv is fused to the 2nd scFv by using the link of too joint. In some embodiments, the length of peptide linker is in about 5 to about 20 (any one of such as from about 5,10,15 or 20, including these values Between any scope) between individual amino acid.In some embodiments, peptide linker includes amino acid sequence GGGGS (and one It is made from it in a little embodiments).In some embodiments, the first scFv is people, humanization or semi-synthetic.One In a little embodiments, the 2nd scFv is people, humanization or semi-synthetic.In some embodiments, the first scFv and Two scFv are to be people, humanization or semi-synthetic.

In some embodiments, connect two-scFv bispecifics anti-E7MC antibody bindings to comprising HPV16-E7 peptides and The complex of MHC I proteinoid, with reference to K_dAbout 0.1pM between about 500nM (such as from about 0.1pM, 1.0pM, 10PM, Any model between any one of 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including these values Enclose).In some embodiments, the anti-E7MC antibody bindings of two-scFv bispecifics of connecting extremely include HPV16-E7 peptides and MHC I The complex of proteinoid, with reference to K_dAbout 1nM between about 500nM (such as from about 1,10,25,50,75,100,150,200, 250th, any scope between any one of 300,350,400,450 or 500nM, including these values).

Chimeric antigen receptor (CAR) and CAR effector cell

In some embodiments, anti-E7MC constructs are the Chimeric antigen receptor (CAR) comprising anti-E7MC antibody moieties (be also known as herein " anti-E7MC CAR ").CAR effector cell's (the example for including the CAR containing anti-E7MC antibody moieties is also provided Such as T cell) (being also known as " anti-E7MC CAR effector cells " herein, for example, " anti-E7MC CAR T cells ").

Anti- E7MC CAR include a) extracellular, and it includes specific binding and includes HPV16-E7 peptides and MHC I albuminoids The anti-E7MC antibody moieties of the complex of matter, and b) intracellular signal transduction domain.Membrane-spanning domain may be present in extracellular with it is intracellular Between domain.

Between anti-E7MC CAR extracellular and membrane-spanning domain, or anti-E7MC CAR intracellular domain and membrane-spanning domain it Between, spacer domain may be present.Spacer domain can be times for the membrane-spanning domain in polypeptide chain to be connected to extracellular or intracellular domain What oligopeptides or polypeptide.Spacer domain may comprise up to about 300 amino acid, including e.g., from about 10 to about 100, or about 25 to about 50 Amino acid.

Membrane-spanning domain can derive from natural origin or synthesis source.Source for it is natural when, the domain can derive from any film knot Hop protein or transmembrane protein.Be specifically used for transmembrane region in the present invention can be derived from φt cell receptor CD28, CD3 ε, CD3 ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154's α, β, δ, γ or ζ chain (that is, comprising at least its transmembrane region).In some embodiments, membrane-spanning domain can be synthesis, in this feelings Under condition, it can mainly include hydrophobic residue, such as leucine and valine.In some embodiments, in synthesis membrane-spanning domain Each end can find the triplet of phenylalanine, tryptophan and valine.In some embodiments, there is such as length about 2 The short oligopeptides or peptide linker of length between about 10 (any one of such as from about 2,3,4,5,6,7,8,9 or 10) individual amino acid Key can be formed between anti-E7MCCAR membrane-spanning domain and intracellular signal transduction domain.In some embodiments, joint is based on sweet Propylhomoserin-serine dyad.

In some embodiments, using naturally related to one of the sequence in anti-E7MC CAR intracellular domain Membrane-spanning domain (if for example, anti-E7MC CAR intracellular domains include CD28 costimulation sequences, anti-E7MC CAR membrane-spanning domain derives From CD28 membrane-spanning domains).In some embodiments, membrane-spanning domain can by 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor select or modify with avoid this class field with The membrane spaning domain of identical or different surface membrane protein matter is combined to cause the interaction with other members of Receptor Complex Minimize.

Cause the normal effect of immunocyte (wherein having inserted anti-E7MC CAR) in anti-E7MC CAR intracellular signal transduction domain Answer the activation of at least one in function.The effector function of T cell for example can be cell lysis activity or auxiliary activity, including divide Secrete cell factor.Therefore, term " intracellular signal transduction domain " refers to transduction effector function signal and guiding cell performs specifically The protein portion of sexual function.Although whole intracellular signal transduction domains can be generally used, as a rule, it is not necessary to make With whole piece chain.As for the degree of the truncation part using intracellular signal transduction domain, as long as such truncation part transduction effect Function signal can be used to substitute complete chain.Term " intracellular signal transduction sequence " therefore it is intended to include being enough effector function of transduceing Any truncation part in the intracellular signal transduction domain of signal.

Example for the intracellular signal transduction domain in the anti-E7MC CAR of the present invention includes common rise and connect in antigen receptor The φt cell receptor (TCR) of initial signal transduction and the cytoplasmic sequences of co-receptor after conjunction, and these sequences are any Derivative or variant and any composition sequence with identical function ability.

It is known to be not enough to complete activating T cell via signal caused by single TCR and also need to two level or costimulation letter Number.Therefore, T cell activation can be described as mediating by the intracellular signal transduction sequence of two kinds of different classifications：Via TCR initial antigens Those (the one stage signal conduction sequences) of the activation of dependence one-level and worked with antigen independent mode to provide two level or common thorn Those (costimulatory signal conduction sequences) of energizing signal.

One stage signal conducts the one-level activation that sequence adjusts TCR complexs with stimulation mode or suppressor mode.With stimulation side The one stage signal conduction sequence that formula works can contain signal transduction motif, the referred to as activation motifs based on immunity receptor tyrosine Or ITAM.In some embodiments, anti-E7MC CAR constructs include one or more ITAM.

The example for the conduction sequence of the one stage signal containing ITAM being specifically used in the present invention includes being derived from TCR ζ, FcR Those of γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d.

In some embodiments, anti-E7MC CAR include the one stage signal conduction sequence derived from CD3 ζ.For example, CAR intracellular signal transduction domain can include CD3 ζ intracellular signal transductions sequence in itself or with suitable for the present invention anti-E7MC CAR Situation under any other required intracellular signal transduction combined sequence.For example, anti-E7MC CAR intracellular domain can wrap The intracellular signal transduction sequences of ζ containing CD3 and costimulatory signal conduction sequence.Costimulatory signal conduction sequence can costimulatory molecules it is thin A part for Intracellular domain, the costimulatory molecules include such as CD27, CD28,4-1BB (CD137), OX40, CD30, CD40, PD- 1st, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, specific binding CD83 Part etc..

In some embodiments, anti-E7MC CAR intracellular signal transduction domain includes CD3 ζ intracellular signal transduction sequence And CD28 intracellular signal transduction sequence.In some embodiments, anti-E7MC CAR intracellular signal transduction domain includes CD3 ζ Intracellular signal transduction sequence and 4-1BB intracellular signal transduction sequence.In some embodiments, anti-E7MC CAR intracellular Signal transduction domain includes CD3 ζ intracellular signal transduction sequence and CD28 and 4-1BB intracellular signal transduction sequence.

Therefore, for example, in some embodiments, there is provided include following anti-E7MC CAR：A) comprising specificity With reference to the extracellular of the anti-E7MC antibody moieties of the complex comprising HPV16-E7 peptides and MHC I proteinoid, b) membrane-spanning domain, And c) intracellular signal transduction domain.In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4). In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I proteinoid is HLA- A*02:01.In some embodiments, intracellular signal transduction domain being capable of activating immune cell.In some embodiments, intracellular Signal transduction domain includes one stage signal conduction sequence and costimulatory signal conduction sequence.In some embodiments, one stage signal Conduction sequence includes CD3 ζ intracellular signal transduction sequences.In some embodiments, costimulatory signal conduction sequence includes CD28 Intracellular signal transduction sequence.In some embodiments, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellulars Signal transduction sequence.In some embodiments, anti-E7MC antibody moieties with it is at least one (at least 2,3,4,5 or 6 Any one) HPV16-E7 peptides comprising MHC I proteinoid and with 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conserved amino acid substitution) The complex cross reaction of variant.In some embodiments, anti-E7MC antibody moieties and at least one (such as at least 2,3,4 or 5 It is any one of individual) the complex cross reaction of the different subtype comprising HPV16-E7 peptides and MHCI proteinoid.

In some embodiments, there is provided include following anti-E7MC CAR：A) HPV16- is included comprising specific binding E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):The extracellular of the anti-E7MC antibody moieties of 01 complex, b) cross-film Domain, and c) intracellular signal transduction domain.In some embodiments, intracellular signal transduction domain being capable of activating immune cell.At some In embodiment, intracellular signal transduction domain includes one stage signal conduction sequence and costimulatory signal conduction sequence.In some implementations In scheme, one stage signal conduction sequence includes CD3 ζ intracellular signal transduction sequences.In some embodiments, costimulatory signal passes Lead sequence and include CD28 intracellular signal transduction sequences.In some embodiments, intracellular domain includes CD3 ζ intracellular signal transductions Sequence and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding Anti- E7MC antibody moieties comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 amino acid sequence, or it takes comprising at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid The variant in generation, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (examples Any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, b) membrane-spanning domain, and c) intracellular signal transduction domain.In some realities Apply in scheme, intracellular signal transduction domain being capable of activating immune cell.In some embodiments, intracellular signal transduction domain includes one Level signal transduction sequence and costimulatory signal conduction sequence.In some embodiments, one stage signal conduction sequence includes CD3 ζ Intracellular signal transduction sequence.In some embodiments, costimulatory signal conduction sequence includes CD28 intracellular signal transduction sequences. In some embodiments, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 Amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 Amino acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence；B) intracellular signal transduction domain. In some embodiments, intracellular signal transduction domain being capable of activating immune cell.In some embodiments, intracellular signal transduction Domain includes one stage signal conduction sequence and costimulatory signal conduction sequence.In some embodiments, one stage signal conduction sequence Include CD3 ζ intracellular signal transduction sequences.In some embodiments, costimulatory signal conduction sequence includes CD28 intracellular signals Conduct sequence.In some embodiments, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transductions Sequence.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variable district, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as Any one of about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence, or it takes comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid The variant in generation, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 Arrange, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable Area, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or It includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 bags The NO of ID containing SEQ:Any one of 141-161 amino acid sequence, or it includes at most about 3 (any in such as from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Appointing in 162-182 and 247-250 The amino acid sequence of one, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； B) membrane-spanning domain, and c) intracellular signal transduction domain.In some embodiments, intracellular signal transduction domain being capable of activating immune cell. In some embodiments, intracellular signal transduction domain includes one stage signal conduction sequence and costimulatory signal conduction sequence.One In a little embodiments, one stage signal conduction sequence includes CD3 ζ intracellular signal transduction sequences.In some embodiments, costimulation Signal transduction sequence includes CD28 intracellular signal transduction sequences.In some embodiments, intracellular domain is believed comprising CD3 ζ intracellulars Number conduction sequence and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it included at most about 5 (appointing in such as from about 1,2,3,4 or 5 One) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；Or it is individual comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in LC-CDR sequences, b) membrane-spanning domain, and c) intracellular signal transduction domain.In some embodiments, Intracellular signal transduction domain being capable of activating immune cell.In some embodiments, intracellular signal transduction domain passes comprising one stage signal Lead sequence and costimulatory signal conduction sequence.In some embodiments, one stage signal conduction sequence includes CD3 ζ intracellular signals Conduct sequence.In some embodiments, costimulatory signal conduction sequence includes CD28 intracellular signal transduction sequences.In some realities Apply in scheme, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 are included SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162- Any one of 182 and 247-250 amino acid sequence；And b) intracellular signal transduction domain.In some embodiments, intracellular is believed Number transduction domain being capable of activating immune cell.In some embodiments, intracellular signal transduction domain includes one stage signal conduction sequence And costimulatory signal conduction sequence.In some embodiments, one stage signal conduction sequence includes CD3 ζ intracellular signal transduction sequences Row.In some embodiments, costimulatory signal conduction sequence includes CD28 intracellular signal transduction sequences.In some embodiments In, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising weight chain variable district, it is included SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or it has at least about 95% (for example, at least about 96%th, any one of 97%, 98% or 99%) variant of sequence identity, and light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence, or its have at least about 95% (including for example, at least about 96%, 97%th, any one of 98% or 99%) variant of sequence identity；B) membrane-spanning domain, and c) intracellular signal transduction domain.At some In embodiment, intracellular signal transduction domain being capable of activating immune cell.In some embodiments, intracellular signal transduction domain includes One stage signal conducts sequence and costimulatory signal conduction sequence.In some embodiments, one stage signal conduction sequence includes CD3 ζ intracellular signal transduction sequences.In some embodiments, costimulatory signal conduction sequence includes CD28 intracellular signal transduction sequences Row.In some embodiments, intracellular domain includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, wrapped comprising specific binding The anti-E7MC antibody moieties of peptide containing HPV16-E7 and the complex of MHC I proteinoid, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, include SEQ ID NO:36-56 and Any one of 238-243 amino acid sequence；B) intracellular signal transduction domain.In some embodiments, intracellular signal transduction Domain being capable of activating immune cell.In some embodiments, intracellular signal transduction domain includes one stage signal conduction sequence and common thorn Energizing signal conducts sequence.In some embodiments, one stage signal conduction sequence includes CD3 ζ intracellular signal transduction sequences.One In a little embodiments, costimulatory signal conduction sequence includes CD28 intracellular signal transduction sequences.In some embodiments, cell Internal area includes CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, b) membrane-spanning domain, and c) include CD3 ζ born of the same parents The intracellular signal transduction domain of interior signal transduction sequence and CD28 intracellular signal transduction sequences.In some embodiments, HPV16- HPV-16 E7 is HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I proteinoid is HLA-A02. In some embodiments, MHC I proteinoid is HLA-A*02:01.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding Include HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):The anti-E7MC antibody moieties of 01 complex, b) cross-film Domain, and c) the intracellular signal transduction domain comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:183 amino acid sequence, or its include at most about 3 (e.g., from about 1,2 Or any one of 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 ammonia Base acid sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, The HC-CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or its include at most about 3 (e.g., from about 1, Any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 is included SEQ ID NO:189 or 190 amino acid sequences, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual amino acid Substituted variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, b) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions The intracellular signal transduction domain of sequence and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 Amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 ammonia Base acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence；Wherein X can be any amino Acid, b) membrane-spanning domain, and the c) intracellular signal transduction comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences Domain.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variable district, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as Any one of about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence, or it takes comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid The variant in generation, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 Arrange, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable Area, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or It includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 bags The NO of ID containing SEQ:Any one of 141-161 amino acid sequence, or it includes at most about 3 (any in such as from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Appointing in 162-182 and 247-250 The amino acid sequence of one, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； B) membrane-spanning domain, and c) the intracellular signal transduction domain comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ IDNO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it included at most about 5 (appointing in such as from about 1,2,3,4 or 5 One) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；Or it is individual comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in LC-CDR sequences；B) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions sequences and CD28 born of the same parents The intracellular signal transduction domain of interior signal transduction sequence.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variabl area sequence, its SEQ ID NO are included comprising HC-CDR1, the HC-CDR1:Any one of 57-77 amino acid sequence；HC-CDR2, the HC- CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC-CDR1, should LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 are included SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162- Any one of 182 and 247-250 amino acid sequence；B) membrane-spanning domain, and c) comprising CD3 ζ intracellular signal transductions sequences and The intracellular signal transduction domain of CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising i) weight chain variable district, it is included SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or it has at least about 95% (for example, at least about 96%th, any one of 97%, 98% or 99%) variant of sequence identity, and light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence, or its have at least about 95% (including for example, at least about 96%, 97%th, any one of 98% or 99%) variant of sequence identity；B) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions The intracellular signal transduction domain of sequence and CD28 intracellular signal transduction sequences.

In some embodiments, there is provided include following anti-E7MC CAR：A) extracellular, it includes specific binding The anti-E7MC antibody moieties of complex comprising HPV16-E7 peptides and MHC I proteinoid, comprising weight chain variable district, it is included SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence；B) membrane-spanning domain, and c) comprising CD3 ζ intracellular signal transductions sequences and The intracellular signal transduction domain of CD28 intracellular signal transduction sequences.

The effector cell's (such as lymphocyte, such as T cell) for expressing anti-E7MC CAR is also provided herein.

Also the method for producing the effector cell for expressing anti-E7MC CAR is provided, this method, which includes to include, encodes anti-E7MC The carrier of CAR nucleic acid is introduced in effector cell.In some embodiments, by carrier be introduced in effector cell include by By carrier transduction effector cell.In some embodiments, carrier is introduced in effector cell to include to transfect by carrier and imitated Answer cell.Any method known in the art can be used to carry out into effector cell for carrier transduction or transfection.

Immunoconjugates

In some embodiments, anti-E7MC constructs include exempts from containing the anti-E7MC antibody moieties for being attached to effector molecule Epidemic disease conjugate (is also known as " anti-E7MC immunoconjugates ") herein.In some embodiments, effector molecule is therapeutic agent, Such as cancer therapeutic agent, it is cytotoxicity, cell growth inhibition or otherwise provide some treatment benefits.At some In embodiment, effector molecule is mark, and it can directly or indirectly produce detectable signal.

In some embodiments, there is provided anti-E7MC immunoconjugates comprising anti-E7MC antibody moieties and therapeutic agent ( Also referred to herein as " antibody-drug conjugates " or " ADC ").In some embodiments, therapeutic agent is toxin, and it is cell Ability that is toxicity, cell growth inhibition or otherwise preventing or reduce target cell division.It is thin using ADC localized deliveries Cellular toxicity agent or cytostatic agent, i.e., in treatment of cancer kill or suppress tumour cell medicine (Syrigos and Epenetos,Anticancer Research 19:605-614(1999)；Niculescu-Duvaz and Springer, Adv.Drg.Del.Rev.26:151-172(1997)；U.S. Patent No. 4,975,278) allow drug moiety targeted delivery extremely Target cell, and intracellular accumulation wherein, wherein these non-binding therapeutic agents of systemic administration for normal cell and can be set The target cell that method eliminates produces unacceptable toxicity level (Baldwin et al., Lancet (on March 15th, 1986):603- 605(1986)；Thorpe,(1985)“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review”,Monoclonal Antibodies'84:Biological And Clinical Applications, A.Pinchera et al. (eds.), the 475-506 pages).And then seek the maximum effect with minimum toxicity. Importantly, due to which E7MC is not presented in most of normal cell in its surface, it can not combine anti-E7MC immunoconjugates, and It is protected from the kill effect of toxin or other therapeutic agents.

Include such as daunomycin, adriamycin, methotrexate (MTX) and Changchun for the therapeutic agent in anti-E7MC immunoconjugates Pungent (Rowland et al., the Cancer Immunol.Immunother.21 in ground:183-187(1986)).It is immunized and sews for anti-E7MC Toxin in compound includes bacteriotoxin, such as diphtheria toxin；Phytotoxin, such as ricin (WA)；Small molecule toxins, as Ge Erde is mould Element (geldanamycin) (Mandler et al., J.Nat.Cancer Inst.92 (19):1573-1581(2000)；Mandler Et al., Bioorganic＆Med.Chem.Letters 10:1025-1028(2000)；Mandler et al., Bioconjugate Chem.13:786-791 (2002)), class maytansine (EP 1391213；Liu et al., Proc.Natl.Acad.Sci.USA 93: 8618-8623 (1996)) and calicheamicin (calicheamicin) (Lode et al., Cancer Res.58:2928(1998)； Hinman et al., Cancer Res.53:3336-3342(1993)).Toxin can be by including tubulin binding, DNA combinations Or its cytotoxicity of the mechanisms play of topoisomerase enzyme level and cell-growth inhibitory effect.Some cytotoxic drugs tend to When being bound to larger antibody or protein receptor ligand to be inactive or weak active.

Workable enzyme activity toxin and its fragment include such as nonbinding active fragments of diphtheria A chains, diphtheria toxin, outer Toxin A chains (coming from Pseudomonas aeruginosa (Pseudomonas aerugiNOa)), ricin A chains, abrin A chains, not Enlightening element A chains, α-sarcin (α-sarcin), tung oil tree (Aleurites fordii) albumen, carnation albumen, dyers' grapes (Phytolaca americana) albumen (PAPI, PAPII and PAP-S), balsam pear (momordica charantia) inhibitor, It is curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibitor, white Tree plain (gelonin), mitogen (mitogellin), restrictocin (restrictocin), phenomycin, enomycin And mycotoxin (tricothecene) (enomycin).The WO 93/21232 announced see, for example, on October 28th, 1993.

Also cover anti-E7MC antibody moieties and one or more small molecule toxins herein, as calicheamicin, class maytansine, Aplysiatoxin (dolastatin), auspicious statin (aurostatin), crescent toxin and CC1065 difficult to understand, and there is this of neurotoxin active The anti-E7MC immunoconjugates of the derivative of a little toxin.

In some embodiments, there is provided the anti-E7MC immunoconjugates comprising the therapeutic agent with intracellular activity.One In a little embodiments, anti-E7MC immunoconjugates, to block the protein of cell to synthesize, cause wherein through internalization and therapeutic agent The cytotoxin of cell death.In some embodiments, therapeutic agent is comprising with the ribosomes not polypeptide of activating activities Cytotoxin, including such as Bai Shusu, Bo Ganning (bouganin), Sha Boning (saporin), ricin (WA), ricin A Chain, bryoidin, diphtheria toxin, restrictocin, Pseudomonas aeruginosa Exotoxin A and its variant.In some embodiments, when controlling Treat agent be comprising with ribosomes not the cytotoxin of the polypeptide of activating activities when, anti-E7MC immunoconjugates must be bound to Through internalization so that protein is cytotoxicity for cell during target cell.

In some embodiments, there is provided destroy the anti-E7MC immunoconjugates of the therapeutic agent of DNA effects comprising it.One In a little embodiments, the therapeutic agent for playing destruction DNA is selected from the group consisted of：Enediyne (such as calicheamicin And Ai Sipeila mycins) and non-enediyne small molecule agent (such as bleomycin methidium propyl group-EDTA-Fe (II)).According to this Shen Other cancer therapeutic agents that please be applicable include but is not limited to daunomycin, adriamycin, Distacin, cis-platinum, mitomycin C, Ecteinascidin, times carcinomycin (duocarmycin)/CC-1065 and bleomycin/send Lay mycin.

The present invention covers in addition is formed at anti-E7MC antibody moieties and compound (such as the ribose core with nuclear decomposition activity Sour enzyme or DNA endonuclease, such as deoxyribonuclease；DNA enzymatic) between anti-E7MC immunoconjugates.

In some embodiments, anti-E7MC immunoconjugates have included the medicament for destroying tubulin effect.Such medicine Agent may include such as nitragin/maytansine, Paclitaxel, vincristine and vincaleukoblastinum, colchicin, Rui Tatinghai difficult to understand Rabbit toxin 10MMAE and peloruside A.

In some embodiments, anti-E7MC immunoconjugates include alkylating agent, including such as Asaley NSC 167780th, AZQ NSC 182986, BCNU NSC 409962, busulfan NSC 750, carboxyl phthalic acid platinum NSC 271674th, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, Chlorambucil NSC 3088, chlorine Urea rhzomorph NSC 178248, cis-platinum NSC 119875, clomesone NSC 338947, cyano group (N- morpholinyls) adriamycin NSC 357704th, inferior (cyclodisone) NSC 348948 of cedi, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, extra large law popularization Nurse (hepsulfam) NSC 329680, hycanthone NSC 142982, melphalan NSC 8806, Methyl CCNU NSC 95441st, mitomycin C NSC 26980, rice support azoles acid amides NSC 353451, mustargen NSC 762, PCNU NSC 95466, piperazine Piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycin NSC 56410, in spiral shell second Uride mustard NSC 172112, teroxirone (teroxirone) NSC 296934, four platinum NSC 363812, thiotepa NSC 6396th, triethylenemelanin NSC 9706, uracil mastard NSC 34462 and Yoshi-864NSC 102627.

In some embodiments, the cancer therapeutic agent part of the anti-E7MC immunoconjugates of the application, which can include to resist, silk Disintegrating agent, including but not limited to allocolchicine NSC 406042, halichondrin B NSC 609395, colchicin NSC 757th, colchicine derivative NSC 33410, aplysiatoxin 10NSC 376128 (NG- auspicious statin derivatives difficult to understand), maytansine NSC 153858th, nitragin NSC 332598, taxol NSC 125973, paclitaxel derivatives NSC 608832, thio colchicum Alkali NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842 and vincristine sulphate NSC 67574。

In some embodiments, anti-E7MC immunoconjugates include topoisomerase I inhibitor, including but not limited to Camptothecine NSC 94600, camptothecine, Na salt NSC 100880, amino camptothecin NSC 603071, camptothecin derivative NSC 95382nd, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, happiness Alkali derivant NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecine is set to derive Thing NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172nd, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, Camptothecin derivative NSC 610457, camptothecin derivative NSC610459, camptothecin derivative NSC 606499, camptothecine spread out Biological NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497 and morpholinyl adriamycin NSC 354646。

In some embodiments, anti-E7MC immunoconjugates include Topoisomerase II inhibitors, including (but it is unlimited In) adriamycin NSC 123127, Amonafide (amonafide) NSC 308847, m-AMSA NSC 249992, anthracene pyrazoles spread out Biological NSC 355644, Pai Laruiding (pyrazoloacridine) NSC 366140, bisantrene (bisantrene) HCL NSC 337766th, daunomycin NSC 82151, deoxy doxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril (menogaril) NSC 269148, N, N- benzhydryl daunomycin NSC 268242, oxanthrazole NSC 349174, Daunomycin phenylhydrazone NSC 164011, VM-26NSC 122819 and VP-16NSC 141540.

In some embodiments, anti-E7MC immunoconjugates include RNA or DNA antimetabolites, including but not limited to L- alanopsin NSC 153353, U-18496 NSC 102816,5 FU 5 fluorouracil NSC 19893, Acivicin (acivicin) NSC 163501, aminopterin-induced syndrome derivative NSC 132483, aminopterin-induced syndrome derivative NSC 184692, amino are talked endlessly Purine derivative NSC 134033, antifol NSC 633713, antifol NSC 623017, bayesian (Baker's) are solvable anti- Folic acid agent NSC 139105, two chlorallyl lawsone NSC 126771, cloth quinoline that (brequinar) NSC 368390, replace Fluorine (prodrug) NSC 148958,5,6- dihydros-U-18496 NSC 264880, methotrexate (MTX) NSC 740, methotrexate (MTX) is added to derive Thing NSC 174121, N- (phosphonoacetyl)-L-Aspartic acid (PALA) NSC 224131, pyrazofurin NSC 143095th, Trimetrexate (trimetrexate) NSC 352122,3-HP NSC 95678,2'- deoxidations -5-FUD NSC 27640th, 5-HP NSC 107392, α-TGDR NSC 71851, glycine aphidicolin NSC 303812, cytarabine NSC 63878th, 5- azepines -2'- deoxycytidines NSC 127716, β-TGDR NSC 71261, ancitabine NSC 145668, guanazole NSC 1895th, hydroxycarbamide NSC 32065, inosine glycolaldehyde NSC 118994, macbecin Il NSC 330500, pyrazolo imidazoles NSC 51143rd, thioguanine NSC 752 and thio-purine NSC 755.

In some embodiments, anti-E7MC immunoconjugates include high radioactive atom.A variety of radio isotopes Available for the antibody for producing radioactivity combination.Example include 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb and Lu radio isotope.

In some embodiments, anti-E7MC antibody moieties can be bound to " acceptor " (such as streptavidin), with In being targetted in advance for tumour, wherein to patient's administration of antibodies-acceptor conjugate, then uncombined is conjugated using scavenger Removed in thing self-loopa, and then apply " part " (such as the antibiosis for being bound to cytotoxic agent (such as radioactive nucleotides) Fibroin).

In some embodiments, anti-E7MC immunoconjugates can include the anti-E7MC antibody portion for being bound to pro-drug activating enzyme Point.In some such embodiments, pro-drug activating enzyme is by prodrug (such as peptidyl chemotherapeutic agent, referring to WO 81/01145) It is converted into active medicine, such as cancer therapy drug.In some embodiments, such anti-E7MC immunoconjugates be applied to antibody according to Rely the prodrug therapy (" ADEPT ") of property enzyme mediation.The enzyme that antibody can be bound to includes but is not limited to alkaline phosphatase, and it is applicable In the prodrug of phosphoric acid ester group is transformed into free drug；Aryl sulfatase, it is applied to turn the prodrug of sulfur-bearing perester radical Become free drug；Cytosine deaminase, it is applied to nontoxic 5-flurocytosine being transformed into cancer therapy drug 5 FU 5 fluorouracil；Egg White enzyme, such as Serratia protease, thermolysin, subtilopeptidase A, carboxypeptidase and cathepsin are (such as Cathepsin B and L), it is transformed into free drug suitable for that will contain peptide prodrug；D- propylamine acyl group carboxypeptidases, it is applied to Prodrug of the transformation containing D- 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor bases；Carbohydrate-cleaving enzyme, such as beta galactose and neuraminidase, it is suitable For glycosylated prodrugs to be transformed into free drug；Beta-lactamase, it is applied to through medicine derived from beta-lactam to change Into free drug；And penicillin amidase, such as Penicillin-V-Amidase and Penicillin-G-amidases, it is applied to respectively to exist Amine nitrogen is transformed into free drug through medicine derived from nitrophenoxyacetyl or phenethyl.In some embodiments, enzyme can be by Recombinant DNA technology covalent binding antibodies part well known in the art.See, for example, Neuberger et al., Nature 312:604- 608(1984)。

In some embodiments, the treatment part of anti-E7MC immunoconjugates can be nucleic acid.Workable nucleic acid includes (but not limited to) antisense RNA, gene or other polynucleotides, including nucleic acid analog, such as thioguanine and thio-purine.

The application provides the anti-E7MC immunoconjugates for including the anti-E7MC antibody moieties for being attached to effector molecule in addition, its Middle effector molecule is mark, and it can directly or indirectly produce detectable signal.These anti-E7MC immunoconjugates can be used for studying Or diagnostic application, as being used for inside cancer detecting.Mark is preferably able to directly or indirectly produce detectable signal.Citing and Speech, mark can be radiopaque or radio isotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I；Fluorescence (fluorescence Group) or chemiluminescence (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or fluorescein；Enzyme, as alkaline phosphatase, Beta galactose or HRPO；Developer；Or metal ion.In some embodiments, labeled as scintigraphy The radioactive atom of research, such as 99Tc or 123I, or for nuclear magnetic resonance (NMR) imaging (being also known as magnetic resonance imaging, MRI) Spin labeling, such as zirconium -89, iodo- 123, iodine -131, indium -111, fluoro- 19, carbon -13, nitrogen -15, oxygen -17, gadolinium, manganese or iron.Zirconium- 89 can be complexed with various metal-chelators and be bound to antibody, such as be imaged (WO 2011/056983) for PET.

In some embodiments, can the anti-E7MC immunoconjugates of indirect detection.For example, E7MC immunoconjugates are resisted Thing has specificity and the secondary antibody containing detectable label can be used for detecting anti-E7MC immunoconjugates.

Therefore, for example, in some embodiments, there is provided include following anti-E7MC immunoconjugates：A) it is special Property combine the anti-E7MC antibody moieties of the complex comprising HPV16-E7 peptides and MHC I proteinoid, and b) effector molecule.One In a little embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4).In some embodiments, MHC I classes Protein is HLA-A02.In some embodiments, MHC I proteinoid is HLA-A*02:01.In some embodiments, Effector molecule is covalently attached to anti-E7MC antibody moieties.In some embodiments, effector molecule is selected from for example by with the following group Into group therapeutic agent：Medicine, toxin, radio isotope, protein, peptide and nucleic acid.In some embodiments, effect point Son is cancer therapeutic agent.In some embodiments, cancer therapeutic agent is chemotherapeutant.In some embodiments, cancer Therapeutic agent is the high radioactive atom selected from the group for example consisted of：²¹¹At、¹³¹I、¹²⁵I、⁹⁰Y、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、³²P and²¹²Pb.In some embodiments, effector molecule is mark, and it can directly or indirectly produce detectable letter Number.In some embodiments, labeled as the radio isotope selected from the group for example consisted of：³H、¹⁴C、³²P、³⁵S 、¹²³I、¹²⁵I and¹³¹I.In some embodiments, anti-E7MC antibody moieties are scFv.In some embodiments, anti-E7MC resists Body portion is the mankind, humanization or semi-synthetic.In some embodiments, anti-E7MC antibody moieties with least one (as extremely It is few any one of 2,3,4,5 or 6) comprising MHC I proteinoid and with a 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, (such as conserved amino acid takes Generation) HPV16-E7 peptides variant complex cross reaction.In some embodiments, anti-E7MC antibody moieties and at least one The complex of the individual different subtype of (such as any one of at least 2,3,4 or 5) comprising HPV16-E7 peptides and MHC I proteinoid Cross reaction.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes HPV16-E7 11-19 peptides (SEQ ID NO:And HLA-A*02 4):The anti-E7MC antibody moieties of 01 complex, and b) effect point Son.In some embodiments, effector molecule is covalently attached to anti-E7MC antibody moieties.In some embodiments, effect point Son is the therapeutic agent selected from the group for example consisted of：Medicine, toxin, radio isotope, protein, peptide and nucleic acid. In some embodiments, effector molecule is cancer therapeutic agent.In some embodiments, cancer therapeutic agent is chemotherapeutant. In some embodiments, cancer therapeutic agent is the high radioactive atom selected from the group for example consisted of：²¹¹At、¹³¹I 、¹²⁵I、⁹⁰Y、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、³²P and²¹²Pb.In some embodiments, effector molecule is mark, and it can be straight Connect or produce detectable signal indirectly.In some embodiments, labeled as the radioactivity selected from the group for example consisted of Isotope：³H、¹⁴C、³²P、³⁵S、¹²³I、¹²⁵I and¹³¹I.In some embodiments, anti-E7MC antibody moieties are scFv.At some It is that anti-E7MC antibody moieties are behaved, humanization or semi-synthetic in embodiment.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, or it is included at most about 3 (in e.g., from about 1,2 or 3 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence Arrange, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or it includes at most about 3 (e.g., from about 1,2 or 3 Any one of) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, include SEQ ID comprising LC-CDR1, the LC-CDR1 NO:189 or 190 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or it includes at most about 3 (e.g., from about 1st, any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188's Amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 ammonia Base acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, and b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variable district, it includes HC- CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence, or it includes at most about 5 (such as from about 1st, any one of 2,3,4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:78-98 Any one of amino acid sequence, or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant, and HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245 Arrange, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable Area, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence, or It includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 bags The NO of ID containing SEQ:Any one of 141-161 amino acid sequence, or it includes at most about 3 (any in such as from about 1,2 or 3 ) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Appointing in 162-182 and 247-250 The amino acid sequence of one, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, And b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 Include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO: The amino acid sequence of any one of 99-119,244 and 245；Or it includes at most about 5 (any in such as from about 1,2,3,4 or 5 ) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；Or it is individual comprising at most about 5 (any one of such as from about 1,2,3,4 or 5) The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in LC-CDR sequences；And b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising i) weight chain variabl area sequence, it is included HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, the HC-CDR2 Include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO: The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC-CDR1, the LC- CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:162-182 With any one of 247-250 amino acid sequence；And b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, comprising weight chain variable district, it includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or its have at least about 95% (for example, at least about 96%, 97%th, any one of 98% or 99%) variant of sequence identity, and light chain variable district, it includes SEQ ID NO:36-56 With any one of 238-243 amino acid sequence, or its have at least about 95% (for example, at least about 96%, 97%, 98% or Any one of 99%) variant of sequence identity, and b) effector molecule.

In some embodiments, there is provided include following anti-E7MC immunoconjugates：A) specific binding includes The anti-E7MC antibody moieties of HPV16-E7 peptides and the complex of MHC I proteinoid, it includes weight chain variable district, includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, include SEQ ID NO:36-56 and Any one of 238-243 amino acid sequence；And b) effector molecule.

Nucleic acid

Also the nucleic acid molecules for encoding anti-E7MC constructs or anti-E7MC antibody moieties are covered.In some embodiments, carry For the nucleic acid (or nucleic acid set) of the anti-E7MC antibody of encoding full leng.In some embodiments, there is provided coding polyspecific resists E7MC molecules (such as the anti-E7MC antibody of polyspecific, the anti-E7MC antibody of bispecific or bispecific T cell joint molecule resist E7MC antibody) or its polypeptide portion nucleic acid (or nucleic acid set).In some embodiments, there is provided encode anti-E7MC CAR's Nucleic acid (or nucleic acid set).In some embodiments, there is provided encode anti-E7MC immunoconjugates, or the nucleic acid of its polypeptide portion (or nucleic acid set).

The application also includes the variant of these nucleotide sequences.For example, variant is included under appropriate stringent hybridization condition It is hybridized to the nucleotide sequence of the anti-E7MC constructs of coding the application or the nucleotide sequence of anti-E7MC antibody moieties.

The present invention also provides the carrier of the nucleic acid inserted with the present invention.

For simplified summary, resist by the natural or synthetic expression of nucleic acid for encoding anti-E7MC constructs or its polypeptide portion E7MC constructs (such as anti-E7MC CAR) or its polypeptide portion can be inserted into appropriate expression vector by by nucleic acid so that core Acid is operably connected to 5' and 3' regulating elements, including for example promoter (such as lymphocyte specific promoter) and 3' are non- Translated region (UTR) and realize.Carrier is suitably adapted for duplication and integration in eukaryotic host cell.Clonotypic and expression vector contain There are transcription and translation termination, the homing sequence and promoter for wanting nucleotide sequence to express suitable for regulation.

The nucleic acid of the present invention also may be used in the nucleic acid immunization and gene therapy of standard gene delivering scheme.Gene Delivering method is known in the art.See, for example, U.S. Patent No. 5,399,346, No. 5,580,859, the 5,589,466th Number, it is incorporated herein in entirety by reference.In some embodiments, the present invention provides gene therapy vector.

Nucleic acid can be cloned into polytype carrier.For example, nucleic acid can be cloned into carrier, including (but it is unlimited In) plastid, phasmid, phage-derived thing, animal virus and clayey body.Especially concerned carrier includes expression vector, answered Carrier, probe generation vectors and sequencing carrier processed.

In addition, expression vector can be supplied to cell in the form of viral vector.It is that viral vector technology is well known in the art and It is described in such as Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manual in.It is suitable for the virus bag of carrier Include (but not limited to) retroviruse, adenovirus, adeno-associated virus, herpesviral and slow virus.In general, it is adapted to carrier to contain Have at least one organism, promoter sequence, convenient limiting acid endo enzyme site and one or more optional labels In effective replication orgin (see, for example, WO 01/96584；WO 01/29058；And U.S. Patent No. 6,326,193).

Multiple systems based on virus are developed into mammalian cell for gene transfer.For example, invert Record virus provides the convenient platform of genes delivery system.Selected gene is inserted into middle carrier and uses skill known in the art Art is packaged in retroviruse particle.Recombinant virus can after through separation and it is in vivo or in vitro be transferred to individual cell in. Multiple retroviral systems are known in the art.In some embodiments, using adenovirus vector.Multiple adenovirus vectors It is known in the art.In some embodiments, using slow virus carrier.From the load of retroviruse (such as slow virus) Body is the suitable instrument for realizing long-term gene transfer, because it allows integration transgenosis steady in a long-term and its biography in daughter cell Broadcast.Slow virus carrier, which has, to be better than deriving from cancer-retroviruse (onco-retrovirus) (such as Murine Leukemia Virus) Carrier additional advantage because its transducible nonproliferating cell, such as liver cell.It is also extra with low immunogenicity Advantage.

Additional Promoters element (for example, enhancer) adjusts transcription initiation frequency.Generally, these elements are positioned at start bit In point upstream 30-110bp areas, although multiple promoters have shown that the functional elements also contained in initiation site downstream recently. The spacing started between daughter element is usually flexible so that when element is inverted or is moved relative to each other and is constantly retained promoter Function.In thymidine kinase (tk) promoter, the spacing started between daughter element can increase to phase before activity starts decline Every 50bp.

An example for being adapted to promoter is early stage cytomegalovirus (CMV) promoter sequence at once.This promoter sequence For high level can be driven to express the strong constitutive promoter sequence of any polynucleotide sequence being operably coupled to thereon. Be adapted to promoter it, another example be the α of the elongation growth factor -1 (EF-1 α).However, other constitutive promoters also can be used Sequence, including but not limited to simian virus 40 (SV40) early promoter, MMTV (MMTV), human immunity lack Weary virus (HIV) LTR (LTR) promoter, MoMuLV promoters, avian leukosis viruses promoter, Ai-bar Er Shi viruses early promoter, rous sarcoma virus promoter (Rous sarcoma virus promoter) at once, Yi Jiren Genoid promoter, such as (but not limited to) actin promoter, Myosin promoter, Hemoglobin promoter and creatine Kinase promoter.In addition, the present invention should be not limited to the use of constitutive promoter.Also inducible promoter is covered as the present invention Part.The use of inducible promoter provides molecular switch, and the molecular switch can open it when needing such expression can The polynucleotide sequence expression being operatively connected or the closing expression when that need not express.The example of inducible promoter includes (but not limited to) metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline promoter.

In order to assess the expression of polypeptide or part thereof, the expression vector in cell to be introduced can also contain optional marker gene Or reporter gene or both is differentiated from the cell colony for trying to transfect or infect through viral vector with promotion and selects expression cell. In in other respects, optional label can be carried in independent DNA sections and be used for cotransfection program.Optional label and report body Gene all can the appropriate regulatory sequence of side joint to allow to express in host cell.Being applicable optional label includes for example resistance to antibiosis Plain gene, such as neo and the like.

Reporter gene be used for differentiate it is potential through transfectional cell and assess regulatory sequence function.In general, reporter gene is Receive to be not present or express in organism or tissue and coding schedule reaches the characteristic (such as enzymatic activity) easily detected by some The gene of the polypeptide shown.The expression of suitable time series analysis reporter gene after DNA is had been introduced into recipient cell.It is adapted to report Accusing gene may include coding fluorescence element enzyme, beta galactose, chloramphenicol acetyltransferase, secreting alkaline phosphorus phytase or green fluorescence Gene (such as Ui-Tel et al., the 2000FEBS Letters 479 of GFP:79-82).It is known to be adapted to expression system And can be used known technology prepare or it is commercially available.In general, the minimum 5' with displaying reporter gene highest expression quantity It is promoter that the construct in side joint area, which differentiates,.Such promoter region is connectable to reporter gene and for assessing regulation promoter The reagent of the transcriptional capability of driving.

Introduced into cell and the method for expressing gene is known in the art.In the case of expression vector, carrier can be easy In being introduced into by any method of this area in host cell (for example, mammal, bacterium, yeast or insect cell).Citing For, expression vector can be transferred in host cell by physics, chemistry or biological mode.

Physical method for being introduced to polynucleotides in host cell includes calcium phosphate precipitation, liposome transfection, grain Sub- bombardment, microinjection, electroporation and its similar approach.For manufacturing the side of the cell comprising carrier and/or Exogenous Nucleic Acid What method was well known in the art.See, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York).In some embodiments, turn by calcium phosphate Contaminate and be introduced in host cell into being about to polynucleotides.

By the biological method that polynucleotides of interest are introduced into host cell including the use of DNA and RNA carriers.Virus carries Body and especially retrovirus vector turn into the most popular method for inserting gene in mammal (for example, mankind) cell. Other viral vectors can derive from slow virus, poxvirus, herpes simplex types 1 virus-virus, adenovirus and adeno-associated virus and its Similar virus.See, for example, U.S. Patent No. No. 5,350,674 and No. 5,585,362.

Chemical mode for being introduced to polynucleotides in host cell includes dispersion system of colloid, and such as macromolecular is answered Zoarium, Nano capsule, microballoon, bead and the system based on lipid, including oil-in-water emulsion, micella, mixing micella and liposome. Exemplary colloid system as external and internal transmission mediator is liposome (such as artificial film bubble).

In the case of using non-viral delivery system, exemplary transmission mediator is liposome.It is expected that prepared using lipid Nucleic acid is introduced to host cell (external, in vitro or internal) by agent.In another aspect, nucleic acid can be relevant with lipid.With lipid Associated nucleic acid can be encapsulated in the aqueous interior of liposome, be interspersed in the double-layer of lipoid of liposome, via with liposome And the associated connection molecule of oligonucleotides is attached to liposome, is coated in liposome, and lipid bluk recombination, be scattered in containing In the solution of lipid, mix with lipid, combined with lipid, is contained in form of suspension in lipid, answered containing micella or with micella Close, or it is otherwise associated with lipid.The lipid associated with composition, lipid/DNA or lipid/expression vector are unlimited Any specific structure in solution.For example, it may be present in double-decker, in micella form or with " collapsing " knot Structure.It simply can be also interspersed in solution, it is possible to create size or the uneven aggregation of shape.It can be naturally to deposit that lipid, which is, In lipid or the fatty material of synthesis lipid.For example, lipid includes naturally occurring fat drop in cytoplasm and contained The compounds category (such as aliphatic acid, alcohol, amine, amino alcohol and aldehyde) of long chain aliphatic hydrocarbons and their derivates.

No matter for Exogenous Nucleic Acid to be introduced into host cell or otherwise makes suppression of the cell exposed to the present invention The method of agent, in order to confirm the presence of recombinant DNA sequence in host cell, a variety of analyses can be carried out.Such analytic approach includes example " molecular biosciences " analytic approach as well known to the skilled person, such as south and Northen traces, RT-PCR and PCR；It is " raw Thing chemistry " analytic approach, such as detects the existence or non-existence of particular peptide, such as by immunization wayses (ELISA and western-blot) Or differentiate the reagent in scope of the invention by analytic approach as described herein.

MHC I proteinoid

MHC I proteinoid is that one of major histocompatibility complex (MHC) molecule of two kinds of primary categories is (another One is MHC II classes) and be found on almost each karyoblast of body.Its function is display into the cell to the egg of T cell White matter fragment；To be neglected one's health cell, and the cell containing extraneous protein will be by immune system attack.Due to MHC I class eggs White matter presents the peptide derived from cell lysis matter protein, and MHC I classes present path and are commonly referred to as cell lysis matter or endogenous path.I classes MHC molecule combines the peptide for the degraded (by proteasome) for being mainly produced from cell lysis matter protein.MHC I:Peptide complex is then It is inserted into the plasma membrane of cell.Peptide is bound to the extracellular part of I class MHC molecules.Therefore, I classes MHC function is by intracellular egg White matter is presented to cytotoxic T cell (CTL).It is produced from however, I classes MHC can also be presented in the method for referred to as cross presentation The peptide of exogenous proteins.

MHC I proteinoid is by two polypeptide chains, α and B2M (β 2M) composition.Two chains are via b2m and the domains of α 3 Interaction non-covalent linking.Only α chains are polymorphic and by HLA gene codes, and b2m subunits are not polymorphic and by β -2 Microglobulin gene encodes.The domains of α 3 interact across plasma membrane and with the CD8 co-receptors of T cell.α 3-CD8 interact MHC I molecules are held in position in, and the φt cell receptor (TCR) on cytotoxic T cell surface combines its α 1- α 2 heterogeneous two Aggressiveness part, and check the antigenicity of coupling peptide.α 1 and the domains of α 2 fold and form groove, are combined for peptide.MHC I proteinoid With reference to the peptide that length is 8-10 amino acid.

Human leukocyte antigens (HLA) gene is mankind's pattern of mhc gene.Three kinds of main MHC I class eggs in human body White matter is HLA-A, HLA-B and HLA-C, and 3 kinds of secondary MHC I proteinoid are HLA-E, HLA-F and HLA-G.HLA-A ranks In human body in the gene with most fast evolution coded sequence.By in December, 2013,2432 kinds of codings, 1740 kinds of active eggs be present White matter and 117 kinds of known HLA-A allele for rejecting formula protein (null protein).HLA-A genes are located at chromosome 6 Galianconism on and coding HLA-A larger α chains composition.The change of HLA-A α-chain is most important for HLA functions.This change promotees The gene diversity entered in colony.Because each HLA has a different affinity for the peptide of some structures, more a variety of HLA mean compared with A variety of antigens ' presentation ' are in the possibility that on cell surface, increase colony's subgroup will have resistance to any given external invader. This reduce the possibility that single pathogen has the ability for eliminating whole human colony.Each individual can express at most two types HLA-A, each a kind of in its parent.Some individuals will inherit same HLA-A from two parents, reduce its individual HLA diversity；However, most of individuals will receive HLA-A two kinds of different duplicates.All HLA groups follow this model identical.Change Yan Zhi, individual can only express one of HLA-A allele person or both known to 2432 kinds.

All allele receive at least four digital sorts, such as HLA-A*02:12.A is represented belonging to allele HLA genes.Many HLA-A allele be present so that make classification eases by the classification of serotype.It is next that numeral is indicated This distribution.For example, HLA-A*02:02、HLA-A*02:04 and HLA-A*02:324 all A2 serotypes member (by Indicated by * 02 prefix).This group is to cause the Main Factors of HLA compatibilities.Hereafter all numerals can not be surveyed by serotype Determine and indicated via gene sequencing.Which kind of HLA protein second group of numeral instruction produces.These with discovery order specify and by In December, 2013,456 kinds of different known HLA-A02 protein be present and (specify title HLA-A*02:01 to HLA-A*02: 456).Most short possible HLA titles include both wholes in these details.Each extension in addition represents to change The nucleotides change of protein.

In some embodiments, anti-E7MC antibody moieties specific binding includes HPV16-E7 peptides and MHC I albuminoids The complex of matter, wherein MHC I proteinoid are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.In some implementations In scheme, MHC I proteinoid is HLA-A, HLA-B or HLA-C.In some embodiments, MHC I proteinoid is HLA- A.In some embodiments, MHC I proteinoid is HLA-B.In some embodiments, MHC I proteinoid is HLA- C.In some embodiments, MHC I proteinoid be HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11、HLA-A19、HLA-A23、HLA-A24、HLA-A25、HLA-A26、HLA-A28、HLA-A29、HLA-A30、HLA- A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74 or HLA-A80.In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I albuminoids Matter is HLA-A*02:Any one of 01-555, such as HLA-A*02:01、HLA-A*02:02、HLA-A*02:03、HLA-A*02: 04、HLA-A*02:05、HLA-A*02:06、HLA-A*02:07、HLA-A*02:08、HLA-A*02:09、HLA-A*02:10、 HLA-A*02:11、HLA-A*02:12、HLA-A*02:13、HLA-A*02:14、HLA-A*02:15、HLA-A*02:16、HLA- A*02:17、HLA-A*02:18、HLA-A*02:19、HLA-A*02:20、HLA-A*02:21、HLA-A*02:22 or HLA-A* 02:24.In some embodiments, MHC I proteinoid is HLA-A*02:01.HLA-A*02:01 be expressed in 39-46% it In all Caucasians, and therefore represent the suitable selection of the MHC I proteinoid for the present invention.

Can be for example based on using known to those skilled in the art suitable for the HPV16-E7 peptides for producing anti-E7MC antibody moieties The proteasome of Computer Prediction model and the HLA-A*02 of immunoproteasome:The presence of 01 binding motif and cracking site and It is determined that.For predicting MHC binding sites, this class model includes but is not limited to IEDB (Vita et al., The immune Epitope database (IEDB) 3.0.Nucleic Acids Res.2014 .pii on October 9:gku938)、 ProPred1 (is described in greater detail in Singh and Raghava, ProPred:prediction of HLA-DR binding sites.BIOINFORMATICS17(12):In 1236-1237,2001) and SYFPEITHI (referring to Schuler et al. SYFPEITHI,Database for Searching and T-Cell Epitope Prediction.Immunoinformatics Methods in Molecular Biology, the 409th (1) volume:75-93, 2007)。

Once identify appropriate peptide, you can peptide symthesis is completed according to scheme well known to those skilled in the art.The present invention's Peptide can directly be synthesized in the solution or on solid carrier due to its relative small size according to known peptide symthesis technology.It is various from Dynamic circuit connector is grown up to be a useful person to be commercially available and can be used according to known arrangement.Synthetic peptide has turned into large-scale production synthetic peptide in solution Authorized program and therefore for prepare the present invention peptide suitable alternative (see, for example, Solid Phase Peptide Synthesis, John Morrow Stewart and Martin et al. Application of Almez-mediated Amidation Reactions to Solution Phase Peptide Synthesis, Tetrahedron Letters Volume 39, the 1517-1520 pages, 1998).

The binding activity of candidate's HPV16-E7 peptides can be used antigen processing defect T2 cell line tests, the cell line when by Increase HLA-A expression during stabilized peptide in antigen presentation groove.T2 cells are enough to make on cell surface through candidate peptide impact HLA-A expresses the stabilized time, and any method measurement known in the art can be used in it, such as has spy by with to HLA-A Fluorescent-tagged mAbs (such as BB7.2) immunostaining of the opposite sex, then carry out fluorescence activated cell sorts (FACS) point Analysis.

Prepare anti-E7MC antibody and anti-E7MC antibody moieties

In some embodiments, anti-E7MC antibody or anti-E7MC antibody moieties are monoclonal antibody.Monoclonal antibody can Such as using hybridoma method, such as Kohler and Milstein, Nature, 256:495 (1975) and Sergeeva et al., Blood,117(16):The method of 4262-4272 descriptions, using the phage display method herein and described in Examples below, or Prepared using recombinant DNA method (see, for example, U.S. Patent No. 4,816,567).

In hybridoma method, hamster, mouse or other appropriate host animals are generally immunized with immunizing agent to produce lymph Cell, it, which produces or can produced, to specifically bind the antibody of immunizing agent.Or lymphocyte can be immunized in vitro.It is immune Agent may include the polypeptide or fusion protein of protein of interest, or include the complex of at least two molecules, such as comprising The complex of HPV16-E7 peptides and MHC I proteinoid.Generally, the if desired cell of human origin, then drenched using peripheral blood Bar cell (" PBL ")；Or if desired non-human mammal source, then using splenocyte or lymph node cells.Lymphocyte connects And merged using suitable fusion agent (such as polyethylene glycol) through immortalized cell line to form hybridoma.See, for example, Goding,Monoclonal Antibodies:Principles and Practice(New York:Academic Press, 1986), the 59-103 pages.Immortalized cell line is usually inverted mammalian cell, in specific words rodent, ox class and The myeloma cell of human origin.Generally use rat or mouse myeloma cell line.Hybridoma can preferably comprise one Kind or a variety of suppression do not merge, cultivated in the suitable culture medium of the material of the growth of immortalized cells or survival.For example, if Parental cell lacks enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the then culture for hybridoma Base is typically included hypoxanthine, aminopterin and thymidine (" HAT culture mediums "), and it prevents the cell growth for lacking HGPRT.

In some embodiments, immortalized cell line effective integration, support that antibody is steady by selected antibody producing cell Determine high content expression and for the culture medium sensitivity of such as HAT culture mediums.In some embodiments, immortalized cell line is muroid Myeloma cell line, its can for example obtained from Salk Institute Cell Distribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia.Describe to be used to produce The human marrow knurl of human monoclonal antibody and the miscellaneous myeloma cell line of mouse-human.Kozbor,J.Immunol.,133: 3001(1984)；Brodeur et al. Monoclonal Antibody Production Techniques and Applications(Marcel Dekker,Inc.:New York, 1987) the 51-63 pages.

Then the culture medium of wherein culture hybridoma, the monoclonal can be analyzed for the presence of monoclonal antibody Antibody system is directed to polypeptide.(radiommunoassay (RIA) or enzyme-linked it can such as exempt from by immunoprecipitation or by external binding analysis Epidemic disease adsorption analysis (ELISA)) measure as caused by hybridoma monoclonal antibody binding specificity.The technology and analysis Method is known in the art.The binding affinity of monoclonal antibody can for example by Munson and Pollard, Anal.Biochem., 107:The Scatchard analysis measure of 220 (1980).

After hybridoma needed for discriminating, clone can be subcloned by limitation dilution program and be given birth to by standard method It is long.Goding, it is foregoing.Suitable culture medium for this purpose includes such as Du Erbeikeshi modified Eagle mediums (Dulbecco's Modified Eagle's Medium) and RPMI-1640 culture mediums.Or hybridoma can be with ascites Form tumor growth is in mammal.

The secreted monoclonal antibody of subclone can be by known immunoglobulin purification procedure (such as protein A-Sepharose Sugar, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography) from culture medium or ascites fluid isolated or purified.

Anti- E7MC antibody or antibody moiety also can be by for combining with antibody screening active needed for one or more Library and differentiate.For example, a variety of methods known in the art are used to produce phage display library and for required combination The such library of antibody screening of feature.Such method survey is in such as Hoogenboom et al., Methods in Molecular Biology 178:In 1-37 (O'Brien et al. is compiled, Human Press, Totowa, NJ, 2001), and it is further described in example Such as McCafferty et al., Nature 348:552-554；Clackson et al., Nature 352:624-628(1991)； Marks et al., J.Mol.Biol.222:581-597(1992)；Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo is compiled, Human Press, Totowa, NJ, 2003)；Sidhu et al., J.Mol.Biol.338 (2):299-310(2004)；Lee et al., J.Mol.Biol.340 (5):1073-1093(2004)；Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；And Lee et al., J.Immunol.Methods 284(1-2):In 119-132 (2004).

In some bacteriophages methods of exhibiting, the pedigree of VH and VL genes is cloned by polymerase chain reaction (PCR) respectively And recombinated at random in phage library, then can be such as Winter et al., Ann.Rev.Immunol.12:In 433-455 (1994) It is described, screened for antigen binding bacteriophage.Bacteriophage typically exhibits antibody fragment, in scFv (scFv) pieces Or in Fab pieces.Library from immune origin provides the former high-affinity antibody of anti-immunity without constructing hybridoma. Or original pedigree (such as from mankind) can be cloned to provide for broad range of non-self-antigen and self-antigen Monospecific antibody source is without any immunity inoculation, such as Griffiths et al., EMBO J, and 12:725-734 (1993) is described.Most Afterwards, primary libraries can also synthesis mode prepare as follows：From the non-rearranged V-genes section of stem cell clone, and using containing random Sequence is with code level variable C DR3 areas and realizes the PCR primer reset in vitro, such as Hoogenboom and Winter, J.Mol.Biol.,227:381-388 (1992) is described.Describing the patent publication of human antibodies phage library is included for example： U.S. Patent No. 5,750,373 and U.S. Patent Publication case the 2005/0079574th, No. 2005/0119455, No. 2005/0266000, No. 2007/0117126, No. 2007/0160598, No. 2007/0237764, the 2007/th No. 0292936 and No. 2009/0002360.

Antibody or its antigen-binding fragment phage display can be used to prepare with for be specific to include HPV16-E7 peptides and The antibody screening library of the complex of MHC I proteinoid.Library can be with least 1 × 10⁹(such as at least about 1 × 10⁹、2.5 ×10⁹、5×10⁹、7.5×10⁹、1×10¹⁰、2.5×10¹⁰、5×10¹⁰、7.5×10¹⁰Or 1 × 10¹¹Any one of) individual only The multifarious mankind scFv phage display libraries of special human antibodies fragment.In some embodiments, library is to construct certainly DNA untreated human library, the DNA extract from the mankind PMBC from healthy donors and spleen, forgive all human heavy chains and Light chain subfamily.In some embodiments, library is to construct the untreated human library from DNA, and the DNA is from various diseases Patient, such as PBMC extractions with the patient of autoimmune disease, cancer patient and patient's separation with infectious diseases. In some embodiments, library is semi-synthetic human library, and wherein heavy chain CDR3 is completely random, all amino acid (in addition to cysteine) be comparably likely to be present in any given position (see, for example, Hoet, R.M. et al., Nat.Biotechnol.23(3):344-348,2005).In some embodiments, the heavy chain CDR3 of semi-synthetic human library Length be about 5 to about 24 (such as from about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 Any one of) individual amino acid.In some embodiments, library is non-human phage display library.

E7MC phage clone is bound to high-affinity to be selected as follows：It is bound to repeatedly by by bacteriophage E7MC, the E7MC are bound to solid carrier (as being used for the bead of solution level movement or being moved for the lactation of cellular level movement Thing cell), then remove non-binding bacteriophage and the elution by specific binding bacteriophage.In the example of solution level movement In, E7MC can be fixed to solid carrier through biotin labeling.The E7MC of biotin labeling and phage library and solid carrier, As streptavidin with reference to it wear promise bead M-280 mixing, and then E7MC- bacteriophages-bead complex through separation.Knot Phage clone is closed then through eluting and for infecting appropriate host cell, such as Escherichia coli XL1-Blue, for expressing and pure Change.In the example of cell elutriation, T2 cells (TAP defects, the HLA-A*02 of E7MC HPV16-E7 peptides are loaded with:01+ lymphs Mother cell cell line) mixed with phage library, cell is collected thereafter and combines clone infects appropriate host through eluting and being used for Cell (is used to express and purify).Elutriation can by solution elutriation, cell elutriation or both combination carry out it is multiple (such as from about 2,3, 4th, any of the above item in 5,6 or 6) bout is specifically bound to E7MC phage clone to be enriched with.Can be by this area Any method known, include the specific binding of the phage clone and E7MC of such as ELISA and FACS test enrichments.

Monoclonal antibody also can be by recombinant DNA method, those systems as described in U.S. Patent No. 4,816,567 .Known procedure can be used (such as can be specifically bound to coding by use for the DNA for encoding the monoclonal antibody of the present invention The oligonucleotide probe of the heavy chain of rodent antibody and the gene of light chain) it is easily separated and is sequenced.Hybridoma as described above Cell or the E7MC specific bacteriophages of the present invention may act as such DNA source.After separation, DNA can be placed in expression vector In, then transfect in host cell (such as monkey COS cells, Chinese hamster ovary (CHO) cell or myeloma cell) (otherwise The host cell will not produce immunoglobulin), obtain the synthesis of monoclonal antibody in recombinant host cell.DNA also can example Such as substitute homologous non-human's class sequence (United States Patent (USP) by with the coded sequence of human heavy chain and light-chain constant domains and/or framework region No. 4,816,567；It is Morrison et al., foregoing) or be total to by by all or part of coded sequence of NIg polypeptide Valency is bonded to immunoglobulin coding sequence and modified.Such NIg polypeptide may replace the constant of antibody of the present invention Domain, or may replace antibody of the present invention an antigen combination site variable region to produce chimeric bivalent antibody.

Antibody can be univalent antibody.Method for preparing univalent antibody is known in the art.For example, a kind of method It is related to the recombination expression of light chain immunoglobulin and the heavy chain through modification.Any point generally in Fc areas truncates heavy chain to prevent Heavy chain is crosslinked.Or relevant cysteines residue substitutes through another amino acid residue or prevents from being crosslinked through lacking.

In-vitro method is also suitable in preparing univalent antibody.Any method known in the art can be used to realize the digestion of antibody To produce its fragment, in specific words Fab fragments.

Antibody variable region (antibody-antigene combination site) with required binding specificity can be fused to immunoglobulin perseverance Domain sequences.It is preferred that merged with the heavy chain immunoglobulin constant domain comprising at least part of hinge, CH2 and CH3 areas. In some embodiments, the first heavy chain constant region (CH1) that required site is combined containing light chain is present in fusions extremely In one item missing.The DNA of encoding immune immunoglobulin heavy chain fusions thing and (if desired) light chain immunoglobulin is inserted into independent expression In carrier, and cotransfection is into Suitable host organisms.Other details for producing bispecific antibody, see, for example, Suresh et al., Methods in Enzymology, 121:210(1986).

The mankind and humanized antibody

Anti- E7MC antibody or antibody moiety can be humanized antibody or human antibodies.Humanization form non-human (such as Muroid) antibody is gomphosis immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F (ab') 2, scFv or anti- Other antigen binding subsequences of body), usually contain the minmal sequence derived from non-human immunoglobulin.Humanized antibody bag Include human immunoglobulin (recipient's antibody), wherein recipient CDR residue is by with wanting specificity, affinity and ability Non-human species (such as mouse, rat or rabbit) CDR (donor antibody) residue substitute.In some cases, the mankind exempt from The Fv Framework residues of epidemic disease globulin are replaced into corresponding non-human residues.Humanized antibody also can be included in recipient's antibody and The residue being not present in introduced CDR or frame sequence.In general, humanized antibody can include it is essentially all, extremely Few one and usual two variable regions, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulin CDR region and all or substantially all FR areas are those FR areas of human immunoglobulin consensus sequence.In some embodiments In, humanized antibody will include at least a portion of constant region for immunoglobulin (Fc), and usually human immunoglobulin is extremely A few part.See, for example, Jones et al., Nature, 321:522-525(1986)；Riechmann et al., Nature, 332: 323-329(1988)；Presta,Curr.Op.Struct.Biol.,2:593-596(1992).

In general, there is humanized antibody one or more to be introduced to amino acid residue therein from nonhuman origin. These non-human amino acid residues commonly referred to as " input " residue, and it is normally taken from " inputting " variable region.According to some embodiment party Case, humanization can substantially follow method (Jones et al., Nature, 321 of Winter and colleague:522-525(1986)； Riechmann et al., Nature, 332:323-327(1988)；Verhoeyen et al., Science, 239:1534-1536 (1988)), rodent CDR or CDR sequence is substituted to carry out by with the corresponding sequence of human antibodies.Therefore, such " people source Change " antibody is antibody (U.S. Patent No. 4,816,567), wherein substantially less than intact human variable domain is from inhuman The corresponding sequence substitution of class species.In fact, humanized antibody be usually some CDR residues and may some FR residues through coming from The human antibodies that the residue in the similar site in rodent animal antibody substitutes.

As the alternative solution of humanization, human antibodies can be produced.For example, it is now possible to produce after immunity inoculation Transgenic animals (the example of the complete pedigree of human antibodies can be produced in the case of in the absence of caused by endogenous immunoglobulin Such as mouse).For example, the homotype for having described antibody heavy chain joining region (JH) gene chimeric and in germline mutants mouse connects Closing missing causes to completely inhibit endogenous antibody generation.Human reproduction system immunoglobulin gene array is transferred to such reproduction It is that will cause to produce human antibodies when antigen is attacked in mutant mice.See, for example, Jakobovits et al., PNAS USA, 90:2551(1993)；Jakobovits et al., Nature, 362:255-258(1993)；Bruggemann et al., Year in Immunol.,7:33(1993)；U.S. Patent No. 5,545,806, No. 5,569,825, No. 5,591,669；5th, No. 545,807；And WO 97/17852.Or can by by human immunoglobulin gene seat introduce transgenic animals (such as Mouse that endogenous immunoglobulin gene has not activated partially or completely) in prepare human antibodies.After attack, observer Antibody-like produces, and it is extremely similar with seen in the mankind in all respects, including gene rearrangement, assembling and antibody pedigree.This Method is described in such as U.S. Patent No. 5,545,807；No. 5,545,806；No. 5,569,825；5,625,126th Number；No. 5,633,425；And the 5th, 661, No. 016, and Marks et al., Bio/Technology, 10:779-783(1992)； Lonberg et al., Nature, 368:856-859(1994)；Morrison,Nature,368:812-813(1994)； Fishwild et al., Nature Biotechnology, 14:845-851(1996)；Neuberger,Nature Biotechnology,14:826(1996)；Lonberg and Huszar, Intern.Rev.Immunol., 13:65-93(1995) In.

Human antibodies also can by Activated in Vitro B cell (referring to United States Patent (USP) 5,567,610 and 5,229,275) or by Produced using various techniques known in the art, including phage display library.Hoogenboom and Winter, J.Mol.Biol.,227:381(1991)；Marks et al., J.Mol.Biol., 222:581(1991).Cole et al. and Boerner et al. technology also can be used for preparing human monoclonal antibody.Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, page 77 (1985) and Boerner et al., J.Immunol., 147 (1): 86-95(1991)。

Multi-specificity antibody

In some embodiments, anti-E7MC constructs are multi-specificity antibody.Manufacture polyspecific (such as it is double special Property) appropriate methodology of antibody is well known in the art.For example, the generation of bispecific antibody can be based on two immune globulins The coexpression of Bai Chonglian/light chain pair, wherein two pairs respectively have not homospecificity, and antibody heterodimer (ginseng is produced after association See such as Milstein and Cuello, Nature, 305:537-539(1983)；WO 93/08829, and Traunecker et al., EMBO J.10:3655(1991)).Due to heavy chain immunoglobulin and the random assortment of light chain, (four sources hybridize these hybridomas Knurl) potential mixtures of ten kinds of different antibodies molecules is produced, wherein only a kind of have appropriate bispecific structure.Appropriate molecule Purifying is generally realized by affinity chromatography step.Similar program is disclosed in WO 93/08829 and Traunecker et al., EMBO,10:In 3655-3659 (1991).Or the combination of heavy chain and light chain (can be joined by using the limited pairing orientation of species See such as Lindhofer et al., J.Immunol., 155:219-225 (1995)) and the pairing of heavy chain can be by using CH3 domains Its " pestle-mortar " engineered orientation is (see, for example, U.S. Patent No. 5,731,168；Ridgway et al., Protein Eng., 9(7):617-621(1996)).Multi-specificity antibody also can manufacture antibody Fc-miscellaneous two by engineered electrostatic steering effect Polymer molecular and be made (see, for example, WO 2009/089004A1).In another method, stable bispecific antibody can by by Control Fab arms and exchange generation, wherein two parental antibodies with the matching point mutation in different antigentic specificity and CH3 domains are also Mixed under old terms to allow to separate, recombinate and reoxidize and form highly pure bispecific antibody.Labrigin et al., Proc.Natl.Acad.Sci.,110(13):5145-5150(2013).This antibody-like of mixture comprising heavy chain/light chain pair It is also known as " heteromultimeric antibody " herein.

Also can to produce, polyspecific is different to be sewed through being chemically crosslinked with different specific antibody or its antigen-binding fragment Compound antibody.For example, respectively have the specific molecule of two F (ab') 2 can be through being connected chemically for not synantigen. Pullarkat et al., Trends Biotechnol., 48:9-21(1999).Immune system can for example be made by having pointed out this antibody-like The non-required cell of cell-targeting (U.S. Patent No. 4,676,980) and for treating HIV.WO 91/00360；WO 92/ 200373；EP 03089.It is expected that the known method in synthetic protein chemistry can be used (including to be related to the side of crosslinking agent for antibody Method) prepare in vitro.For example, immunotoxin can be used disulfide bond exchange reaction or be constructed by thioether bond is formed.Go out Include imino group mercaptan alcohol ester and 4- sulfydryls butyryl imines methyl esters in the example of the suitable reagent of this purpose and be disclosed in such as U.S. Those in patent the 4,676,980th.

In some embodiments, recombinant DNA technology can be used to prepare for multi-specificity antibody.For example, bispecific Antibody can by fusion two scFv, such as merged by through peptide linker, produce connect scFv and it is engineered.Connect scFv An example be bispecific T cell joint molecule.Bispecific T cell joint molecule connects by by AntiCD3 McAb scFv To the surface antigen of target cell, as tumor associated antigen (TAA) has specific scFv, T cell is caused to be redirected to target Cell and be made.Mack et al., Proc.Natl.Acad.Sci., 92:7021-7025(1995)；Brischwein et al., Mol.Immunol.,43(8):1129-1143(2006).By the peptide linker length shortened between two variable regions, it can prevent Only self-assembly and force with the second polypeptide on domain match, produce be referred to as bifunctional antibody (Db) compact bispecific resist Body.Holliger et al., Proc.Natl.Acad.Sci., 90:6444-6448(1993).Db two polypeptides it is each it is self-contained by By too short the joint of the pairing between two domains in same chain can not be allowed to be connected to VL VH.Therefore, polypeptide VH and VL domains are forced to match with the complementary VL and VH domains of another polypeptide, and then form two antigen binding sites.With this pattern Modification, two polypeptides connect by another peptide linker, produce Single-chain bifunctional antibody (scDb).In another modification of Db patterns In, double affinity target (DART) bispecific antibody again can be by introducing between the cysteine residues of the C-terminal of each polypeptide Disulfide bond, optionally produced including the domain before the C-terminal cysteine residues of the assembling of heterodimer structure needed for driving. Veri et al., Arthritis Rheum., 62 (7):1933-1943(2010).This area also known Double variable regions immune globulin (DVD-Ig in vain^TM), the target combination variable region of two of which monoclonal antibody is via naturally occurring splice combinations to produce four Valency, bispecific antibody.Gu and Ghayur, Methods Enzymol., 502:25-41(2012).In another pattern, by Using the peptide (AD2) by the grappling domain derived from mankind A kinase anchoring proteins (AKAP) to derived from mankind's cAMP dependences The peptide (DDD2) of the regulation subunit of protein kinase (PKA) carries out dimerization and prepares depressed place lock (DNL), bispecific antibody.Rossi etc. People, Proc.Natl.Acad.Sci., 103:6841-6846(2006).

Also describe directly from recombinant cell culture thing manufacture and separate the various technologies of bispecific antibody fragment.Citing and Speech, produce bispecific antibody using leucine zipper.Kostelny et al., J.Immunol., 148 (5):1547-1553 (1992).The method also can be used for producing antibody morphism dimer.

Anti- E7MC variants

In some embodiments, the amino acid sequence variation of antibody moiety provided in this article is covered.For example, may be used The binding affinity and/or other biological characteristic of improvement antibody moiety can be needed.The amino acid sequence variation of antibody moiety can be by By appropriate modification is introduced into the nucleotide sequence for encoding the antibody moiety or prepared by peptide symthesis.Such modification is included for example The missing of residue in the amino acid sequence of antibody moiety and/or insertion and/or substitution.It can be lacked, inserted and be substituted Any combinations to obtain final construct, its restrictive condition be final construct there is desired characteristics, such as antigen binding.

In some embodiments, there is provided there is the antibody moiety variant of one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.For substituting The related locus that type mutation induces includes HVR and FR.49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can be introduced in associated antibodies part and for required living Property screening product, such as reservation/raising antigen binding, reduce immunogenicity or improved ADCC or CDC product.

Conservative replacement is shown in table 5 below.

Table 5：Conservative replacement

Amino acid can be grouped into different classes of according to common side chain properties：

A. hydrophobicity：Nor-leucine, Met, Ala, Val, Leu, Ile；

B. Neutral hydrophilic：Cys、Ser、Thr、Asn、Gln；

C. it is acid：Asp、Glu；

D. it is alkaline：His、Lys、Arg；

E. the residue of chain orientation is influenceed：Gly、Pro；

F. aromatics：Trp、Tyr、Phe.

Non-conservative substitutions changes certainty into another classification with by a kind of member in these classifications.

Exemplary substituted type variant is affinity maturation antibody moiety, and it can be for example, use the parent based on phage display Advantageously produced with power mature technology.In short, make one or more CDR residue mutations and antibody variants are presented on bacteriophage Partly and for particular organisms active (such as binding affinity) screening.Changing (such as substitution) can be carried out with for example in HVR Improve antibody moiety affinity.Such change can be in HVR " focus " (that is, by undergoing high-frequency during body cell maturation Residue coded by the codon of mutation is (see, for example, Chowdhury, Methods Mol.Biol.207:179-196 (2008)), and/or specificity determining residue (SDR)) in carry out, wherein test gained variant VH or VL binding affinity.By By constructing two level library and having been described in Hoogenboom's et al. from two level library reselection to reach affinity maturation Methods in Molecular Biology 178:1-37 (O'Brien et al. is compiled, Human Press, Totowa, NJ, (2001) in).

In some embodiments of affinity maturation, by a variety of methods (such as fallibility PCR, chain reorganization or few nucleosides The mutation of acid guiding induces) any of diversity be introduced into be chosen in ripe variable gene.Then produce two Level storehouse.The storehouse is then screened to differentiate any antibody moiety variant with required affinity.Introduce multifarious another method It is related to HVR guided pathways, wherein some HVR residues (such as a 4-6 residue) are grouped at random.It is related to antigen binding HVR residues specific can differentiate, such as is induced or modeled to differentiate using Alanine scanning mutagenesis.In specific words, it is usually targeted CDR-H3 and CDR-L3.

In some embodiments, substituting, insert or lacking can occur in one or more HVR, as long as these change The ability of not essentially decreased antibody moiety combination antigen.For example, not essentially decreased combination can be carried out in HVR The conservative change (such as conservative replacement as herein provided) of affinity.Such change can be outside HVR " focus " or SDR. In some embodiments of variation VH and VL sequence presented above, each HVR do not change or containing no more than one, two or Three 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

The residue for being used to differentiate antibody moiety of mutation induction can be targetted or the usability methods in region are referred to as " Alanine-scanning Mutation induces ", such as by Cunningham and Wells (1989) Science, 244:Described by 1081-1085.In this method, Differentiate the group of residue or target residues (such as charged residues, such as arg, asp, his, lys and glu) and by neutral or negatively charged Amino acid (such as alanine or polyalanine) displacement is to determine the phase of antibody moiety and antigen mutually with whether being affected.Can Other substitutions are introduced at the amino acid position to initial substitution display function sensitiveness.Besides or furthermore, Ag-Ab part The crystal structure of complex can differentiate the contact point between antibody moiety and antigen after measured.Such contact residues and neighbouring residual Base can be used as the target of substitution candidate or exclude substituting outside candidate.Variant can be screened to judge it whether containing required Characteristic.

Amino acid sequence insertion is included in a residue to the polypeptide length model containing 100 or more than 100 residues Enclose in interior amino and/or c-terminus fusions, and the sequence with single or multiple amino acid residues and insert.End is inserted Example include with N-terminal first thiamines acyl residue antibody moiety.Other insertion variants of antibody moiety include antibody portion The N-terminal or C-terminal and enzyme (such as ADEPT) or the fusions of the polypeptide for the serum half-life for extending antibody moiety divided.

Fc region variants

In some embodiments, one or more amino acid modified anti-E7MC of total length provided in this article that are introduced to resist In Ti Fc areas, and then produce Fc region variants.In some embodiments, Fc region variants have the antibody dependent cellular of enhancing Toxicity (ADCC) effector function, it is related generally to being bound to Fc acceptors (FcR).In some embodiments, Fc region variants have There are the ADCC effector functions of reduction.In the presence of multiple examples of change or the mutation of the Fc sequences that can change effector function.Citing and Speech, WO00/42072 and Shields et al. J Biol.Chem.9 (2):6591-6604 (2001) descriptions are with raising or decline And FcR combination antibody variants.The content of these publications is specifically incorporated to herein by reference.

The cytotoxicity (ADCC) of antibody dependent cellular mediation is mechanism of action of the therapeutic antibodies to tumour cell. ADCC is cell-mediated immune defense, and thereby the effector cell of immune system actively cracks target cell (such as cancer cell), The film surface antigen of the cell combines with specific antibody (such as anti-E7MC antibody).Typical ADCC is related to NK cells by anti- The activation of body.NK cells are expressed as the CD16 of Fc acceptors.This Receptor recognition is bound to the Fc parts of the antibody of target cells, and It is bound to the Fc parts.Most common Fc acceptors are referred to as CD16 or Fc γ RIII on NK cell surfaces.The Fc of Fc acceptors and antibody The combination in area causes NK cell activations, the release of cell particles and thing followed target cell apoptosis.ADCC is to tumour cell The contribution of kill can measure by the specific test of the NK-92 cells using high-affinity FcR transfections.As a result compared to FcR wild type NK-92 cells are not expressed.

In some embodiments, the present invention cover comprising with some but and the Fc areas of not all effector function it is anti- E7MC construct variants, this cause its for anti-E7MC constructs Half-life in vivo it is important, and some effector functions (such as CDC and ADCC) the required candidate of unnecessary or harmful application.The analysis of external and/or in vivo cytotoxicity can be carried out to confirm CDC And/or reduction/elimination of ADCC activity.For example, Fc acceptors (FcR) binding analysis can be carried out to ensure antibody deficiency Fc γ R combines (therefore may lack ADCC activity), but retains FcRn binding abilities.For mediating ADCC primary cell NK cells only Expression Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Expression general introductions of the FcR on hematopoietic cell In Ravetch and Kinet, Annu.Rev.Immunol.9:In 457-492 (1991) table 3 of page 464.Assess of interest Molecule ADCC activity analyzed in vitro non-limiting examples be described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc.Nat'l Acad.Sci.USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.Nat'l Acad.Sci.USA 82:1499-1502(1985)；U.S. Patent No. 5,821,337 (referring to Bruggemann, M. et al., J.Exp.Med.166:1351-1361 (1987)) in.Or on-radiation analysis side can be used Method is (see, for example, the ACTI for flow cytometry^TMNon-radioactive cell oxicity analysis (CellTechnology, Inc.Mountain View,Calif.；And CytoToxNon-radioactive cell oxicity analysis (Promega, Madison, Wis.).Include peripheral blood monocytes (PBMC) and natural killer (NK) cell suitable for the effector cell of this alanysis.Or Person or in addition, the ADCC activity of molecule of interest can be assessed in vivo, such as such as it is being disclosed in Clynes et al. Proc.Nat'l Acad.Sci.USA 95:In animal model in 652-656 (1998).Also C1q binding analysis can be carried out to confirm that antibody can not Lack with reference to C1q and therefore CDC activity.See, for example, the C1q and C3c in WO 2006/029879 and WO 2005/100402 With reference to ELISA.In order to assess complement activation, can carry out CDC analyses (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996)；Cragg, M.S. et al., Blood 101:1045-1052(2003)；And Cragg, M.S. and M.J.Glennie, Blood 103:2738-2743(2004)).Also methods known in the art can be used (see, for example, Petkova, S.B. et al., Int'l.Immunol.18 (12):1759-1769 (2006)) carry out FcRn combinations and Internal clearance rate/half-life period measure.

The antibody of effector function with reduction is included with Fc areas residue 238,265,269,270,297,327 and 329 In one or more those substituted (U.S. Patent No. 6,737,056).Such Fc mutant is included in amino acid position Putting has the Fc mutant of substitution at more than both in 265,269,270,297 and 327 or both, including residue 265 and 297 It is substituted by so-called " DANA " the Fc mutant (U.S. Patent No. 7,332,581) of alanine.

Some antibody variants of the description with combination improve or reduce and FcR.(see, for example, U.S. Patent No. 6, No. 737,056；WO 2004/056312, and Shields et al., J.Biol.Chem.9 (2):6591-6604(2001)).

In some embodiments, there is provided include the variant Fc regions containing one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors for improving ADCC Anti- E7MC constructs (such as the anti-E7MC antibody of total length) variant.In some embodiments, variant Fc regions include one or more ADCC 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is improved, wherein substitution is in the position 298,333 and/or 334 of variant Fc regions (the EU numberings of residue) Place.In some embodiments, anti-E7MC constructs (such as the anti-E7MC antibody of total length) variant include its variant Fc regions in Lower 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor：S298A, E333A and K334A.

In some embodiments, the C1q for carrying out causing to change (that is, improve or reduce) in Fc areas is combined and/or mended The change of body dependent cellular cytotoxicity (CDC), such as such as U.S. Patent No. 6,194,551, WO 99/51642 and Idusogie Et al., J.Immunol.164:Described in 4178-4184 (2000).

In some embodiments, there is provided the anti-E7MC structures comprising the variant Fc regions containing one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Body (such as the anti-E7MC antibody of total length) variant, one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor increase half-life period and/or improvement and neonate The combination of Fc acceptors (FcRn).The antibody of with increased half-life period and improvement and FcRn combination is described in US2005/ In 0014934A1 (Hinton et al.).Those antibody include the Fc areas with one or more substitutions, wherein Fc areas and FcRn Combination improved.Such Fc variants include those substituted with one or more places in following Fc areas residue： 238、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、 413rd, 424 or 434, such as the substitution (U.S. Patent No. 7,371,826) of Fc areas residue 434.

On other examples of Fc region variants, also referring to Duncan and Winter, Nature 322:738-40(1988)； U.S. Patent No. 5,648,260；U.S. Patent No. 5,624,821；And WO 94/29351.

Cover comprising any one of Fc variants as described herein, or (such as total length resists the anti-E7MC constructs of its combination E7MC antibody).

Glycosylation variants

In some embodiments, provided herein is anti-E7MC constructs through changing to increase or decrease anti-E7MC constructs Through glycosylated degree.For the glycosylation site addition of anti-E7MC constructs or missing can by change anti-E7MC constructs or The amino acid sequence of its polypeptide portion so that produce or remove one or more glycosylation sites and advantageously realize.

In the case where anti-E7MC constructs include Fc areas, the carbohydrate being attached to can be changed.Moved by lactation Natural antibody caused by thing cell generally comprises the double antennary oligosaccharides of branched chain, and it is typically by the CH2 domains of N key connections to Fc areas Asn297.See, for example, Wright et al. TIBTECH 15:26-32(1997).Oligosaccharides may include various carbohydrate, such as Mannose, N- acetyl glucosamines (GlcNAc), galactolipin and sialic acid, and be connected in " trunk " of double tactile oligosaccharide structures GlcNAc trehalose.In some embodiments, can carry out the present invention anti-E7MC constructs in oligosaccharides modification with Produce the anti-E7MC constructs variant of the characteristic with some improvement.

In some embodiments, there is provided anti-E7MC constructs (the anti-E7MC antibody of such as total length) variant comprising Fc areas, its In be attached to Fc areas carbohydrate structure there is reduced trehalose or without trehalose, it can improve ADCC functions. In specific words, the amount relative to the trehalose in wild-type CHO cells in caused identical anti-E7MC constructs is contemplated herein The anti-E7MC constructs of trehalose with reduction.That is, it is characterized in that the amount of trehalose having than it thin by natural CHO Will in the case of caused by born of the same parents' (such as producing the Chinese hamster ovary celI of native glycosylation pattern, the Chinese hamster ovary celI as contained natural FUT8 genes) The amount having in addition is low.In some embodiments, anti-E7MC constructs for wherein be less than about 50%, 40%, 30%, 20%, 10% or 5% includes the construct of trehalose in the bonded glycan of N- thereon.For example, the sea in such anti-E7MC constructs The amount of algae sugar can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%.In some embodiments, resist E7MC constructs are none includes trehalose, i.e. its moderate resistance E7MC constructs completely not wherein in the bonded glycan of N- thereon Containing trehalose, or without trehalose or through going fucosylated construct.The amount of trehalose by calculate relative to such as by All sugared structures (such as compound, hybridization and the high mannose knot that attach to Asn 297 measured by MALDI-TOF mass spectral analyses Structure) summation, the average magnitude of the trehalose in sugar chain at Asn297 determines, as described in such as WO 2008/077546. Asn297 refers to the asparagine residue at pact position 297 (the Eu numberings of Fc areas residue) place in Fc areas；However, due to antibody In minor sequence change, Asn297 may also be at the amino acid of the upstream of position 297 or downstream about ± 3, i.e., position 294 with Between 300.Such fucosylated variant can have the ADCC functions of improvement.See, for example, U.S. Patent Publication case US No. 2003/0157108 (Presta, L.)；US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).With The example of the related publication of " going fucosylated " or " trehalose shortage " antibody variants includes：US 2003/0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119； WO 2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140； Okazaki et al. J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki et al. Biotech.Bioeng.87: 614(2004).The example that the cell line of fucosylated antibody can be produced is fucosylated including lacking protein Lec13CHO cells (Ripka et al., Arch.Biochem.Biophys.249:533-545(1986)；U.S. Patent Application No. US 2003/0157108A1, Presta, L；And WO 2004/056312A1, Adams et al., especially example 11), and base Because of knockout cell system, such as α -1,6- fucosyltransferases gene, FUT8, gene knockout Chinese hamster ovary celI (see, for example, Yamane-Ohnuki et al., Biotech.Bioeng.87:614(2004)；Kanda, Y. et al., Biotechnol.Bioeng.,94(4):680-688(2006)；And WO2003/085107).

Anti- E7MC constructs (the anti-E7MC antibody of such as total length) variant has in addition divides oligosaccharides (bisected equally Oligosaccharide two antennary oligosaccharides for), such as being wherein attached to the Fc areas of anti-E7MC constructs are divided equally by GlcNAc. Such anti-E7MC constructs (the anti-E7MC antibody of such as total length) variant can have fucosylated and/or improvement the ADCC work(reduced Energy.The example of such antibody variants is described in such as WO 2003/011878 (Jean-Mairet et al.)；U.S. Patent No. 6, No. 602,684 (Umana et al.)；US 2005/0123546 (Umana et al.) and Ferrara et al., Biotechnology and Bioengineering,93(5):In 851-861 (2006).It is also provided to be attached in the oligosaccharides in Fc areas and has at least one Anti- E7MC constructs (the anti-E7MC antibody of such as total length) variant of individual galactose residue.Such anti-E7MC constructs variant, which can have, to be changed Good CDC functions.Such antibody variants are described in such as WO 1997/30087 (Patel et al.)；WO 1998/58964 (Raju,S.)；And in WO 1999/22764 (Raju, S.).

In some embodiments, the anti-E7MC constructs comprising Fc areas (the anti-E7MC antibody of such as total length) variant can combine To Fc γ RIII.In some embodiments, the anti-E7MC constructs comprising Fc areas (the anti-E7MC antibody of such as total length) variant is in people There is ADCC activity or compared to the other identical anti-E7MC structures comprising human wild type IgG1Fc areas in the presence of class effector cell Build body (the anti-E7MC antibody of such as total length) has increased ADCC activity in the presence of mankind effector cell.

The engineered variant of cysteine

In some embodiments, it may be necessary to which producing the engineered anti-E7MC constructs of cysteine, (such as total length resists E7MC antibody), wherein one or more amino acid residues substitute through cysteine residues.In some embodiments, it is substituted Residue be present at the accessible site of anti-E7MC constructs.Substitute those residues, reactive mercaptan by with cysteine Base and then it is positioned at the accessible site of anti-E7MC constructs and available for anti-E7MC constructs are bound into other parts, such as Drug moiety or linker-drug part, to produce anti-E7MC immunoconjugates (as described further herein).Cysteine work Journey is transformed anti-E7MC constructs and can produced (such as the anti-E7MC antibody of total length) as described in such as U.S. Patent No. 7,521,541 It is raw.

Derivative

In some embodiments, anti-E7MC constructs presented herein can contain this area through further modification Known and readily available extra non-protein portion.Being suitable for the part of anti-E7MC constructs derivatization is included (but not It is limited to) water-soluble polymer.The non-limiting examples of water-soluble polymer include but is not limited to polyethylene glycol (PEG), second two Alcohol/propylene glycol copolymers, carboxymethyl cellulose, polydextrose, polyvinyl alcohol, PVP, poly- 1,3- dioxanes Pentane, poly- 1,3,6- trioxanes, ethene/maleic anhydride multipolymer, polyaminoacid (homopolymer or random copolymer) and poly- It is glucose or poly- (n- ethene Pyrrolizidines ketone) polyethylene glycol, propropylene glycol homopolymers, PPOX/ethylene oxide copolymer, poly- Oxygen ethene polyalcohol (such as glycerine), polyvinyl alcohol and its mixture.Methoxy PEG-propionaldehyde can because of its stability in water With manufacture advantage.Polymer can have any molecular weight, and can be branched chain or non-branch.It is connected to anti-E7MC constructs The quantity of polymer can change, and if connect more than one polymer, it can be identical or different molecule.In general, For the polymer of derivatization number and/or type can based on including but not limited to wait to improve the spy of anti-E7MC constructs Determine whether characteristic or function, anti-E7MC constructs derivative will determine for Considerations such as the therapies under qualifications.

In some embodiments, there is provided anti-E7MC constructs with can be by exposed to the non-egg that selectively heats of radiation The conjugate of white matter part.In some embodiments, non-protein portion be CNT (Kam et al., Proc.Natl.Acad.Sci.USA 102:11600-11605(2005)).Any wavelength can be had by radiating, and including (but not It is limited to) ordinary cells are not damaged but are heated to non-protein portion to kill anti-E7MC constructs-non-protein portion nearside The wavelength of the temperature of cell.

It is prepared by CAR effector cell

In one aspect, the present invention provides the effector cell's (such as lymphocyte, such as T cell) for expressing anti-E7MC CAR. Provided herein is effector cell's (such as T cell) (the anti-E7MC CAR effector cells, such as anti-E7MC CAR for preparing the anti-E7MC CAR of expression T cell) exemplary methods.

In some embodiments, anti-E7MC CAR effector cells (such as T cell) can by will include anti-E7MC CAR (such as CAR comprising anti-E7MC antibody moieties and CD28 and CD3 ζ intracellular signal transduction sequences) carrier (including for example slow virus carry Body) it is introduced to generation in effector cell's (such as T cell).In some embodiments, anti-E7MC CAR effector cells of the invention (such as T cell) can replicate in vivo, cause that HPV16-E7 positive diseases (such as cancer, such as squamous cell carcinoma, cervical carcinoma can be caused Or cancer of anus) lasting control long-term persistence.

In some embodiments, the present invention relates to apply the genetic modification T cell for expressing anti-E7MC CAR with using leaching Bar cell infusion therapies suffer from HPV16-E7 positive diseases or the patient under with HPV16-E7 positive diseases risks.One In a little embodiments, autologous leukocytes are transfused for treating.Autologous PBMC uses collected from the patient and T cell for needing to treat The activation of described herein and methods known in the art and amplification, and be then transfused back in patient.

In some embodiments, anti-E7MC CAR T cells expression includes the anti-E7MC CAR of anti-E7MC antibody moieties (being also known as " anti-E7MC CAR T cells " herein).In some embodiments, anti-E7MC CAR T cells expression includes Extracellular containing anti-E7MC antibody moieties and the intracellular domain of the intracellular signal transduction sequence comprising CD3 ζ and CD28 it is anti- E7MC CAR.The anti-E7MC CAR T cells of the present invention can undergo firm internal T cell amplification and can produce in blood and marrow In persistently extend the E7MC specific memory cells that time quantum is held under high content.In some embodiments, it is infused to trouble The E7MC that anti-E7MC CAR T cells of the invention in person can eliminate in the patient with HPV16-E7 positive diseases in vivo is in Delivery cell, as E7MC presents cancer cell.In some embodiments, the anti-E7MC CAR T of the invention being infused in patient are thin Born of the same parents can be difficult to at least one known treatment come the patient for the HPV16-E7 positive diseases treated in vivo eliminate E7MC Cancer cell is presented in delivery cell, such as E7MC.

Before T cell amplification and genetic modification, T cell source is obtained from individual.T cell available from a variety of sources, including Peripheral blood monocytes, marrow, lymph node tissue, Cord blood, thymic tissue, the tissue from sites of infection, ascites, pleura Hydrops, spleen tissue and tumour.In some embodiments of the present invention, the available T cell in any number of this area can be used System.In some embodiments of the present invention, T cell is available from any number of technology well known by persons skilled in the art of use (such as Ficoll^TMSeparation) from the blood unit of individual collection.In some embodiments, obtained by removing art from individual Blood circulation cell.Remove art product and usually contain lymphocyte, including T cell, monocyte, granulocyte, B cell, Other nucleation white blood cell, red blood cell and blood platelets.In some embodiments, by remove art collect cell can it is scrubbed with Remove blood plasma fractions and cell is placed in appropriate buffer solution or culture medium for subsequent processing steps.In some embodiments In, cell is washed with phosphate buffered saline (PBS) (PBS).In some embodiments, wash solution lacks calcium and may lack magnesium Or many (and if not all) bivalent cations may be lacked.Such as general technology, person will be apparent that, washing step can be by this Known to art personnel method realize, such as by according to manufacturer specification using semi-automatic " circulation " centrifugation (such as The cellular processors of Cobe 2991, Baxter CytoMate or Haemonetics cells conservator 5).After wash, cell It can be resuspended in various biocompatible buffer, such as Ca²⁺Free, Mg²⁺Free PBS, PlasmaLyte A or other have Or the normal saline solution without buffer.Or removable non-want the component for removing art sample and cell is direct again It is suspended in culture medium.

In some embodiments, by cracking red blood cell and for example by through PERCOLL^TMGradient centrifugation or by adverse current Centrifugal elutriation exhausts monocyte and from periphery blood separation of lymphocytes T cell.T cell specific subset (such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+T cell) can further it be separated by positive selection or negative selection technology.Citing For, in some embodiments, by the bead combined with AntiCD3 McAb/anti- CD28 (that is, 3 × 28) (such asM-450CD3/CD28 T) cultivate together and be enough positive selection and want the time of T cell thin to separate T Born of the same parents.In some embodiments, the period is about 30 minutes.In some embodiments, the period is at 30 minutes to 36 hours or more In the range of long and all integer values therebetween.In some embodiments, the period is at least 1,2,3,4,5 or 6 hour.At some In embodiment, the period is 10 to 24 hours.In some embodiments, it is 24 hours to cultivate the period.In order to suffer from white blood certainly The patient of disease separates T cell, and the longer cultivation time (such as 24 hours) of use can improve cell yield.Compared to other cells Type, which exists, can be used longer cultivation temporal separation T cell in any situation of seldom T cell, such as from tumor tissues or immune Insufficiency individual separation tumor infiltrating lymphocyte (TIL).In addition, it is thin to improve seizure CD8+ T using the longer cultivation time The efficiency of born of the same parents.Therefore, T cell is allowed to be bound to the time of CD3/CD28 beads and/or by increase by only shortening or extension Or reduce bead in the ratio of T cell, can for or for cultivate originate when or method during other times point preferentially select Select T cell subgroup.In addition, by increaseing or decreasing the ratio of AntiCD3 McAb and/or anti-CD28 antibody in bead or other surfaces, it is right In or for cultivate originate when or at other want time point can prioritizing selection T cell subgroup.Those skilled in the art will recognize Know in the context of the present invention and multiple selection bouts also can be used.In some embodiments, it may be necessary to carry out selection journey Sequence and use " unselected " cell in activation and amplification procedure." unselected " cell can also carry out other and select back Close.

The antibody of the unique surface markers of the cell for being directed to negative selection can be used by negative selection enrichment T cell colony Combine to realize.A kind of method is that the negative magnetic immuno of warp sticks or flow cytometry sorts and/or selection cell, and this is negative Magnetic immuno sticks or flow cytometry uses the Dan Ke for being directed to cell surface marker thing present on negative selection cell Grand mixtures of antibodies.For example, in order to generally include CD by negative selection enrichment CD4+ cells, monoclonal antibody mixed liquor 14th, CD20, CD11b, CD 16, HLA-DR and CD8 antibody.In some embodiments, it may be necessary to which enrichment or positive selection are adjusted T cell is saved, it is often expressed as CD4+, CD25+, CD62Lhi, GITR+ and FoxP3+.Or in some embodiments, T is adjusted Ganglion cell by anti-CD25 combinations bead or other exhausted similar to system of selection.

In order to want cell colony by positive selection or negative selection separation, cell concentration and surface (such as particle, such as pearl Grain) alterable.In some embodiments, it may be necessary to be substantially reduced bead and mixing with cells together volume (that is, increase Add cell concentration), to ensure the Maximum Contact of cell and bead.For example, in some embodiments, using about 2,000,000,000 The concentration of cells/ml.In some embodiments, using the concentration of about 1,000,000,000 cells/mls.In some embodiments In, use greater than about 100,000,000 cells/mls.In some embodiments, using about 10,000,000,15,000, 000th, 20,000,000,25,000,000,30,000,000,35,000,000,40,000,000,45,000,000 or 50, The concentration of any one of 000,000 cells/ml.In some embodiments, using about 75,000,000,80,000, 000th, the concentration of any one of 85,000,000,90,000,000,95,000,000 or 100,000,000 cells/mls. In some embodiments, using about 125,000,000 or about 150, the concentration of 000,000 cells/ml.Use high concentration Increased cell yield, cell activation and cell amplification can be caused.In addition, high cell concentration is used make it that more effective catch may The cell (such as CD28 negative T cells) of weak expression target antigen of interest or exist certainly many tumour cells sample (that is, Leukemic blood, tumor tissues etc.) more effectively catch cell.Such cell colony can have therapeutic value and will need to obtain. For example, the more effective selection generally CD8+ T cells with weaker CD28 expression are allowed using high cell concentration.

In some embodiments of the present invention, T cell is obtained directly from the patient after treatment.Thus, observed To after some treatments of cancer, in specific words use and destroy after the drug therapy of immune system, in patient generally by autonomy Treat during the period recovered after treatment soon, the T cell quality of acquisition can most preferably or its ability expanded in vitro is improved. Equally, after being manipulated in vitro using approach described herein, the enhancing transplanting and amplification in vivo of these cells can be in preferred State.Therefore, it is thin that collection haemocyte (including T cell), dendron shape during this Restoration stage are covered in context of the invention Other of born of the same parents or hematopoietic lineage cell.In addition, in some embodiments, mobile (such as being moved with GM-CSF) and regulation scheme Available for establishing intraindividual condition, wherein preferably breeding again, recycling, regenerating during formulation time window especially after the treatment And/or amplification particular cell types.Illustrative cell type includes its of T cell, B cell, BMDC and immune system His cell.

No matter before or after genetic modification T cell is with anti-E7MC CAR needed for expressing, T cell can typically use such as example Such as the method activation and amplification described in the following：U.S. Patent No. 6,352,694；6,534,055；6,905,680；6, 692,964；5,858,358；6,887,466；6,905,681；7,144,575；7,067,318；7,172,869；7,232, 566；7,175,843；5,883,223；6,905,874；6,797,514；No. 6,867,041；And Patent Application Publication No. 20060121005.

In general, the present invention T cell by make connected surface with stimulate CD3/TCR composite bulk phase OFF signals Medicament and stimulate the ligand contact of costimulatory molecules on T cell surface and expand.In specific words, T cell colony can be such as By with anti-cd 3 antibodies or its antigen-binding fragment, or be fixed on surface anti-CD2 antibody contact, or by with calcium from Subcarrier with reference to protein kinase c activator (such as bryostatin) contact and stimulate.In order to auxiliary on costimulation T cell surface Molecule is helped, uses the part for combining accessory molecule.For example, T cell colony can be with anti-cd 3 antibodies and anti-CD28 antibody suitable Contacted under conditions of stimulating T cell to breed.In order to stimulate the propagation of CD4+ T cells or CD8+ T cells, anti-cd 3 antibodies and Anti-CD28 antibody.The example of anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,France), may be used (Berg et al., Transplant Proc.30 (8) typically can be used in other method as generally known in the art:3975- 3977,1998；Haanen et al., J.Exp.Med.190 (9):13191328,1999；Garland et al., J.Immunol Meth.227(1-2):53-63,1999)。

It is prepared by immunoconjugates

Any method known in the art can be used to prepare for anti-E7MC immunoconjugates.See, for example, WO 2009/ 067800th, WO 2011/133886 and Patent Application Publication the 2014322129th, it is in entirety by reference simultaneously Enter herein.

The anti-E7MC antibody moieties of anti-E7MC immunoconjugates " can be attached to " effector molecule, anti-E7MC by any method Antibody moiety can be associated with effector molecule by this method, or is connected to effector molecule.For example, anti-E7MC immunoconjugates The anti-E7MC antibody moieties of thing can be attached to effector molecule by chemistry or recombination method.Prepare the chemistry of fusions or conjugate Method is known in the art and can be used for preparing anti-E7MC immunoconjugates.For combining anti-E7MC antibody moieties and effector molecule Method allow for making in the case of the ability for the antigen for not disturbing associated proteins to be bound on target cell associated proteins with Effector molecule engages.

The anti-E7MC antibody moieties of anti-E7MC immunoconjugates can be indirectly connected to effector molecule.For example, anti-E7MC If the anti-E7MC antibody moieties of immunoconjugates may be connected directly to the liposome of the effector molecule containing one of dry type. Effector molecule and/or anti-E7MC antibody moieties can also be bound to the surface of solids.

In some embodiments, anti-the E7MC antibody moieties and effector molecule of anti-E7MC immunoconjugates are protein And technology well known in the art can be used to combine.Hundreds of available crosslinking agents for combining two kinds of protein be present.(see, for example, " Chemistry of Protein Conjugation and Crosslinking”.1991,Shans Wong,CRC Press, Ann Arbor).Crosslinking agent is generally basede on reactive functional that is available or inserting on anti-E7MC antibody moieties and/or effector molecule Base selects.In addition, if reactive group is not present, it can be used light can activatable crosslinking agent.In certain circumstances, it may be necessary to Include interval base between anti-E7MC antibody moieties and effector molecule.Crosslinking agent known to technique includes homotype difunctionality medicine Agent：Glutaraldehyde, diimine are for dimethyl adipate and double (diazo benzidines) and allograft difunctionality medicament：Between maleic two Acylimino benzoyl-N-hydroxysuccinimide and sulfonic group-maleimide benzoyl-N-hydroxyl fourth Imidodicarbonic diamide.

In some embodiments, the anti-E7MC antibody moieties of anti-E7MC immunoconjugates can change through specific residue engineering Make to be connected chemically effector molecule.Specific residue known in the art for chemical linker molecule is included from propylhomoserin and half Guang Propylhomoserin.Based on inserting on anti-E7MC antibody moieties and available reactive functional group selective cross-linking agent on effector molecule.

Anti- E7MC immunoconjugates also recombinant DNA technology can be used to prepare.In the case, anti-E7MC antibody moieties are encoded DNA sequence dna be fused to the DNA sequence dna of coded actions molecule, produce chimeric dna molecule.Gomphosis DNA array, which is transfected to expression, to be melted In the host cell of hop protein.Fusion protein can be reclaimed from cell culture and purified using techniques known in the art.

Effector molecule (it is mark) is connected into protein-bonded example includes Hunter et al., Nature 144:945 (1962)；David et al., Biochemistry 13:1014(1974)；Pain et al., J.Immunol.Meth.40:219 (1981)；Nygren,J.Histochem.and Cytochem.30:407(1982)；Wensel and Meares, Radioimmunoimaging And Radioimmunotherapy,Elsevier,N.Y.(1983)；And Colcher et al., “Use Of Monoclonal Antibodies As Radiopharmaceuticals For The Localization Of Human Carcinoma Xenografts In Athymic Mice”,Meth.Enzymol.,121:In 802-16 (1986) Described method.

Radioactivity or other marks can be incorporated in immunoconjugates in a known way.For example, peptide can biosynthesis or It can synthesize by chemical amino acid, be synthesized using suitable amino acid precursor (including for example fluoro- 19 replacement hydrogen).Such as 99Tc or 123I, 186Re, 188Re and 111In mark can connect via the cysteine residues in peptide.Yttrium-90 can be via residual from propylhomoserin Base connects.IODOGEN methods (Fraker et al. Biochem.Biophys.Res.Commun.80:49-57 (1978)) it can be used to It is incorporated to iodo- 123.”Monoclonal Antibodies in Immunoscintigraphy”(Chatal,CRC Press 1989) other method is described in detail.

It can be used a variety of bifunctional protein coupling agents that the immunoconjugates of antibody moiety and cytotoxic agent are made, it is described Bifunctional protein coupling agent such as N- succinyl imino group -3- (2- pyridyidithios) propionic ester (SPDP), succinyl are sub- Amino -4- (N- maleimidomethyls) hexamethylene -1- formic acid esters (SMCC), iminothiolane (IT), Asia (such as the succinyl of suberic acid two is sub- for the dual-function derivative (such as diimine is for dimethyl adipate HCI) of amino ester, active ester Amino ester), aldehyde (such as glutaraldehyde), double repeatedly nitrogen base compounds (such as double (to repeatedly nitrogen base benzoyl) hexamethylene diamines), dual nitrogen Derivative (such as double (to diazoniumbenzoyl) ethylenediamines), diisocyanate (such as toluene 2,6- diisocyanate) and dual-active Property fluorine compounds (the fluoro- 2,4- dinitro benzenes of such as 1,5- bis-).For example, ricin immunotoxin can such as Vitetta People, Science 238:Prepared described in 1098 (1987).The two sub- second of 1- isothiocyano benzyl -3- methyl that carbon 14 marks Base pentaacetic acid (MX-DTPA) is for by the exemplary chelating agent of radioactive nucleotides binding antibody.See, for example, WO94/ 11026.Joint can be to promote the cytotoxic drug in cell to discharge its " cleavable joint ".For example, acid can be used not Stable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker or joint containing disulfide bond (Chari et al., Cancer Research 52:127-131(1992)；U.S. Patent No. 5,208,020).

The anti-E7MC immunoconjugates of the present invention clearly cover the ADC of (but not limited to) crosslinking agent reagent preparation： BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo group-EMCS, Sulfo group-GMBS, sulfo group-KMUS, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC and sulfo group-SMPB, and SVSB (succinyl imido Base-(4- vinyl sulfones) benzoic ether), its is commercially available (such as from Pierce Biotechnology, Inc., Rockford,IL.,U.S.A).Referring to the 467-498 pages, 2003-2004Applications Handbook and Catalog。

Pharmaceutical composition

The composition comprising anti-E7MC constructs is also provided herein (such as pharmaceutical composition, to be also known as prepare herein Agent).In some embodiments, composition further comprising the cell related to anti-E7MC constructs (such as effector cell, such as T cell).In some embodiments, there is provided the drug regimen comprising anti-E7MC constructs and pharmaceutically acceptable supporting agent Thing.In some embodiments, pharmaceutical composition further comprising the cell related to anti-E7MC constructs (such as effector cell, Such as T cell).

The suitable preparaton of anti-E7MC constructs by by the anti-E7MC constructs with required purity with optionally existing Pharmaceutically acceptable load excipient or stabilizer (Remington's Pharmaceutical Sciences the 16th edition, Osol, A. compile (1980)) mixed and obtained in the form of freeze-dried formulation or the aqueous solution., can under used dosage and concentration The supporting agent of receiving, excipient or stabilizer are nontoxic to recipient, and it includes buffer, such as phosphate, citrate and its His organic acid；Antioxidant, including ascorbic acid and methionine；Preservative (such as chlorination octadecyldimethyl benzyl Ammonium；Hexamethonium chloride；Benzalkonium chloride；Benzethonium chloride；Phenol；Butanol or phenmethylol；P-hydroxybenzoic acid alkyl ester, such as to hydroxyl Yl benzoic acid methyl esters or propylparaben；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols and metacresol)；Low molecule Measure (being less than about 10 residues) polypeptide；Protein, such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer, it is all Such as PVP；Amino acid, such as glycine, bran amic acid, asparagine, histidine, arginine or from propylhomoserin；It is single Sugar, disaccharide and other carbohydrate, including glucose, mannose or dextrin；Chelating agent, such as EDTA；Sugar, such as sucrose, Mannitol, trehalose or D-sorbite；Into salt relative ion, such as sodium；Metal complex is (for example, Zn- protein is compound Body)；And/or non-ionic surfactant, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).Exemplary preparaton It is described in WO98/56418, during the patent is expressly incorporated herein by reference.Lyophilized suitable for subcutaneous administration is prepared Agent is described in WO97/04801.Such freeze-dried formulation can be restored by suitable diluent to increased protein concentration and recovered Preparaton can subcutaneous administration to individual to be treated herein.Liposome (Lipofectin/liposome) can be used for sending out this Bright anti-E7MC constructs are transferred in cell.

Preparaton herein is also containing one or more reactive compounds in addition to anti-E7MC constructs (depending on treatment Specific adaptations disease needs), preferably have can not adversely affect one another complementary activity those.For example, may Need to further provide for anti-superfluous raw agent, growth inhibitor, cytotoxic agent or the chemotherapeutant in addition to anti-E7MC constructs. This quasi-molecule to the effective amount of expected purpose preferably to be present in combination.The effective dose of other such medicaments is depending on being present in preparaton In amount, disease or the illness of anti-E7MC constructs or the type for the treatment of and other factors as described above depending on.These medicines Agent typically uses with about the 1% to 99% of same dose as described herein and route of administration or the dosage used so far.

Also anti-E7MC constructs for example can be embedded in prepared microcapsules by condensation technique or by interfacial polymerization In, such as hydroxy-methyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules are embedded in gluey medicine respectively In thing transmission system (such as liposome, albumi microspheres, microemulsion, nano-particle and Nano capsule) or in huge emulsion. Such technology is disclosed in Remington's Pharmaceutical Sciences the 16th edition, and Osol, A. are compiled in (1980).Can Prepare extended release preparation.

The extended release preparation of anti-E7MC constructs can be prepared.The suitable example of extended release preparation includes containing antibody The semi-permeable matrix of the solid hydrophobic polymers of (or its fragment), the matrix are in formed article, such as film or microcapsules shape Formula.The example of sustained-release matrix includes polyester, hydrogel (for example, poly- (HEMA) or poly- (ethene Alcohol)), polylactide (U.S. Patent No. 3,773,919), L- brans propylhomoserin and ethyl-L- bran propylhomoserins copolymer, can not drop Ethane-acetic acid ethyenyl ester, such as LUPRON DEPOT TM are solved (by lactic acid-ethanol copolymer and Leuprorelin acetate (leuprolide acetate) form Injectable microspheres body) degradable lactic acid-ethanol copolymer and poly- D- (-) -3- Hydroxybutyric acid.Although the polymer of such as ethane-acetic acid ethyenyl ester and lactic acid-ethanol allows to be continued above release point in 100 days Son, but some hydrogels continue shorter time period release protein.If be encapsulated antibody for a long time reside in vivo, its can because It is denatured or assembles exposed to moisture at 37 DEG C, causes bioactivity loss and the change of possible immunogenicity.It may depend on phase Shutdown is made and designs the anti-stabilized rational strategy of E7MC constructs.For example, if finding aggregation of multiple via the sulphur of sulfenyl-two Compound is exchanged and forms intermolecular S -- S, then stabilizing can freeze by modification sulfhydryl residue, from acid solution, control moisture Content, reached using suitable additives and exploitation specific aggregation base composition.

In some embodiments, anti-E7MC constructs allocate in comprising citrate, NaCl, acetate, succinate, In glycine, polysorbate80 (Tween 80) or foregoing any combination of buffer solution.In some embodiments, resist E7MC constructs are allocated in the buffer solution comprising about 100mM to about 150mM glycine.In some embodiments, anti-E7MC Construct is allocated in the buffer solution comprising about 50mM to about 100mM NaCl.In some embodiments, anti-E7MC constructs Allocate in the buffer solution comprising about 10mM to about 50mM acetates.In some embodiments, anti-E7MC constructs allocate in In buffer solution comprising about 10mM to about 50mM succinates.In some embodiments, anti-E7MC constructs allocate in comprising In the buffer solution of about 0.005% to about 0.02% polysorbate80.In some embodiments, anti-E7MC constructs allocate in In buffer solutions of the pH between about 5.1 and 5.6.In some embodiments, anti-E7MC constructs are allocated in comprising 10mM lemons Hydrochlorate, 100mM NaCl, 100mM glycine and 0.01% polysorbate80 buffer solution in, wherein preparaton is in pH 5.5 Under.

The preparaton applied in vivo is necessary for sterile.This via aseptic filtration membrane filtration by for example easily realizing.

Use the treatment method of anti-E7MC constructs

Anti- E7MC constructs of the invention and/or composition can be applied to individual (such as mammal, such as mankind) to control Treat and the disease of abnormal high HPV16-E7 expression and/or illness (being also known as " HPV16-E7 is positive " disease or illness herein) Correlation, including such as HPV16-E7 positive cancers (such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, penis Cancer, head and neck cancer or oropharyngeal cancer).Therefore the application provides the HPV16-E7 positive diseases in treatment individual in some embodiments The method of (such as cancer, such as squamous cell carcinoma), it includes the including containing anti-E7MC antibody moieties using effective dose to individual The composition (such as pharmaceutical composition) of any one of anti-E7MC constructs, anti-E7MC constructs as described herein.At some In embodiment, composition further includes the cell (such as effector cell) related to anti-E7MC constructs.In some embodiment party In case, cancer is selected from the group for example consisted of：Squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, penis Cancer, head and neck cancer or oropharyngeal cancer

For example, in some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is wrapped It is special containing the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties that effective dose is applied to individual, the antibody moiety Property combines the complex comprising HPV16-E7 peptides and MHC I proteinoid.In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19(SEQ ID NO:4).In some embodiments, MHC I proteinoid is HLA-A02.In some realities Apply in scheme, MHC I proteinoid is HLA-A*02:01.In some embodiments, anti-E7MC constructs are non-naturally-occurring 's.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are more Specific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some implementations In scheme, anti-E7MC constructs are immunoconjugates.In some embodiments, composition is further included and built with anti-E7MC The cell (such as effector cell) that body phase is closed.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiment party In case, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer. In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The peptides of 11-19 containing HPV16-E7 (SEQ ID NO:And HLA-A*02 4):01 complex.In some embodiments, anti-E7MC Construct is full length antibody.In some embodiments, anti-E7MC constructs are polyspecific (such as bispecific) molecule.One In a little embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments, anti-E7MC constructs are sewed to be immune Compound.In some embodiments, composition further includes the cell (such as effector cell) related to anti-E7MC constructs. In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer be such as squamous cell carcinoma, Cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, or its include at most about 3 (e.g., from about 1, Any one of 2 or 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 ammonia Base acid sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, The HC-CDR3 includes SEQ ID NO:Any one of 186-188 amino acid sequence, or its include at most about 3 (e.g., from about 1,2 Or any one of 3) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 is included SEQ ID NO:189 or 190 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual amino The variant of acid substitution, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.In some embodiments, anti-E7MC constructs are non- It is naturally occurring.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC structures It is polyspecific (such as bispecific) molecule to build body.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor. In some embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, composition is further included with resisting The related cell (such as effector cell) of E7MC constructs.In some embodiments, HPV16-E7 positive diseases are cancer.One In a little embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or Oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is The mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188's Amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:189 or 190 ammonia Base acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino acid sequence.In some embodiments, resist E7MC constructs are non-naturally occurring.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiment party In case, anti-E7MC constructs are polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are embedding Close antigen receptor.In some embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, composition Further include the cell (such as effector cell) related to anti-E7MC constructs.In some embodiments, HPV16-E7 is positive Disease is cancer.In some embodiments, cancer be such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, Carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some realities Apply in scheme, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence, or its include at most The variant of about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence, or its include at most about 5 (any one of e.g., from about 1,2,3,4 or 5) it is individual The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 99-119,244 and 245 Amino acid sequence；Or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And Ii) light-chain variable sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 Amino acid sequence, or it includes the variant of at most about 5 (any one of e.g., from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence, or its include at most about 3 The variant of (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And LC-CDR3, the LC-CDR3 include SEQ ID NO: Any one of 162-182 and 247-250 amino acid sequence；Or it included at most about 5 (appointing in e.g., from about 1,2,3,4 or 5 One) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.In some embodiments, anti-E7MC constructs are non-naturally occurring.In some realities Apply in scheme, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are that polyspecific is (such as double Specificity) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments, resist E7MC constructs are immunoconjugates.In some embodiments, composition is further comprising related to anti-E7MC constructs Cell (such as effector cell).In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer Disease is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some realities Apply in scheme, cancer is hepatocellular carcinoma.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.At some In embodiment, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, should HC-CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it includes at most about 5 (e.g., from about 1,2,3,4 or 5 Any one of) variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual HC-CDR sequences；And ii) light-chain variable sequence, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC- CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；Or it includes at most about 5 (in e.g., from about 1,2,3,4 or 5 Any one) 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in individual LC-CDR sequences variant.In some embodiments, anti-E7MC constructs are non-day It is so existing.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC structures Body is polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.One In a little embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, composition is further included with resisting The related cell (such as effector cell) of E7MC constructs.In some embodiments, HPV16-E7 positive diseases are cancer.One In a little embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or Oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is The mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include：I) weight chain variabl area sequence, It includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 amino acid sequence；HC-CDR2, should HC-CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence；And HC-CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light-chain variable sequence, it includes LC- CDR1, the LC-CDR1 include SEQ ID NO:Any one of 120-140 and 246 amino acid sequence；LC-CDR2, the LC- CDR2 includes SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence.In some embodiments, anti-E7MC constructs are non-day It is so existing.In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC structures Body is polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.One In a little embodiments, anti-E7MC constructs are immunoconjugates.In some embodiments, composition is further included with resisting The related cell (such as effector cell) of E7MC constructs.In some embodiments, HPV16-E7 positive diseases are cancer.One In a little embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or Oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is The mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include weight chain variable district, and it is included SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or it has at least about 95% (for example, at least about 96%th, any one of 97%, 98% or 99%) variant of sequence identity, and light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence, or its have at least about 95% (for example, at least about 96%, 97%, Any one of 98% or 99%) variant of sequence identity.In some embodiments, anti-E7MC constructs are deposited for non-natural .In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are Polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some realities Apply in scheme, anti-E7MC constructs are immunoconjugates.In some embodiments, composition further includes and anti-E7MC structures Build the cell (such as effector cell) of body phase pass.In some embodiments, HPV16-E7 positive diseases are cancer.In some implementations In scheme, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharynx Cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the anti-E7MC constructs containing anti-E7MC antibody moieties of effective dose, antibody moiety specific binding bag The complex of peptide containing HPV16-E7 and MHC I proteinoid, its moderate resistance E7MC antibody moieties include weight chain variable district, and it is included SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence.In some embodiments, anti-E7MC constructs are deposited for non-natural .In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, anti-E7MC constructs are Polyspecific (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some realities Apply in scheme, anti-E7MC constructs are immunoconjugates.In some embodiments, composition further includes and anti-E7MC structures Build the cell (such as effector cell) of body phase pass.In some embodiments, HPV16-E7 positive diseases are cancer.In some implementations In scheme, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharynx Cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments of any one of the method for the treatment of HPV16-E7 positive diseases described above, resist E7MC constructs are bound to cell (such as immunocyte, such as T cell) before being applied to individual.Therefore, for example, there is provided The method of HPV16-E7 positive diseases in treatment individual, it is included a) by any one of anti-E7MC constructs as described herein Cell (such as immunocyte, such as T cell) is bound to form anti-E7MC constructs/cell conjugates, and b) is applied to individual The composition for including anti-E7MC constructs/cell conjugates of effective dose.In some embodiments, it is cell-derived from individual. In some embodiments, cell is not derived from individual.In some embodiments, anti-E7MC constructs are coupled to by covalent bond Molecule on cell surface and be bound to cell.In some embodiments, anti-E7MC constructs are coupled to carefully by non-covalent bond Molecule on cellular surface and be bound to cell.In some embodiments, anti-E7MC constructs are by by a part of anti-E7MC structures Body is built to be inserted into epicyte and be bound to cell.In some embodiments, anti-E7MC constructs are non-naturally occurring. In some embodiments, anti-E7MC constructs are full length antibody.In some embodiments, how special anti-E7MC constructs be Property (such as bispecific) molecule.In some embodiments, anti-E7MC constructs are Chimeric antigen receptor.In some embodiments In, anti-E7MC constructs are immunoconjugates.In some embodiments, HPV16-E7 positive diseases are cancer.In some realities Apply in scheme, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharynx Cancer.In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, individual for mammal (such as the mankind, non-human primate, rat, mouse, Cow, horse, pig, sheep, goat, dog, cat etc.).In some embodiments, individual is the mankind.In some embodiments, it is individual Body is clinical patients, clinical test volunteer, experimental animal etc..In some embodiments, individual age is less than about 60 years old (bag Such as age is included less than about any one of 50,40,30,25,20,15 or 10 years old).In some embodiments, individual age Greater than about 60 years old (including such as age is greater than about any one of 70,80,90 or 100 years old).In some embodiments, it is individual Diagnosis suffer from or gene on be susceptible to suffer from one or more (such as squamous cell carcinoma, cervical carcinoma, anuses in disease or illness as described herein Door cancer, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer).In some embodiments, individual has and one kind or more One or more risk factors of kind disease as described herein or illness correlation.

In some embodiments, the application is provided anti-E7MC constructs (in anti-E7MC constructs as described herein Any one) method of cell that is transferred in individual, the cell presents comprising HPV16-E7 peptides and MHC I classes in its surface The complex of protein, this method include the composition applied to individual and include anti-E7MC constructs.In some embodiments, Anti- E7MC constructs to be passed are related with cell (such as effector cell, such as T cell).

It is known in the art to be used for HPV16-E7 positive cancers (such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, vulva Cancer, carcinoma of penis, head and neck cancer or oropharyngeal cancer) or show abnormal HPV16-E7 expression any other disease many diagnostic methods and The clinical delimitation of those diseases.Such method includes but is not limited to such as immunohistochemistry, PCR and FISH (FISH)。

In some embodiments, anti-E7MC constructs of the invention and/or composition and second, third or the 4th medicament (including such as antitumor agent, growth inhibitor, cytotoxic agent or chemotherapeutant) is administered in combination is related to HPV16- to treat The disease or illness of E7 expression.In some embodiments, anti-E7MC constructs and the expression of increase MHC I proteinoid and/or The pharmaceutical agent combinations presented by the surface of MHC I proteinoid enhancing HPV16-E7 peptides are applied.In some embodiments, medicament Including such as IFN receptor stimulating agents, Hsp90 inhibitor, p53 expression facilitators and chemotherapeutant.In some embodiments, Medicament is IFN receptor stimulating agents, including such as IFN γ, IFN β and IFN α.In some embodiments, medicament suppresses for Hsp90 Agent, including such as KOS-953 (tanespimycin；17-AAG), Ah's spiramvcin (alvespimycin；17-DMAG)、 His auspicious mycin (retaspimycin；IPI-504)、IPI-493、CNF2024/BIIB021、MPC-3100、Debio 0932 (CUDC-305), PU-H71, Jia Litepi (Ganetespib；STA-9090)、NVP-AUY922(VER-52269)、HSP990、 KW-2478, AT13387, SNX-5422, DS-2248 and XL888.In some embodiments, medicament is p53 expression facilitators, Including such as 5 FU 5 fluorouracil and nutlin-3.In some embodiments, medicament is chemotherapeutant, including for example topology is replaced Health, Etoposide, cis-platinum, Paclitaxel and vincaleukoblastinum.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, wherein expressing HPV16-E7 cell does not present generally in its surface, or is presented with relative low content and include HPV16-E7 protein and MHC The complex of I proteinoid, method include the table that MHC I proteinoid is applied comprising anti-E7MC constructs and increased to individual Reach and/or by MHC I proteinoid strengthen HPV16-E7 peptides surface present medicament composition.In some embodiments In, medicament includes such as IFN receptor stimulating agents, Hsp90 inhibitor, p53 expression facilitators and chemotherapeutant.In some implementations In scheme, medicament is IFN receptor stimulating agents, including such as IFN γ, IFN β and IFN α.In some embodiments, medicament is Hsp90 inhibitor, including for example KOS-953 (17-AAG), Ah's spiramvcin (17-DMAG), his auspicious mycin (IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305), PU-H71, Jia Litepi (STA- 9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248 and XL888. In some embodiments, medicament is p53 expression facilitators, including such as 5 FU 5 fluorouracil and nutlin-3.In some embodiment party In case, medicament is chemotherapeutant, including such as topotecan, Etoposide, cis-platinum, Paclitaxel (paclitaxel) And vincaleukoblastinum.

Treatment of cancer can for example by, tumor regression, tumor weight or dimensional contraction, evolution time, survival the duration, Progresson free survival phase, overall reaction rate, duration of the reaction, quality of the life protein expression and/or Activity Assessment.It can be used and determine The method of therapy effect, including for example measure and react via radiophotography.

In some embodiments, therapeutic efficiency is measured in the form of Tumor growth inhibition percentage (TGI%), its user Program 100- (T/C × 100) is calculated, and wherein T is the mean relative tumor volume through treating tumour, and C is not treat tumour Mean relative tumor volume.In some embodiments, TGI% be about 10%, about 20%, about 30%, about 40%, about 50%, About 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% or more than 95%.

Using the dosage and method of anti-E7MC constructs composition

The dosage for the anti-E7MC constructs composition applied to individual (such as mankind) can with particular composition, mode of administration and The type of the disease for the treatment of and change.In some embodiments, the amount of composition is for causing goal response (such as partial reaction Or reaction completely) effectively.In some embodiments, the amount of anti-E7MC constructs composition is enough to cause complete anti-in individual Should.In some embodiments, the amount of anti-E7MC constructs composition is enough to cause the partial reaction in individual.In some implementations In scheme, the amount (such as when being administered alone) of the anti-E7MC constructs composition of administration is enough to combine with anti-E7MC constructs Thing treatment population of individuals in produce greater than about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%th, any one of 65%, 70%, 75%, 80%, 85% or 90% overall reaction rate.Individual is to side described herein The reaction of the treatment of method can be for example fixed containing measurement based on RECIST.

In some embodiments, the amount of composition is enough the progresson free survival phase for extending individual.In some embodiments In, the amount of composition is enough the total survival period for extending individual.In some embodiments, the amount of composition (such as is applied when individually Used time) it is enough to produce greater than about 50%, 60%, 70% or 77% in the population of individuals with anti-E7MC constructs composition treatment Any one of clinical benefit.

In some embodiments, it is as follows individually or with the amount of the composition of second, third and/or the 4th pharmaceutical agent combinations Amount：Compared to corresponding tumor size, cancer cell count or the tumor growth rate in the same individual before treatment or compared to Do not receive the corresponding activity in other individuals for the treatment of, it is sufficient to reduce tumor size, reduce cancer cell count or reduce tumour life Any in long speed at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% .Standard method can be used for the value for measuring this effect, such as by the analyzed in vitro of purifying enzyme, the analysis based on cell, animal Model or people's class testing.

In some embodiments, (such as the anti-E7MC antibody of total length, polyspecific resist the anti-E7MC constructs in composition E7MC molecules, anti-E7MC CAR or anti-E7MC immunoconjugates) amount (that is, be clinically subjected to less than toxicological effect is induced Toxicity level more than effect) content or in when applying composition to individual, can control or allow potential side effect Content.

In some embodiments, maximum tolerated dose of the amount of composition close to the composition for following identical dosage regimen (MTD).In some embodiments, any one of about 80%, 90%, 95% or 98% of the amount of composition more than MTD.

In some embodiments, (such as the anti-E7MC antibody of total length, polyspecific resist the anti-E7MC constructs in composition E7MC molecules, anti-E7MC CAR or anti-E7MC immunoconjugates) amount be included in the range of about 0.001 μ g to about 1000 μ g.

In some embodiments of any one of the above, anti-E7MC constructs in composition (such as total length The anti-E7MC molecules of anti-E7MC antibody, polyspecific, anti-E7MC CAR or anti-E7MC immunoconjugates) effective dose it is total in per kilogram The μ g of body weight 0.1 are to about in the range of 100mg.

Anti- E7MC constructs composition can be applied via various approach to individual (such as mankind), including for example intravenous, dynamic In arteries and veins, intraperitoneal, intrapulmonary, be administered orally, by inhalation, in vesica, intramuscular, tracheal strips, subcutaneous, intraocular, it is intrathecal, through mucous membrane and percutaneous. In some embodiments, the sustained continuous release preparaton of composition can be used.In some embodiments, composition is through quiet Applied in arteries and veins.In some embodiments, applied in composition trans-portal vein.In some embodiments, composition is through artery Interior administration.In some embodiments, composition is applied through intraperitoneal.In some embodiments, composition in liver through applying. In some embodiments, composition is applied by hepatic artery infusion.

Anti- E7MC CAR effector cells therapy

The application is also provided to be redirected to the specificity of effector cell's (such as naive T cells) using anti-E7MC CAR and included The method of HPV16-E7 peptides and the complex of MHC I proteinoid.Therefore, the present invention is also provided to stimulate and is directed in mammal Targeted cell population or comprising E7MC in delivery cell tissue effector cell's mediated responses (as T cell mediate immune response) Method, its include to mammal apply effector cell's (such as T cell) for expressing anti-E7MC CAR the step of.

Recipient in need can be infused to by expressing anti-E7MC CAR anti-E7MC CAR effector cells (such as T cell).It is defeated The E7MC that the cell of note can kill in recipient is in delivery cell.In some embodiments, different from antibody therapy, anti-E7MC CAR effector cell's (such as T cell) can replicate in vivo, cause the long duration that continued tumor can be caused to control.

In some embodiments, anti-E7MC CAR effector cells are that can undergo firm internal T cell amplification and can keep Extend the anti-E7MC CAR T cells of time quantum.In some embodiments, anti-E7MC CAR T cells of the invention develop into The specific memory T cell that any extra tumour is formed or grown can be suppressed through being re-activated.

The present invention anti-E7MC CAR T cells can also function as in mammal Ex vivo immunization inoculation and/or in vivo A kind of vaccine of therapy.In some embodiments, mammal is the mankind.

It is inoculated with Ex vivo immunization, to before mammal dosed cells, carries out at least one in the following in vitro：i) Amplifying cells, ii) nucleic acid and/or iii for encoding anti-E7MC CAR are introduced into cell) Cord blood cell.

In vitro program is well known in the art and discussed more fully below.In short, cell is from mammal (preferably people Class) separate and through expression anti-E7MC CAR disclosed herein carrier genetic modification (that is, ex vivo transduction or transfection).Can be to Mammalian subject applies anti-E7MC CAR cells to provide treatment benefit.Mammalian subject can be the mankind and anti-E7MC CAR cells can be autologous on recipient.Or cell can be allogeneic, homology or xenogenesis on recipient.

Program description in vitro amplifying candidate stem cell and progenitor cells is in the U.S. being incorporated herein by reference In state's patent the 5th, 199,942, and it can be applied to the cell of the present invention.Other appropriate methodologies are known in the art, therefore this The method that invention is not limited to any specific in vitro amplifying cells.In short, the cultured in vitro and expanding packet of T cell contain：(1) by Periphery blood collection or marrow explant collect CD34+ candidate stem cells and progenitor cells from mammal；And (2) expand this in vitro Class cell.Outside the Porcine HGF described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3 And the other factors of c- kit ligands can be used for cultivating and expanding the cell.

Except in addition to being used in terms of Ex vivo immunization inoculation based on the vaccine of cell, the present invention is also provided for vivo immunization Inoculation is with the composition and method for the antigen initiation immune response in patient.

The present invention anti-E7MC CAR effector cells (such as T cell) can individually or as with diluent and/or other components, Pharmaceutical composition as IL-2 or other cell factors or cell mass combine is applied.In short, the pharmaceutical composition of the present invention can Comprising anti-E7MC CAR effector cells (such as T cell), and it is one or more pharmaceutically or physiologically acceptable supporting agent, Diluent or excipient.Such composition can include buffer, such as neutral buffered saline, phosphate buffered saline (PBS) and its similar Thing；Carbohydrate, such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid, it is all Such as glycine；Antioxidant；Chelating agent, such as EDTA or the sweet peptide of bran Guang；Adjuvant (for example, aluminium hydroxide)；And preservative.One In a little embodiments, anti-E7MC CAR effector cells (such as T cell) composition is formulated to be used to intravenously apply.

The precise volume of anti-E7MC CAR effector cells (such as T cell) composition of the invention to be administered can be considered by doctor The individual difference of age, body weight, tumor size, infection or metastasis of cancer degree and patient (individual) patient's condition and determine.In some realities Apply in scheme, the pharmaceutical composition comprising anti-E7MC CAR effector cells (such as T cell) is with per kilogram of body weight about 104 to about 109 Individual cell, such as per kilogram of body weight about 104 to about 105, about 105 to about 106, about 106 to about 107, about 107 to about 108 or about 108 Dosage to any one of about 109 cells (including all integer values in the range of those) is applied.Anti- E7MC CAR effects Cell (such as T cell) composition can also these dosage apply it is multiple.Cell can be by using commonly known in immunotherapy Infusion techniques are applied (referring to such as Rosenberg et al., New Eng.J.of Med.319:1676,1988).Particular patient Optimal dosage and therapeutic scheme can be held by the person that is familiar with medical science by the disease indication and corresponding adjustment for the treatment of of monitoring patient Change places determination.

In some embodiments, it may be necessary to the anti-E7MC CAR T cells of individual administration of activated, and subsequent basis Drawn blood again from it (or being purged art), activating T cell of the invention, and patient is transfused these activation and the T cell expanded again. This process can be multiple per several Zhou Jinhang.In some embodiments, the blood drawing activation that T cell can be from 10cc to 400cc.At some In embodiment, T cell activates from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc or 100cc blood drawing.

The administration of anti-E7MC CAR effector cells (such as T cell) can be carried out in any convenient manner, including by atomization Suction, injection, intake, infusion, implantation or transplanting.Can subcutaneous, intracutaneous, intra-tumor, in tubercle, in marrow, it is intramuscular, intravenous (i.v.) to patient compositions described herein is applied in injection or peritonaeum.In some embodiments, it is of the invention anti- E7MC CAR effector cells (such as T cell) composition by it is intracutaneous or be subcutaneously injected to patient apply.In some embodiments In, anti-E7MC CAR effector cells (such as T cell) composition of the invention is applied by intravenous injection.Anti- E7MC CAR effects Answer cell (such as T cell) composition can direct injection into tumour, lymph node or sites of infection.

Therefore, for example, in some embodiments, there is provided the side of the HPV16-E7 positive diseases in treatment individual Method, it includes the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR that effective dose is applied to individual, and this is anti- E7MC CAR are included：A) extracellular, it includes complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Anti- E7MC antibody moieties, b) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences Intracellular signal transduction domain.In some embodiments, HPV16-E7 peptides are HPV16-E7 11-19 (SEQ ID NO:4). In some embodiments, MHC I proteinoid is HLA-A02.In some embodiments, MHC I proteinoid is HLA-A* 02:01.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous Cell cancer, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer Disease is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes specific binding and includes HPV16-E711-19 peptides (SEQ ID NO:And HLA-A*02 4):01 it is compound The anti-E7MC antibody moieties of body, b) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences The intracellular signal transduction domain of row.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, Cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.At some In embodiment, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variabl area sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, Or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, the HC-CDR2 bags The NO of ID containing SEQ:184 or 185 amino acid sequence, or it includes at most about 3 (any one of e.g., from about 1,2 or 3) individual ammonia The variant of base acid substitution, and HC-CDR3, the HC-CDR3 include SEQ ID NO:Any one of 186-188 amino acid sequence, Or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And ii) light chain variable district, its SEQ ID NO are included comprising LC-CDR1, the LC-CDR1:189 or 190 amino acid sequence, or its include at most about 3 (such as Any one of about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 Amino acid sequence, or it includes the variant of at most about 3 (any one of e.g., from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, b) cross-film Domain, and c) the intracellular signal transduction domain comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.At some In embodiment, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous cell carcinoma, uterine neck Cancer, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer HPV16-E7 Positive squamous cell cancer.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variabl area sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:183 amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:184 or 185 amino acid sequence, and HC-CDR3, the HC-CDR3 are included SEQ ID NO:Any one of 186-188 amino acid sequence；And ii) light chain variable district, it includes LC-CDR1, the LC-CDR1 Include SEQ ID NO:189 or 190 amino acid sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:191 amino Acid sequence, b) membrane-spanning domain, and the c) intracellular signal comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences Transduction domain.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squama Shape cell cancer, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, Cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variable district, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 ammonia Base acid sequence, or it includes the variant of at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, The HC-CDR2 includes SEQ ID NO:Any one of 78-98 amino acid sequence, or its include at most about 5 (such as from about 1,2,3, Any one of 4 or 5) variant of individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and HC-CDR3, the HC-CDR3 include SEQ ID NO:99-119、244 With any one of 245 amino acid sequence, or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual amino acid Substituted variant；And ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:120-140 and 246 Any one of amino acid sequence, or it includes at most about 5 (any one of such as from about 1,2,3,4 or 5) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Variant, LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence, or it includes The variant of at most about 3 (any one of such as from about 1,2 or 3) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence, or it is included at most about 5 (in such as from about 1,2,3,4 or 5 Any one) individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor variant；B) membrane-spanning domain, and c) include CD3 ζ intracellular signal transductions sequences and CD28 intracellular signals Conduct the intracellular signal transduction domain of sequence.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiment party In case, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer. In some embodiments, cancer is HPV16-E7 positive squamous cell cancers.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variabl area sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 Amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC- CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it is included extremely The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in more about 5 (any one of such as from about 1,2,3,4 or 5) individual HC-CDR sequences；And ii) light chain variable Region sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid sequence Row；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, should LC-CDR3 includes SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；Or it includes at most about 5 The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in (any one of such as from about 1,2,3,4 or 5) individual LC-CDR sequences；B) membrane-spanning domain, and c) include The intracellular signal transduction domain of CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, the moon Road cancer, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cells Cancer.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variabl area sequence, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 Amino acid sequence；HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence；And HC- CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；And ii) light chain can Become region sequence, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Any one of 120-140 and 246 amino acid Sequence；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence；And LC-CDR3, The LC-CDR3 includes SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；B) membrane-spanning domain, and c) wrap The intracellular signal transduction domain of the intracellular signal transduction sequences of ζ containing CD3 and CD28 intracellular signal transduction sequences.In some embodiments In, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, anus Cancer, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamas Shape cell cancer.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising i) weight chain variable district, it includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or its Variant with least about 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity, and gently Chain variable region, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or its have at least about The variant of 95% (for example, at least any one of about 96%, 97%, 98% or 99%) sequence identity；B) membrane-spanning domain, and c) Intracellular signal transduction domain comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.In some embodiments In, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, anus Cancer, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamas Shape cell cancer.In some embodiments, individual is the mankind.

In some embodiments, there is provided the method for the HPV16-E7 positive diseases in treatment individual, it is included to individual Using the composition for including the effector cell's (such as T cell) for expressing anti-E7MC CAR of effective dose, the anti-E7MC CAR are included a) Extracellular, it includes the anti-E7MC antibody portion of complex of the specific binding comprising HPV16-E7 peptides and MHC I proteinoid Point, comprising weight chain variable district, it includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain Variable region, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence；B) membrane-spanning domain, and c) include The intracellular signal transduction domain of CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences.In some embodiments, HPV16-E7 positive diseases are cancer.In some embodiments, cancer is such as squamous cell carcinoma, cervical carcinoma, cancer of anus, the moon Road cancer, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.In some embodiments, cancer is HPV16-E7 positive squamous cells Cancer.In some embodiments, individual is the mankind.

Cancer

In some embodiments, anti-E7MC constructs and anti-E7MC CAR cells are applicable to treat the HPV16-E7 positives Cancer.The cancer of any one of approach described herein treatment can be used to include non-vascularization, or not yet generally blood vessel The tumour of change, and vascularized tumors.Cancer can include non-solid tumors (such as neoplastic hematologic disorder, such as leukaemia and lymthoma) or Entity tumor can be included.Treating the type of the cancer by anti-E7MC constructs and anti-E7MC CAR cell therapies of the invention includes (but not limited to) carcinoma, blastoma and sarcoma, and some leukaemia or lymphoid malignancy, benign and malignant tumour, and dislike Venereal disease, for example, sarcoma, carcinoma and melanoma.Adult's lesion/cancer disease and tumors in children/cancer are also included.

Blood cancer is the cancer of blood or marrow.The example of blood (or courageous and upright) cancer includes leukaemia, including acute white blood Disease (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia and haematogonium, Promyelocyte, bone marrow mononuclear cell, monocarpotic cellularity and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukaemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), true property erythremia, lymph Knurl, lymphogranulomatosis (Hodgkin's disease), non Hodgkin lymphom (intractable and advanced form), multiple bone Myeloma, Walden Si Telunshi macroglobulinemias (Waldenstrom's macroglobulinemia), heavy chain disease, marrow Depauperation disease group, hairy cell leukemia and osteomyelodysplasia.

Entity tumor is the abnormal structure's block for being typically free of tumour or liquid regions.Entity tumor can be benign or dislike Property.Different types of entity tumor names (such as sarcoma, carcinoma and lymthoma) on forming its cell type.Entity tumor The example of (such as sarcoma and carcinoma) includes fibrosarcoma, myxosarcoma, sarcolipoma, chondrosarcoma, osteosarcoma and other sarcomas, cunning Film knurl, celiothelioma, You Wenshi tumours (Ewing's tumor), leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignant Tumour, cancer of pancreas, breast cancer, lung cancer, oophoroma, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal-cell carcinoma, gland cancer, Syringocarcinoma, medullary carcinoma of thyroid gland, papillary thyroid carcinoma, pheochromocytoma carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, marrow Property cancer, bronchiolar carcinoma, clear-cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wei Ermushi tumours (Wilms'tumor), cervical carcinoma (example Such as cervical dysplasias before cervical carcinoma and intrusion is bad), cancer of anus, carcinoma of anal canal or anal orifice and rectal intestine cancer, carcinoma of vagina, carcinoma of vulva (such as squama Shape cell cancer, intraepithelial carcinoma, gland cancer and fibrosarcoma), carcinoma of penis, oropharyngeal cancer, head cancer (such as squamous cell carcinoma), neck cancer (example Such as squamous cell carcinoma), carcinoma of testis (such as seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Lai Dixi Cytoma (Leydig cell tumor), fibroma, adenofibroma, adenomatoid tumor and lipoma), carcinoma of urinary bladder, melanoma, son Palace cancer (such as carcinoma of endometrium), bladder transitional cell carcinoma (such as squamous cell carcinoma, transitional cell carcinoma, gland cancer, carcinoma of ureter and bladder Cancer) and cns tumor (such as neuroglia knurl (such as brain stem neuroglia knurl and composite nerve glioma), spongioblastoma (also known as For glioblastoma multiforme) astrocytoma, CNS lymthomas, enblastoma, medulloblastoma, neurinoma cranium Pharyngeal canal knurl, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, oligodendroglioma, meningioma, nerve are female thin Born of the same parents' knurl, retinoblastoma and metastatic encephaloma).

Diagnosis and imaging method using anti-E7MC constructs

Anti- E7MC antibody moieties and its derivative of mark and the like (it specifically binds the E7MC on cell surface) Available for diagnostic purpose to detect, diagnose or monitor the disease and/or illness related to HPV16-E7 expression, including institute above Any one of disease and illness for stating, such as cancer.For example, anti-E7MC antibody moieties of the invention can be used in situ, body In interior, in vitro and vitro diagnosis assays or imaging analysis.

Other embodiments of the present invention are included in diagnosis individual (such as mammal, such as mankind) with HPV16-E7's Expression or the method for the relevant disease of unconventionality expression or illness.The E7MC that method is included in detection individual is in delivery cell.At some In embodiment, there is provided the disease or disease related to HPV16-E7 expression in diagnosis individual (such as mammal, such as mankind) The method of disease, it includes (a) and resisted to individual using the mark according to any one of embodiment as described above of effective dose E7MC antibody moieties；And the labelled content in (b) measure individual so that the level of mark suffers from higher than threshold level instruction individual Disease or illness.Threshold level can determine by various methods, including for example suffer from by according to diagnostic method described above Have in first group of individual of disease or illness and second group of individual not with disease or illness and detect mark, and threshold value is set The fixed content for extremely allowing to be distinguish between first and second group.In some embodiments, threshold level zero, and method Include the existence or non-existence of the mark in measure individual.In some embodiments, method is further contained in step of applying (a) intervals are waited to permit at the site for marking anti-E7MC antibody moieties preferentially in expression E7MC individual after Concentration (and for removing the non-binding anti-E7MC antibody moieties of mark).In some embodiments, method is further included and subtracted The background content of mark.Background content can determine by various methods, including for example apply mark anti-E7MC antibody moieties it Mark in preceding detection individual, or by according in the individual of diagnostic method detection described above not with disease or illness Mark.In some embodiments, disease or illness are cancer.In some embodiments, cancer is selected from for example by with the following group Into group：Squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer.

The anti-E7MC antibody moieties of the present invention may be used in method known to those skilled in the art analysis biological sample In E7MC be in delivery cell content.Suitable antibody labeling is known in the art and marked including enzyme, such as glucose oxidase；Put Injectivity isotope, as iodine (¹³1I、¹²⁵I、¹²³I、¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹⁵mIn、¹¹³mIn、¹¹²In、¹¹¹In), technetium (⁹⁹Tc、⁹⁹MTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga、⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), samarium (¹⁵³Sm), lutetium (¹⁷⁷Lu), gadolinium (¹⁵⁹Gd), promethium (¹⁴⁹Pm), lanthanum (¹⁴⁰La), ytterbium (¹⁷⁵Yb), holmium (¹⁶⁶Ho), yttrium (⁹⁰Y), scandium (⁴⁷Sc)、 Rhenium (¹⁸⁶Re、¹⁸⁸Re), praseodymium (¹⁴²Pr), rhodium (¹⁰⁵Rh) and ruthenium (⁹⁷Ru)；Luminol (luminol)；Fluorescence labeling, such as fluorescein and Rhodamine；And biotin.

Techniques known in the art can be applied to the anti-E7MC antibody moieties of mark of the present invention.Such technology is included (but not Be limited to) using difunctionality bonding agent (see, for example, U.S. Patent No. 5,756,065；No. 5,714,631；5,696,239th Number；No. 5,652,361；No. 5,505,931；No. 5,489,425；No. 5,435,990；No. 5,428,139；5th, No. 342,604；No. 5,274,119；No. 4,994,560；And No. 5,808,003).In addition to above-mentioned analysis, this area Various internal and in vitro analysis can be used in technical staff.For example, the cell in individual body can be exposed to and optionally passed through Detectable label, such as the anti-E7MC antibody moieties of labelled with radioisotope, and can for example by extraneous radiation scanning or Commented by analysis derived from the individual sample (such as biopsy or other biological sample) for being previously exposed to anti-E7MC antibody moieties Estimate the combination of anti-E7MC antibody moieties and cell.

Product and kit

In some embodiments of the present invention, there is provided containing suitable for treat HPV16-E7 positive diseases, such as cancer (example Such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer), anti-E7MC built Body is transferred to products of the E7MC in the cell for presenting E7MC on the surface, or separation or detection individual in the material of delivery cell. The product can include the mark or package insert that on container and container or container is enclosed.Suitable container include such as bottle, Bottle, syringe etc..Container can be formed by various materials, such as glass or plastic cement.In general, container is accommodated for treatment originally The effective composition of disease or illness described in text, and can have sterile discrepancy port (such as container can be intravenous solution bag Or with can by be subcutaneously injected needle-penetration plug bottle).At least one of composition activating agent is the anti-of the present invention E7MC constructs.Mark or package insert instruction said composition are used to treat very pathology.Mark or package insert will be another It is outer to include the specification that anti-E7MC constructs composition is applied to patient.Also the system for including combination treatment described herein is covered Product and kit.

Package insert refers to generally included containing being related to indication, use, dosage, apply in the encapsulation of commercially available treatment product With, the information of the taboo relevant with the use of such treatment product and/or the specification of warning.In some embodiments, medicine Product specification indication composition be used for treat HPV16-E7 positive cancers (such as squamous cell carcinoma, cervical carcinoma, cancer of anus, carcinoma of vagina, Carcinoma of vulva, carcinoma of penis, head and neck cancer or oropharyngeal cancer).

In addition, product can further include second container, it includes pharmaceutically acceptable buffer solution, such as biocidal property Water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution (Ringer's solution) and dextrose solution.It can Further comprise with regard to business and user's viewpoint for needed for other materials, including other buffer solutions, diluent, filter, Pin and syringe.

Also provide suitable for various purposes kit, such as treat HPV16-E7 positive diseases as described herein or Illness, the E7MC being transferred to anti-E7MC constructs in the cell for presenting E7MC on the surface, or separation or detection individual are presented Cell, optionally with article combination.The kit of the present invention includes one or more containers, and it is combined comprising anti-E7MC constructs Thing (or unit dosage forms and/or product), and in some embodiments, additionally comprise appointing in approach described herein Another medicament (medicament as described herein) and/or operation instructions of one.Kit can further include selection and be suitable for The individual explanation for the treatment of.The specification supplied in kit of the present invention is (for example, reagent generally in mark or package insert The scraps of paper that box includes) on printed instructions, but machine readable specification is (for example, magnetization or optical storage disk are loaded with Specification) also to be acceptable.

For example, in some embodiments, kit includes that (such as the anti-E7MC of total length resists containing anti-E7MC constructs The anti-E7MC molecules of body, polyspecific (the anti-E7MC antibody of such as bispecific) or anti-E7MC immunoconjugates) composition.At some In embodiment, kit includes the composition for a) including anti-E7MC constructs, and b) other at least one medicaments of effective dose, Wherein other medicaments increase MHC I proteinoid expression and/or enhancing HPV16-E7 peptides by MHC I proteinoid (such as IFN γ, IFN β, IFN α or Hsp90 inhibitor) surface present.In some embodiments, kit is included a) comprising anti- The composition of E7MC constructs, and b) anti-E7MC constructs composition is applied to treat HPV16-E7 positive diseases to individual, wrap Include such as squamous cell carcinoma, cervical carcinoma or cancer of anus.In some embodiments, kit includes and a) includes anti-E7MC constructs Composition, b) effective dose other at least one medicaments, wherein the expression of other medicaments increase MHC I proteinoid and/or Strengthen HPV16-E7 peptides to present by the surface of MHC I proteinoid (such as IFN γ, IFN β, IFN α or Hsp90 inhibitor), And anti-E7MC constructs composition and other medicaments c) are applied to treat HPV16-E7 positive diseases, including such as squamous to individual Cell cancer, cervical carcinoma or cancer of anus.Anti- E7MC constructs and other medicaments may be present in autonomous container or single container.Citing For, kit can include a kind of different composition or two or more composition, and one of which composition includes anti- E7MC constructs and another composition includes another medicament.

In some embodiments, kit includes a) (such as the anti-E7MC antibody of total length, more special comprising anti-E7MC constructs Different in nature anti-E7MC molecules (the anti-E7MC antibody of such as bispecific) or anti-E7MC immunoconjugates) composition, and b) combination is anti- E7MC constructs include anti-E7MC constructs/cell with cell (as derived from the cell of individual, such as immunocyte) to be formed The composition of conjugate and anti-E7MC constructs/cell conjugates composition is applied to treat HPV16-E7 positive diseases to individual The specification of (including such as squamous cell carcinoma, cervical carcinoma or cancer of anus).In some embodiments, kit is included and a) included The composition of anti-E7MC constructs, and b) cell (such as cytotoxic cell).In some embodiments, kit, which includes, a) wraps Composition containing anti-E7MC constructs, b) cell (such as cytotoxic cell), and c) anti-E7MC constructs are combined with cell with shape Into the composition comprising anti-E7MC constructs/cell conjugates and anti-E7MC constructs/cell conjugates are applied to individual to combine Thing is to treat the specification of HPV16-E7 positive diseases (including such as squamous cell carcinoma, cervical carcinoma or cancer of anus).In some realities Apply in scheme, kit includes the composition containing the anti-E7MC constructs combined with cell (such as cytotoxic cell).At some In embodiment, kit includes the composition for a) including the anti-E7MC constructs combined with cell (such as cytotoxic cell), And composition b) is applied to treat HPV16-E7 positive diseases (including such as squamous cell carcinoma, cervical carcinoma or cancer of anus) to individual Specification.In some embodiments, with reference to being the molecule that is bound to by anti-E7MC constructs on cell surface.At some In embodiment, association system is inserted into epicyte by by a part for anti-E7MC constructs.

In some embodiments, kit includes that to encode anti-E7MC constructs (such as the anti-E7MC antibody of total length, how special The anti-E7MC molecules (the anti-E7MC antibody of such as bispecific) of property, anti-E7MC CAR or anti-E7MC immunoconjugates) or its polypeptide portion Nucleic acid (or nucleic acid set).In some embodiments, kit, which includes, a) encodes anti-E7MC constructs or its polypeptide portion Nucleic acid (or nucleic acid set), and the b) host cell (such as effector cell) of express nucleic acid (or nucleic acid set).In some implementations In scheme, kit includes and a) encodes anti-E7MC constructs or the nucleic acid (or nucleic acid set) of its polypeptide portion, and b) specification, It is on the anti-E7MC constructs in i) expression host cell (such as effector cell, such as T cell), ii) prepare include anti-E7MC The composition of the host cell of construct or the anti-E7MC constructs of expression, and iii) applied to individual comprising anti-E7MC constructs or The composition of the host cell of anti-E7MC constructs is expressed to treat HPV16-E7 positive diseases, including such as squamous cell carcinoma, Cervical carcinoma or cancer of anus.In some embodiments, host cell is derived from individual.In some embodiments, kit bag Containing the nucleic acid (or nucleic acid set) for a) encoding anti-E7MC constructs or its polypeptide portion, b) place of express nucleic acid (or nucleic acid set) Chief cell (such as effector cell), and c) specification, it is on the anti-E7MC constructs in i) expression host cell, ii) prepare bag The composition of host cell containing anti-E7MC constructs or the anti-E7MC constructs of expression, and iii) applied to individual and include anti-E7MC The composition of the host cell of construct or the anti-E7MC constructs of expression is to treat HPV16-E7 positive diseases, including such as squamous Cell cancer, cervical carcinoma or cancer of anus.

In some embodiments, kit includes the nucleic acid for encoding anti-E7MC CAR.In some embodiments, reagent Box includes the carrier containing the nucleic acid for encoding anti-E7MC CAR.In some embodiments, kit includes a) anti-comprising coding The carrier of E7MC CAR nucleic acid, and b) specification, it by carrier on i) being introduced to effector cell, as derived from the T of individual In cell, ii) prepare the composition for including anti-E7MC CAR effector cells, and iii) thin to the anti-E7MC CAR effects of individual administration Born of the same parents' composition is to treat HPV16-E7 positive diseases, including such as squamous cell carcinoma, cervical carcinoma or cancer of anus.

The kit of the present invention is tied up in suitable packaging.Suitable packaging include but is not limited to bottle, bottle, tank, Flexible package (such as sealing Mylar or plastic bag) and the like.Kit can optionally provide the extra of such as buffer Component and illustrative information.Therefore the application also provides product, it includes bottle (such as sealed vial), bottle, tank, flexible bag Dress and its fellow.

To anti-E7MC constructs composition using related specification generally include the dosage on desired treatment, The information of time-histories and route of administration is administered.Container can be unit dosage, (such as multiple-unit container) in bulk or secondary unit dose.Lift For example, it is possible to provide kit, it contains anti-E7MC constructs (such as the anti-E7MC of total length of sufficient dosage as disclosed herein The anti-E7MC molecules of antibody, polyspecific (the anti-E7MC antibody of such as bispecific), anti-E7MC CAR or anti-E7MC immunoconjugates) with Persistently extend the period, such as one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 The moon, 5 months, 7 months, 8 months, 9 months or 9 months, any of the above item provided effective treatment of individual.Kit also may be used Anti- E7MC constructs and pharmaceutical composition and operation instructions including multiple unit doses and with pharmacy, such as hospital Enough amount packagings for storage and use in pharmacy and dispensary.

Exemplary embodiment

Embodiment 1. is in some embodiments, there is provided the anti-E7MC constructs of separation, it includes specific binding and wrapped The complex of (HPV16) HPV-16 E7 of hypotype containing human papilloma virus 16 and ajor histocompatibility (MHC) I proteinoid The antibody moiety of (HPV16-E7/MHC I class complexs, or E7MC).

In some other embodiments of embodiment 1, HPV16-E7/MHC I classes complex exists embodiment 2. In on cell surface.

In some other embodiments of embodiment 1, HPV16-E7/MHC I classes complex exists embodiment 3. In on cancer cell surfaces.

In some other embodiments of any one of embodiment 1 to 3, MHC I proteinoid is embodiment 4. Human leukocyte antigens (HLA)-A.

For embodiment 5. in some other embodiments of embodiment 4, MHC I proteinoid is HLA-A02.

In some other embodiments of embodiment 5, MHC I proteinoid is selected from the group embodiment 6.：HLA- A*02:01、HLA-A*02:02、HLA-A*02:06、HLA-A*02:07 and HLA-A*02:11.

For embodiment 7. in some other embodiments of embodiment 6, MHC I proteinoid is HLA-A*02:01.

Embodiment 8. in some other embodiments of any one of embodiment 1 to 7, antibody moiety with comprising HPV16-E7 peptides and with the HLA allele different from MHC I proteinoid the 2nd MHC I proteinoid complex hand over Fork reaction.

Embodiment 9. is in some other embodiments of any one of embodiment 1 to 8, HPV16-E7 peptide length For 8 to 12 amino acid.

In some other embodiments of any one of embodiment 1 to 9, HPV16-E7 peptides spread out embodiment 10. It is born from the E7 albumen HPV16-E7 of human papilloma virus hypotype 16.

Embodiment 11. is in some other embodiments of any one of embodiment 1 to 10, HPV16-E7 peptides tool Have selected from by SEQ ID NO:The amino acid sequence of the group of 3-14 compositions.

For embodiment 12. in some other embodiments of embodiment 10, HPV16-E7 peptides have YMLDLQPET (SEQ ID NO:4) amino acid sequence.

Embodiment 13. in some other embodiments of embodiment 12, the anti-E7MC constructs of separation with comprising With YMLDVQPET (SEQ ID NO:11) variant of HPV16-E7 peptides of amino acid sequence and answering for MHC I proteinoid Fit cross reaction.

Embodiment 14. in some other embodiments of any one of embodiment 1 to 13, behave by antibody moiety Class, humanization or semi-synthetic.

For embodiment 15. in some other embodiments of any one of embodiment 1 to 14, antibody moiety is total length Antibody, Fab, Fab', (Fab') 2, Fv or scFv (scFv).

Embodiment 16. is in some other embodiments of any one of embodiment 1 to 15, and antibody moiety is with about 0.1pM to about 500nM equilibrium dissociation constant (Kd) is bound to HPV16-E7/MHC I class complexs.

Embodiment 17. is in some other embodiments of any one of embodiment 1 to 16, the anti-E7MC of separation Construct is bound to HPV16-E7/MHC I class complexs with about 0.1pM to about 500nM Kd.

Embodiment 18. is in some other embodiments of any one of embodiment 1 to 17, antibody moiety bag Contain：

I) weight chain variable district, it includes complementary determining region of heavy chain (HC-CDR) 1, includes amino acid sequence G-F/G/Y-S/T- F-S/T-S-Y-A/G(SEQ ID NO:183), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, the HC- CDR2 includes amino acid sequence I-N/I-P-X-X-G-G/T/I-T/A/P or I-S-X-S/D-G/N-G/S-N-T/I/K (SEQ ID NO:184 or 185), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC-CDR3 include amino acid Sequence A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D, A-R-G-X-X-X-Y-Y/G/S or A-R-G-X-X-Y-Q/W-W-S-X- D-D(SEQ ID NO:186-188)；Or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And

Ii) light chain variable district, it includes complementary determining region of light chain (LC-CDR) 1, includes amino acid sequence N-I-G-S-N/K Or L-R-S/N-X-Y (SEQ ID NO:189 or 190), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and LC- CDR3, the LC-CDR3 include amino acid sequence A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V (SEQ ID NO:191)；Or it includes the variant of at most 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, wherein

X can be any amino acid.

In some other embodiments of any one of embodiment 1 to 17, antibody moiety includes embodiment 19.：

I) weight chain variable district, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 ammonia Base acid sequence, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, HC-CDR2, the HC-CDR2 include SEQ ID NO:78- Any one of 98 amino acid sequence, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and HC-CDR3, the HC- CDR3 includes SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it includes at most about 5 amino The variant of acid substitution；And

Ii) light chain variable district, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:Appointing in 120-140 and 246 The amino acid sequence of one, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence, or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and LC- CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence；Or it is included extremely The variant of more about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

Embodiment 20. is in some other embodiments of any one of embodiment 1 to 17, antibody moiety bag Contain：

I) heavy chain (HC) variable region, it includes HC-CDR1, and the HC-CDR1 includes SEQ ID NO:Any one of 57-77 Amino acid sequence, HC-CDR2, the HC-CDR2 include SEQ ID NO:Any one of 78-98 amino acid sequence, and HC- CDR3, the HC-CDR3 include SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245；Or it is included extremely The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in more about 5 HC-CDR areas；And

Ii) light chain (LC) variable region, it includes LC-CDR1, and the LC-CDR1 includes SEQ ID NO:In 120-140 and 246 Any one amino acid sequence, LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid Sequence, and LC-CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence； Or it includes the variant of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor at most about 5 LC-CDR areas.

Embodiment 21. is in some other embodiments of embodiment 19 or 20, and antibody moiety is comprising a) heavy chain can Become area, it includes SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, or itself and SEQ ID NO:15- Any one of 35 and 233-237 have the variant of at least about 95% sequence identity；And b) light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence, or itself and SEQ ID NO:In 36-56 and 238-243 Any one has the variant of at least about 95% sequence identity.

For embodiment 22. in some other embodiments of embodiment 21, antibody moiety includes weight chain variable district, its Include SEQ ID NO:Any one of 15-35 and 233-237 amino acid sequence, and light chain variable district, it includes SEQ ID NO:Any one of 36-56 and 238-243 amino acid sequence.

Embodiment 23. is in some other embodiments of any one of embodiment 1 to 22, the anti-E7MC structures of separation It is full length antibody to build body.

Embodiment 24. is in some other embodiments of any one of embodiment 1 to 23, the anti-E7MC of separation Construct is monospecific.

Embodiment 25. is in some other embodiments of any one of embodiment 1 to 23, the anti-E7MC of separation Construct is polyspecific.

For embodiment 26. in some other embodiments of embodiment 25, the anti-E7MC constructs of separation are double special The opposite sex.

In some other embodiments of embodiment 25 or 26, the anti-E7MC constructs of separation are embodiment 27. Series connection scFv, bifunctional antibody (Db), Single-chain bifunctional antibody (scDb), double affinity target (DART) antibody, double variable again Area (DVD) antibody, pestle-mortar (KiH) antibody, depressed place lock (DNL) antibody, chemical crosslinking antibody, heteromultimeric antibody or different conjugate Antibody.

Embodiment 28. in some other embodiments of embodiment 27, the anti-E7MC constructs of separation be comprising Two scFv by peptide linker connection series connection scFv.

For embodiment 29. in some other embodiments of embodiment 28, peptide linker includes amino acid sequence GGGGS。

Embodiment 30. is in some other embodiments of any one of embodiment 25 to 29, the anti-E7MC of separation Construct further includes the secondary antibody part of the second antigen of specific binding.

For embodiment 31. in some other embodiments of embodiment 30, the second antigen is anti-on T cell surface It is former.

For embodiment 32. in some other embodiments of embodiment 31, the second antigen is selected from what is consisted of Group：CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.

For embodiment 33. in some other embodiments of embodiment 31, the second antigen is CD3 ε, and is separated anti- E7MC constructs are specific comprising having to HPV16-E7/MHC I classes complexs with specific N-terminal scFv and to CD3 ε C-terminal scFv series connection scFv.

For embodiment 34. in some other embodiments of embodiment 31, T cell is selected from the group consisted of： Cytotoxic T cell, helper cell and natural killer T cell.

Embodiment 35. in some other embodiments of embodiment 30, the second antigen be constant killer cell, in Antigen on property granulocyte, monocyte, macrophage or surface of dendritic cells.

Embodiment 36. is in some other embodiments of any one of embodiment 1 to 22, the anti-E7MC structures of separation It is Chimeric antigen receptor to build body.

For embodiment 37. in some other embodiments of embodiment 36, Chimeric antigen receptor includes portion containing antibody Extracellular, membrane-spanning domain and the intracellular signal comprising CD3 ζ intracellular signal transductions sequences and CD28 intracellular signal transduction sequences divided Transduction domain.

Embodiment 38. is in some other embodiments of any one of embodiment 1 to 22, the anti-E7MC structures of separation It is the immunoconjugates comprising antibody moiety and effector molecule to build body.

For embodiment 39. in some other embodiments of embodiment 37, effector molecule is selected from consisting of Group therapeutic agent：Medicine, toxin, radio isotope, protein, peptide and nucleic acid.

For embodiment 40. in some other embodiments of embodiment 39, therapeutic agent is medicine or toxin.

For embodiment 41. in some other embodiments of embodiment 38, effector molecule is mark.

Embodiment 42. is in some embodiments, there is provided the separation comprising any one of embodiment 1 to 40 resists The pharmaceutical composition of E7MC constructs.

Embodiment 43. is in some embodiments, there is provided the separation of any one of expression embodiment 1 to 41 resists The host cell of E7MC constructs.

Embodiment 44. is in some embodiments, there is provided the separation of any one of coding embodiment 1 to 41 resists The nucleic acid of the polypeptide fractions of E7MC constructs.

Embodiment 45. is in some embodiments, there is provided the carrier of the nucleic acid comprising embodiment 44.

Embodiment 46. is in some embodiments, there is provided the anti-E7MC structures of the separation of expression embodiment 36 or 37 The effector cell of body.

For embodiment 47. in some other embodiments of embodiment 46, effector cell is T cell.

Embodiment 48. is in some embodiments, there is provided detection presents include HPV16-E7 peptides and MHC I on the surface The method of the cell of the complex of proteinoid, it, which is included, makes cell be contacted with the anti-E7MC constructs of the separation of embodiment 41 And the presence of the mark on detection cell.

Embodiment 49. is in some embodiments, there is provided individual method of the treatment with HPV16-E7 positive diseases, It includes the pharmaceutical composition for the embodiment 42 that effective dose is applied to individual.

Embodiment 50. is in some embodiments, there is provided individual method of the treatment with HPV16-E7 positive diseases, It includes the effector cell for the embodiment 46 or 47 that effective dose is applied to individual.

Embodiment 51. is in some embodiments, there is provided individual method of the diagnosis with HPV16-E7 positive diseases, It is included

A) the anti-E7MC constructs of the separation of the embodiment 41 of effective dose are applied to individual；And

B) mark level in individual is determined, it is positive to suffer from HPV16-E7 for wherein mark level individual on threshold level Disease.

Embodiment 52. is in some embodiments, there is provided individual method of the diagnosis with HPV16-E7 positive diseases, It is included

A) sample from individual is made to be contacted with the anti-E7MC constructs of the separation of embodiment 41；And

B) quantity of the fixed cell combined with the anti-E7MC constructs of the separation in sample, wherein with the anti-E7MC structures that separate Build body combination cell quantitative value on threshold level individual suffer from HPV16-E7 positive diseases.

In embodiment 49-52 some other embodiments, HPV16-E7 positive diseases are embodiment 53. HPV16-E7 positive cancers.

For embodiment 54. in some other embodiments of embodiment 53, HPV16-E7 positive diseases are that squamous is thin Born of the same parents' cancer.

Embodiment 54. in some other embodiments of embodiment 53, HPV16-E7 positive diseases be cervical carcinoma, Anogenital cancer, head and neck cancer or oropharyngeal cancer.

Embodiment

It would be recognized by those skilled in the art that in scope of the invention and spirit, some embodiments are possible.It is existing The present invention will be more fully described with reference to following non-limiting examples.Embodiments below further illustrates the present invention, but works as It so should not be construed in any way as limiting its category.

Material

Cell sample, cell line and antibody

Cell line includes：CaSki(ATCC CRL-1550；HLA-A2⁺、HPV16⁺), Head and neck squamous cell carcinoma cell line UM- SCC-104(Millipore；HLA-A2⁺、HPV16⁺), liver gland cell system SK-HEP-1 (ATCC HTB-52；HLA-A2⁺, HPV16^-), cervical squamous cell carcinoma cell line SiHa (ATCC HTB-35；HLA-A2^-、HPV16⁺), cervical cancer tumer line C33A (ATCC HTB-31；HLA-A2⁺、HPV16^-), cervical cancer tumer line MS-751 (ATCC HTB-34；HLA-A2⁺、HPV16^-), cervical gland Cancerous cell line HeLa (ATCC CCL-2；HLA-A2^-、HPV16^-), hepatocellular carcinoma cells system Hep3B (ATCC HB-8064；HLA- A2^-、HPV16^-), hepatocellular carcinoma cells system HepG2 (ATCC HB-8065；HLA-A2^-、HPV16^-), lymphoma cell line Raji (ATCC CCL-86；HLA-A2^-、HPV16^-), T cell Leukemia Cell Lines Jurkat (ATCC TIB-152；HLA-A2^-、 HPV16^-), lymphoma cell line Daudi (ATCC CCL-213；HPA-A2^-、HPV16^-), Leukemia Cell Lines K562 (ATCC CCL-243；HLA-A2^-、HPV16^-) and lymphoblastoid cell line T2 (ATCC CRL-1992；HLA-A2⁺、HPV16^-).T2 is TAP Deficient cells system.Cell line is in 37 DEG C/5%CO₂Be supplemented with 5%FCS, penicillin, streptomysin, 2mmol/L glutamine with And cultivated in the RPMI 1640 of 2 mercapto ethanol.

All peptides pass through Genemed Synthesis, Inc. (San Antonio, Tex.) purchases and synthesis.Peptide it is pure Degree>90%.Peptide is dissolved in DMSO and freezed with 5mg/mL normal saline dilution and at -180 DEG C.Biotin labeling is single-stranded HPV16-E7 peptides/HLA-A*02:01 and control peptide/HLA-A*02:01 complex by with restructuring HLA-A02 and β -2 microballoon eggs In vain (β 2M) (~2M) refolding peptide and synthesize.11 kinds combine HLA-A*02:01 control peptide (SEQ ID NO:193-203) certainly 10 kinds of genes produce below：BCR、BTG2、CALR、CD247、CTSG、DDX5、HLA-E、IFI30、PPP2R1B、SSR1.

Embodiment 1. produces biotin labeling HPV16-E7/HLA-A*02:01 complex monomer

Biotin labeling HPV16-E7/HLA-A*02:01 complex single mass system is according to standard scheme (John D.Altman And Mark M., Davis, Current Protocols in Immunology 17.3.1-17.3.33,2003) prepare.Letter Yan Zhi, the DNA of encoding full leng mankind beta-2 microglobulin (β 2m) are synthesized and are cloned into carrier PET-27b by Genewiz.BirA Peptide substrate (BSP) is added to HLA-A*02:The C-terminal of 01 extracellular (ECD).Encode HLA-A*02:01ECD-BSP DNA is also Synthesized and be cloned into carrier pET-27b by Genewiz.Express the mankind β 2m and HLA-A*02:01ECD-BSP carrier difference Transition is into e. coli bl21 cell, and the albumen expressed is separated with inclusion bodies from bacterial cultures.Peptide ligand HPV16-E7 peptides 11-19 is by the mankind β 2m and HLA-A*02:01ECD-BSP refoldings are to form HPV16-E7 peptides/HLA-A* 02:01 complex monomer.Fold peptide/HLA-A*02:01 monomer by be concentrated by ultrafiltration and it is further via the exclusive chromatography of size Purifying.HiPrep 26/60Sephacryl S-300HR clone (Hyclone) Du Erbeikeshi by the sea of 1.5 tubing string volumes Phosphate buffered salt solution (Thermo Scientific, catalog number (Cat.No.) SH3002802) balances.Non- purification of samples it is loaded and Eluted with 1 tubing string volume.Eluting substantially at 111mL corresponding to the first peak of false folding aggregation, folded corresponding to appropriate The peak of MHC complexs is observed at 212mL, and is observed corresponding to free β 2M peak at 267mL (Fig. 1).Peptide/HLA-A*02: 01 monomer purifies via the BirA enzyme reaction biotin labelings mediated and then by high-res anion-exchange chromatography.Biology Element mark peptide/HLA-A*02:01 monomer is stored in PBS at -80 DEG C.

The SDS-PAGE of HPV16-E7 peptides/MHC complexs can be carried out purifying to determine lipidated protein.For example, 1 μ g eggs White matter complex and 2.5 μ L NuPAGE LDS sample buffers (Life Technologies, NP0008) mix and use is gone Ionized water reaches 10 μ L.Sample heats 10 minutes at 70 DEG C, is then supported on gel.Gel electrophoresis 1 is carried out in 180V Hour.

Embodiment 2. is selected and characterized to HPV16-E7/HLA-A*02:01 complex has specific scFv.

A collection of mankind scFv antibody phage display libraries (diversity=10 constructed by Eureka Therapeutics × 1010) it is specific to HPV16-E7/HLA-A*02 for selection:01 mankind mAb.Use in 15 whole mankind bacteriophage scFv libraries In relative to HPV16-E7/HLA-A*02:01 complex elutriation.In order to reduce plastic cement table is fixed to by by protein complex The topographical variations of the MHCI complexs introduced on face, known disc elutriation is substituted using solution elutriation and cell elutriation.Washed in a pan in solution To choose, biotin labeling antigen mixes with mankind's scFv phage libraries first after by the extension of PBS washing, And then, antigen-scFv antibody phages composite wears promise bead M-280 via magnetic track by what streptavidin combined Leave behind.With reference to clone then through eluting and being used for ehec infection XL1-Blue cells.In cell elutriation, it is loaded with The T2 cells of HPV16-E7 peptides mix with mankind's scFv phage libraries first.T2 cells are TAP defects, HLA-A*02:01+ drenches Bar mother cell cell line.In order to load peptide, T2 cells are through the serum-free containing peptide (50 μ g/ml) in the presence of 20 μ g/ml β 2M RPMI1640 culture main pulses are overnight.After being washed by PBS extension, there is the peptide with reference to scFv antibody phages to load T2 cells through quick centrifugation.With reference to clone then through eluting and being used for ehec infection XL1-Blue cells.It is expressed in Phage clone in bacterium is then purified.Carried out by solution elutriation, cell elutriation or the combination of solution and cell elutriation Elutriation 3-4 bouts specifically bind HPV16-E7/HLA-A*02 to be enriched with:01 scFv phage clones.

Streptavidin ELISA disks are coated respectively with biotin labeling HPV16-E7 11-19 peptides/HLA-A*02:01 Complex monomer or biotin labeling C3 control peptides/HLA-A*02:01 monomer.C3 peptides are people's nucleoprotein p68 derived peptides YLLPAIVHI(SEQ ID NO:193).For HPV16-E711-19 peptides/HLA-A*02:01 complex carrys out self enrichment phagocytosis Indivedual phage clones in body display elutriation pond are cultivated in coating pan.The anti-M13 that the combination of phage clone combines by HRP Antibody test and use HRP Substrate developments.Absorptivity is read at 450nm.145 positives are pure to wash in a pan via 1272 from bacteriophage The ELISA screenings of the phage clone of enrichment are selected to differentiate.Fig. 2 is provided and biotin labeling HPV16- is bound in elisa assay E7/HLA-A*02:The example of the phage clone of 01 monomer.41 Unique clones are by 145 ELISA positive phage clones DNA sequencing differentiate.By flow cytometry (facs analysis), spy is further tested using the HPV16-E7 work T2 cells loaded The opposite sex and Unique clones and the HLA-A*02 on liver cell surface:The combination of 01/ peptide complex.Fig. 3 proves exemplary positive gram The grand T2 cells for being bound to the load of HPV16-E7 11-19 peptides or the T2 cells of control peptide (C3 peptides) load.Control includes not loading The conjugated horse anti-mouse IgG controls (only secondary antibody) of the T2 cells (only cell) and R-PE of peptide.In short, it is loaded with HPV16- The T2 cells of E7 11-19 peptides or C3 peptides dye first by purified scFv phage clones, then using the anti-M13mAb of mouse The second dyeing is carried out, and the horse anti-mouse IgG (coming from Vector Labs) combined using R-PE carries out the 3rd dyeing.Each dyeing Step between 30-60 minutes on ice carry out and cell washed twice between dyeing.In 41 clone's tests, 24 spies The T2 cells of opposite sex identification HPV16-E7 loads.This 24 phage clones are specifically bound to the T2 cells of HPV16-E7 loads And in HLA-A*02:Nonrecognition is loaded with the T2 cells of C3 peptides in the case of 01, or does not load the T2 cells of peptide.

The sign of embodiment 3.FACS positive HPV16-E7 specific bacteriophages clone

With the cross reactivity of mouse HPV16-E7 peptide 11-19 variants

The HPV16-E7 11-19 peptides selected in the present invention are highly conserved in a variety of HPV16 bacterial strains.In a research, It was found that this peptide is completely conservative in 16 kinds of HPV16-E7 protein sequences known to 17 kinds.Identified in remaining sequence Single amino acids mutation in E7 11-19.Then in 35 HPV16 of analysis cervical carcinomas infected or 1 of virus in patients with cervicitis The middle discovery variant (Zhang G.L. etc., Database (Oxford) .2014:bau031,2014).Selected from for HPV16-E7 The clone of the FACS binding analysis of the T2 cells of peptide 11-19 loads is loaded by facs analysis using variant HPV16-E7 peptides T2 cells living are on for HPV16-E7 peptides 11-19 variants/HLA-A*02 on liver cell surface:The intersection of 01 complex is anti- Answering property further characterizes.The variant peptides are different from the conservative HPV16-E7 11-19 peptides for 5 in single amino acids.It is described Variant peptide sequence is YMLDVQPET (SEQ ID NO:11), the conservative HPV16-E7 11-19 sequences are YMLDLQPET (SEQ ID NO:4)。

In short, T2 cell loadings have conservative HPV16-E7 11-19 peptides, variant HPV16-E7 11-19 peptides or β 2M. By the T2 cells of peptide load first by purifying scFv phage clones dyeing, then the second dye is carried out using the anti-M13mAb of mouse Color, and the horse anti-mouse IgG (coming from Vector Labs) being conjugated using R-PE carries out the 3rd dyeing.Each staining procedure is in 30-60 Minute is carried out on ice, and cell washes twice between each dyeing.

The epitope mapping walked by alanine

Identified to accurately study epitope for mAb, the HPV16-E7 peptide arteries and veins that will there is alanine to substitute at 1 and 8 On punching to T2 cells.Then the combination of the T2 cells loaded by facs analysis test antibody phage clone in these peptides.Often FACS average fluorescent strengths (MFI) value of kind FACS measure is displayed in Table 6.K07 helper phages are negative controls, are shown in The single bacteriophage presented without scFv on phage particle surface is not tied with any one in the T2 groups of cells of peptide load Close.Antibody BB7.2 identifies HLA-A02 α chains.The MHC complexs of the stable cell surface of the combination of peptide and MHC complexs.Therefore, with The T2 cells for not loading peptide are compared, and being loaded with the T2 cells of MHC binding peptides has the BB7.2 binding signals of enhancing (such as table 6 Shown in the first row).BB7.2 combinations data represent that the peptide of alanine substitution remains able to combine the HLA-A* on T2 cell surfaces 02:01 molecule.Although the small configuration epitope that all test antibody identifications are formed by HPV16-E7 peptides and its surrounding MHC α chain residues, It is quite different from the crucial peptide residue of various antibody interaction.For example, prediction clone #4 is bound to the N of HPV16-E7 peptides Half portion is held, because the alanine substitution at position 1 or 5 substantially reduces and the combination of the T2 cells of peptide load.By contrast, position Alanine substitution at 8 does not change identical clone and HLA-A*02:The combination of 01 complex.On the other hand, #11 contrapositions are cloned The alanine substitution put at 1 and 8 is insensitive, but its combination is completely eliminated in the alanine substitution at position 5.Fig. 4 provides FACS The example of analysis, show phage clone #11 and be loaded with the combination of the T2 cells of various HPV16-E7 peptides.Control includes not having The T2 cells (only cell) and the horse anti-mouse IgG conjugated with R-PE of peptide load compare (only secondary antibody).Determined by FACS Test the antibody that the T2 cells of peptides different from being loaded with these combine.

Table 6

Assessed for the antibody binding specificity of endogenous peptide

On average, each karyoblast expression about 500,000 different peptides/MHC I class complexs in human body.In order to by anti-peptide/ The exploitation of MHC I classes recombinant antibody is the cancer therapy drug with high specific and therapeutic index, to antibody it is essential that spy Opposite sex identification target peptide/MHC I complexs, rather than MHC I molecules itself, or it is bound to other peptides presented on cell surface MHC I molecules.For current research, related MHC I molecules are HLA-A*02:01.In the morning of our bacteriophage elutriations and screening During stage phase, we eliminate and are bound to single HLA-A*02:The antibody of 01 molecule (see, for example, Fig. 2 and Fig. 3).Also it is directed to 11 kinds of endogenous HLA-A*02:Phage clone at the top of 01 peptide screening, the peptide derived from be often expressed as in it is polytype into The protein of core human cell, such as hyperglobulinemia α chains, β chains, nucleoprotein p68 and the like.With 11 endogenous peptides (table 7, SEQ ID NO:193-203) recombinant peptide/the HLA-A*02 folded:01 complex is respectively coated on streptavidin disk Above and via elisa assay determine antibody binding.In short, by indivedual phage clones in peptide/HLA-A*02：01 complex applies Incubated on the disk of cloth.By the combination of the HRP anti-M13 antibody tests phage clones being conjugated, and developed the color using HRP substrates. Absorbance is read at 450nm.As shown in Figure 5, HPV16-E7 peptides/HLA-A*02:01 specific antibody phage clone combines HPV16-E7/HLA-A*02:01 complex, without combining the HLA-A*02 folded with endogenous peptide:01 complex.We draw The antibody of discriminating is specific to HPV16-E7 peptides/HLA-A*02:01 complex, and nonrecognition is bound to other HLA-A*02:01 limit The HLA-A*02 of the peptide of system:The conclusion of 01 molecule.

Table 7

The engineered bispecific antibody of embodiment 4.

Use HPV16-E7/HLA-A*02:The scFv sequences of 01 specific bacteriophage clone produce bispecific antibody (BsAb).BsAb is single chain bispecific antibody, and it includes HPV16-E7/HLA-A*02 in N-terminal:01 specific bacteriophage is cloned ScFv sequences and include anti-human CD3 ε mouse monoclonals scFv (Brischwein, K. et al., Molecular in C-terminal Immunology 43:1129-1143,2006).Encode HPV16-E7scFv and anti-human CD3 ε scFv DNA fragmentation by Genewiz is synthesized and is subcloned using standard DNA techniques into You Lika (Eureka) mammalian expression vectors pGSN-Hyg. Six histamine labels insert C-terminal for antibody purification and detection.Chinese hamster ovary (CHO) cell turns through BsAb expression vectors Dye, and 7 days are then incubated for produce BsAb antibody.Collect the Chinese hamster ovary celI supernatant of the HPV16-E7BsAb molecules containing secretion. Using HisTrapHP tubing strings (GE Healthcare) BsAb is purified by FPLC AKTA systems.In short, Chinese hamster ovary celI culture Thing is clarified and is loaded under low imidazole concentration (20mM) on tubing string, and then isocratic high imidazole concentration elution buffer (500mM) is used for elution of bound BsAb protein.Purify HPV16-E7 BsAb purity and molecular weight under the reducing conditions by Gel electrophoresis determines.4 μ g proteins and 2.5 μ LNuPAGE LDS sample buffers (Life Technologies, NP0008) are mixed Close and it is added up 10 μ L by deionized water.Sample heats 10 minutes at 70 DEG C, is then loaded on gel.In 180V Lower progress gel electrophoresis 1 hour.About 50KD bands are observed the master tape (Fig. 6) on gel.

Antibody aggregation can be assessed by the exclusive chromatography of size (SEC).For example, 50 μ L samples are by Dulbecco's phosphoric acid The buffer solution of salt buffer salt solution (Fisher Scientific, SH30028.FS) and 0.2M arginine composition (is adjusted to pH 7.0) sprayed when flowing into SEC tubing strings (such as Agilent, BioSEC-3,300A, 4.6 × 300mm).Selection, which has, to be less than The BsAb of 10% HMW aggregation is used to further characterize.

The sign of embodiment 5.HPV16-E7BsAb antibody

The binding affinity of HPV16-E7 BsAb antibody

HPV16-E7 BsAb antibody and restructuring HPV16-E7/HLA-A*02:The binding affinity of 01 complex for example by Surface plasma resonant (BiaCore) measures.HPV16-E7 BsAb and HPV16-E7/HLA-A*02:Combination between 01 complex Parameter is according to manufacturer's scheme for multi-cycle kinetic measurement for example using His Captur kits (GE Healthcare, catalog number (Cat.No.) 28995056) measured on Biacore X100 (GE Healthcare).For all of analysis Protein is diluted using HBS-E buffer solutions.For example, 1 μ g/mL HPV16-E7 BsAb are by making solution continue 2 with 2 μ L/min Minute flows through flow cell 2 and is fixed on the sensor wafer by the pre- functionalization of anti-polyhistidine antibody.Towards HPV16-E7/A* 02:01 complex is incorporated in, such as is analyzed at 0.19,0.38,7.5,15 and 30 μ g/mL, is respectively run by 3 under 30 μ L/min Minute association and 3 minutes dissociation compositions.At the end of circulation, surface is buffered using the regeneration from His Capture kits Liquid regenerates.After kinetic measurement, surface is regenerated using the actified solution from kit.Data are by BiaCore X-100 Assess software and use 1:1 binding site pattern analysis.Then calculations incorporated parameter (association rate constants k_a, dissociation constant k_dIt is and flat The dissociation constant that weighs K_d)。

The T cell killing analysis of the T2 cells of peptide impact

Cytotoxicity is analyzed by LDH cytotoxicity analysis (Promega).It is thin purchased from AllCells mankind T Born of the same parents wear promise bead (Invitrogen) according to manufacturer's scheme activation and amplification by CD3/CD28.Activating T cell (ATC) is having There is 10%FBS plus culture in 100U/ml IL-2 RPMI1640 culture mediums and maintain, and used at the 7-14 days.Pass through Facs analysis, the CD3 of T cell⁺For>99%.Activating T cell (effector cell) and target peptide load T2 cells using 1 μ g/ml or 0.2 μ g/ml BsAb is with 5:1 ratio co-cultures 16 hours.By using 50 μ g/ml target HPV16-E7 11-19 peptides (YMLDLQPET,SEQ ID NO:Or negative control AFP158 peptides (FMNKFIYEI, SEQ ID NO 4):192) T2 cells are incubated Peptide load T2 cells are prepared overnight.Also negative control AFP158/HLA-A*02 is included:01 specific b sAb.Cultivated by determining LDH activity in supernatant determines cytotoxicity.As shown in fig. 7, clone #2, #5, #17 and #40 are with selectivity and agent The mode that amount relies on kills HPV16-E7 peptides load T2 cells.The anti-AFP158/HLA-A*02 of negative control:01BsAb is especially oriented Kill AFP158 peptides load T2 cells.

The T cell of cancerous cell line kills analysis

Cytotoxicity determines by LDH CTAs (Promega).Human T-cell is by CD3/CD28 Dynabeads (Invitrogen) purchased from AllCells and is activated and expanded according to the scheme of manufacturer.Activating T cell (ATC) cultivate and keep in the RPMI1640 culture mediums for adding 30U/ml IL-2 with 10%FBS, and used at the 7-14 days. Activating T cell (effector cell) and target cancer cells are in different BsAb antibody concentrations (including such as 0.2,0.04,0.008 and 0.0016 μ g/ml BsAb) under with 5:1 ratio co-cultures 16 hours.Then by measuring LDH activity in culture supernatants And determine cytotoxicity.

The target cancer cells of test include cervical cancer tumer line CaSki (ATCC CRL-1550；HLA-A2⁺, HPV16⁺) and palace Neck cancer cell line MS-751 (ATCC HTB-34；HLA-A2⁺,HPV16^-).As shown in Figure 8 A, it is more in 0.2 μ g/ml BsAb Individual clone (such as 2,41 and US-7) mediation HPV-16⁺Cell line CaSki is to HPV-16^-Cell line MS-751 selectivity is killed. As seen in fig. 8b, the target positive CaSki of BsAb mediations kill is dose dependent.

The other target cancer cells that can be tested include head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore；HLA-A2⁺,HPV16⁺), liver gland cell system SK-HEP-1 (ATCC HTB-52；HLA-A2⁺,HPV16^-), palace Carcinoma of neck cell line SiHa (ATCC HTB-35；HLA-A2^-,HPV16⁺), cervical cancer tumer line C33A (ATCC HTB-31；HLA- A2⁺,HPV16^-), cervix adenocarcinoma cell line HeLa (ATCC CCL-2；HLA-A2^-,HPV16^-), hepatocellular carcinoma cells system Hep3B (ATCC HB-8064；HLA-A2^-,HPV16^-), hepatocellular carcinoma cells system HepG2 (ATCC HB-8065；HLA-A2^-, HPV16^-), lymphoma cell line Raji (ATCC CCL-86；HLA-A2^-,HPV16^-), T cell Leukemia Cell Lines Jurkat (ATCC TIB-152；HLA-A2^-,HPV16^-), lymphoma cell Daudi (ATCC CCL-213；HPA-A2^-,HPV16^-) and it is white Blood disease cell line k562 (ATCC CCL-243；HLA-A2^-,HPV16^-)。

HPV16-E7 BsAb antibody is directed to the cross reactivity human MHC I molecules of a variety of HLA-A02 allele by 6 classes Formed with work(allograft thing HLA-A ,-B ,-C ,-E ,-F and G.HLA-A ,-B and-C heavy chain genes are highly polymorphous.It is right In carrying out each same work(allograft thing, HLA genes are grouped in addition according to the similitude of sequence of heavy chain.For example, HLA-A is divided into not Iso-allele, such as HLA-A01 ,-A02 ,-A03.For HLA-A02 allele, a variety of hypotypes, such as HLA-A*02 be present: 01、A*02:02 etc..Between the different subtype of HLA-A02 groups, sequence difference is only limitted to some amino acid.Therefore in many feelings Under condition, HLA-A*02 is bound to:The peptide of 01 molecule also can form complex with multiple hypotypes of HLA-A02 allele.Such as table 8 (http://www.allelefrequencies.net/) shown in, although HLA-A*02:01 in Caucasian colony is excellent Gesture HLA-A02 hypotypes, in Asia, A*02:05、A*02:06、A*02:07 and A*02:11 be also common HLA-A02 hypotypes. HPV16-E7 antibody is not only in HLA-A*02:01, but HPV16-E7 peptides are also identified in the case of HLA-A02 other hypotypes Ability broadens the PATIENT POPULATION for making that the treatment of HPV16-E7 antibody drugs may can be benefited from significantly.In order to determine cross reaction Property, the HPV16-E7/MHC I classes complexs and test HPV16-E7/ of other hypotypes of the generation with HLA-A02 allele HLA-A*02:Binding affinity of 01 specific antibody for these other complexs.For example, use ForteBio Octet QK determines binding affinity.In short, 5 μ g/mL biotin labelings has the HPV16-E7 peptides/HLA-A02MHC for changing hypotype Complex is loaded on streptavidin biology sensor.After excessive antigen is washed off, BsAb surveys in such as 10 μ g/mL Examination association and Dissociation.Use 1:1 binding site, partial fitting model calculations incorporated parameter.

Table 8

Embodiment 6. produces HPV16-E7/HLA-A*02:01 specific chimeric antigen receptor presents T cell (CAR-T)

Chimeric antigen receptor therapy (CAR-T therapies) is the novel form of targeting immunotherapy.It merges monoclonal antibody Exquisite targeting specific with by cytotoxic T cell provide strength cytotoxicity and long-term persistence.This technology causes T Cell can gather long-term novel antigen specificity independently of endogenous TCR.Clinical test neuroblastoma (Louis, C.U. et al., Blood 118 (23):6050-6056,2011), B-ALL (Maude, S.L. et al., N.Engl.J.Med.371 (16):1507-1517,2014), CLL (Brentjens, R.J. et al., Blood.118 (18):4817-4828,2011) and B Cell lymphoma (Kochenderfer, J.N. et al., Blood.116 (20):Display CAR-T therapies in 4099-4102,2010) Clinically significant antitumor activity.In a research, report uses 30 trouble for suffering from B-ALL that CD19-CAR T therapies are treated 90% complete remission rate (Maude S.L. et al., foregoing) in person.

In order to further probe into HPV16-E7/HLA-A*02:The efficiency of 01 specific antibody, construction expression CAR's HPV16-E7scFv and transduceed into T cell.For example, HPV16-E7/HLA-A*02:01 specific C AR uses slow virus CAR expression vectors are constructed.Anti- HPV16-E7/HLA-A*02:01scFv is grafted to with cis engineered CD28 signals Transduction domain and TCR ζ second generation CAR (Mackall C.L. et al., Nat.Rev.Clin.Oncol.11 (12):693-703, 2014) to provide intracellular T cell stimulus signal and activating T cell on.Fig. 9 provides the schematic of anti-HPV16-E7 CAR constructs Explanation.

The sign of embodiment 7.HPV16-E7 CAR-T cells

The vitro cytotoxicity research of HPV16-E7 CAR-T cells

Contain HPV16-E7/HLA-A*02:The slow virus of 01 specific chimeric antigen receptor by CAR carriers for example by turning 293T cells are contaminated to produce.Human T cells be used for using CD3/CD28 beads (Invitrogen 2) are stimulated Transduceed after it in the presence of the 30U/ml white element -2 of Jie.Concentration slow virus is coated to the Retronectin containing T cell (Takara) 6 porose disc of coating 72 hours.Using the biotinylation HPV16-E7 tetramers and PE be conjugated streptavidin by Transduction efficiency is assessed by FACS.Thereafter facs analysis is repeated at 72 hours and per 3-4 days.

The functional assessment of transduction T cell (HPV16-E7 CAR-T cells) is carried out using LDH CTAs.Use Effector：Target ratio includes, such as 5:1 and 10:1.Target cell system can include, such as cervical cancer tumer line CaSki (ATCC CRL-1550；HLA-A2⁺,HPV16⁺), head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore；HLA-A2⁺, HPV16⁺), liver gland cell system SK-HEP-1 (ATCC HTB-52；HLA-A2⁺,HPV16^-), cervical squamous cell carcinoma cell line SiHa (ATCCHTB-35；HLA-A2^-,HPV16⁺), cervical cancer tumer line C33A (ATCC HTB-31；HLA-A2⁺,HPV16^-), uterine neck Cancerous cell line MS-751 (ATCC HTB-34；HLA-A2⁺,HPV16^-), cervix adenocarcinoma cell line HeLa (ATCC CCL-2；HLA- A2^-,HPV16^-), hepatocellular carcinoma cells system Hep3B (ATCC HB-8064；HLA-A2^-,HPV16^-), hepatocellular carcinoma cells system HepG2(ATCC HB-8065；HLA-A2^-,HPV16^-)；Lymphoma cell line Raji (ATCC CCL-86；HLA-A2^-, HPV16^-), T cell Leukemia Cell Lines Jurkat (ATCC TIB-152；HLA-A2^-,HPV16^-), lymphoma cell Daudi (ATCC CCL-213；HPA-A2^-,HPV16^-), and Leukemia Cell Lines K562 (ATCC CCL-243；HLA-A2^-,HPV16^-)。 As control, SK-HEP-1-MiniG is produced by using the HPV16-E7 peptides transduction SK-HEP-1 of expression mini gene box, this causes HPV16-E7/HLA-A*02：The high-caliber cell surface expression of 01 complex.It is determined that expression HPV16-E7 CAR T cell is killed Hinder the specificity and efficiency of target positive cancer cell.

Embodiment 8. produces and characterized total length IgG1HPV16-E7 antibody

The total length IgG 1 of selected phage clone, such as in HEK293 and Chinese hamster ovary (CHO) cell line Produce, such as described (Tomimatsu, K. et al., Biosci.Biotechnol.Biochem.73 (7):1465-1469, 2009).In short, antibody variable region is subcloned to the mankind λ or the κ constant region of light chain with matching and the constant region of IgG 1 In the mammalian expression vector of sequence.Using identical Strategies For The Cloning, generation is with the heavy chain of mouse IgG 1 and constant region of light chain Chimeric HPV16-E7 full length antibodies.Under reduction and non reducing conditions, the molecule of the total length IgG antibody of purifying is measured by electrophoresis Amount.The SDS-PAGE of the HPV16-E7 mouse chimera IgG1 antibody purified is to determine lipidated protein.In short, 2 μ g eggs White matter mixes with 2.5 μ L NuPAGE LDS sample buffers (Life Technologies, NP0008) and by deionized water It is set to add up 10 μ L.Sample heats 10 minutes at 70 DEG C, is then loaded on gel.Gel electrophoresis 1 is carried out under 180V Hour.

The knot of IgG1 antibody and HPV16-E7 presentation SK-HEP1 cells is fitted together to by flow cytometry test HPV16-E7 Close.SK-HEP1 is HLA-A*02:01 positive and HPV16-E7 negative cells system.HPV16-E7 mini gene boxes are transfected to SK-HEP1 HPV16-E7 is produced in cell and presents SK-HEP1-miniG cells.10 μ g/mL antibody continue 1 hour thin added on ice Born of the same parents.After wash, anti-mouse IgG (H+L) (carrier Labs#EI-2007) that R-PE is combined is added to detect antibody binding.By By the binding affinity of ForteBio Octet QK measure mouse chimera IgG1HPV16-E7 antibody.By 5 μ g/mL biotinylations HPV16-E7 peptides/HLA-A*02:01 complex is loaded on avidin chain enzyme biology sensor.Washing excessive antigen off Afterwards, mouse chimera full length antibody tests combination and Dissociation under 10 μ g/mL.Use 1:1 binding site, partial fitting Model calculations incorporated parameter.

In elisa assay HPV16-E7 specificity and negative control (such as ET901) mouse chimera IgG1 test for HPV16-E7/HLA-A*02:01st, the combination of HPV16-E7 recombinant proteins and free HPV16-E7 peptides.Antibody is in such as 3 × company Continuous dilution is lower to be tested, and since 100ng/mL, continues 8 concentration altogether.Biotinylation HPV16-E7/A*02:01MHC is with 2 μ g/ ML is applied on streptavidin disk, and HPV16-E7 protein is coated with 2 μ g/mL and HPV16-E7 peptides are applied with 40ng/mL Cloth.Confirm total length HPV16-E7/HLA-A*02:01 antibody identifies HPV16-E7 peptides only in the case of HLA-A02, and does not combine Recombinate HPV16-E7 protein or free HPV16-E7 peptides.

The in vivo efficacy research of embodiment 9.

HPV16-E7 CAR-T cells handle mouse

The generation HLA-A02 in SCID- taupe brown mouse (non-functional T, B, NK cell)⁺/HPV16-E7⁺Cancerous cell line (such as CaSki or UM-SCC-104) subcutaneous (s.c.) heteroplastic transplantation model.Reach 200mm in average s.c. gross tumor volumes³When, move Thing is random.24 hours before CART administrations, animal is handled with 60mg/kg endoxan (via intraperitoneal routes).Will Mouse is divided into 4 groups (n=8-10 mouse/group), and it receives one below：(i) (ii) 10 is not handled⁷The CAR T for simulating transduction are thin Born of the same parents, 1x/ weeks, continue 4 weeks (iii) 10⁷Anti- E7MC CAR T cells, 1x/ weeks, continue 4 weeks or (iv) 2x10⁶Anti- E7MC CAR T Cell, 1x/ weeks, continue 4 weeks.The gross tumor volume of monitoring each group animal, adverse reaction, Human cytokine's overview, CAR T cells People CD3 in the tumour and organ of infiltration⁺Tumor tissue pathology, serum HPV16-E7, body weight and the general health shape of cell Condition (feed, walking, daily routines).

The affinity maturation of the anti-HPV16-E7 antibody reagents of embodiment 10.

The present embodiment confirms the affinity maturation of anti-HPV16-E7 antibody reagents.Specifically, the embodiment specifically confirm by A series of antibody variants are generated by random mutation is incorporated into representative anti-HPV16-E7 antibody reagents (clone US-7), are then sieved Select and characterize the antibody variants.

The generation of variant phage library

According to the specification of manufacturer, kit (Agilent is induced using GeneMorph II random mutations Technologies the DNA that US-7scFv) is cloned to encoding anti-HPV16-E7 carries out random mutagenesis.After mutagenesis, by DNA sequence dna gram About 5x10 is contained with structure in the grand phagemid vector to expression scFv⁸The variant human antibody bacteriophage of individual unique phage clone Library.Typically, compared with the anti-HPV16-E7 clones of parent, variant clone has two coding mutations, each scFv sequences Coding mutation is 1 to 4.

Cell elutriation

Using the people bacteriophage scFv libraries with mutant generated from clone US-7 for described in embodiment 2 HPV16-E7 11-19 peptides/HLA-A*02:01 complex carries out elutriation.Especially, using cell elutriation.People scFv is bitten first Phage library and 20 kinds of different endogenous peptide (P20, SEQ ID NOs for being loaded with 50 μ g/ml:The T2 cells in pond 193-212) Mixing is used as negative control elutriation.Then by people scFv phage libraries that negative control exhausts and HPV16-E7 11- are loaded with The T2 mixing with cells of 19 peptides (first round 1.5ug/ml, the second wheel 0.8ug/ml, third round 0.4ug/ml).In order to load peptide, T2 Cell is stayed overnight in the presence of 20 μ g/ml β 2M in serum-free RPMI1640 culture mediums using peptide shock pulse.Using PBS's Extend after washing, there is the T2 cells that the peptide with reference to scFv antibody phages loads through quick centrifugation (spun down).Then Combining clone elutes and is used for ehec infection XL1-Blue cells.Then the phage clone that will be expressed in bacterium Purifying.Elutriation 3 is carried out to take turns to be enriched with specific binding HPV16-E7 11-19 peptides/HLA-A*02:01 scFv phage clones.

Streptavidin ELISA disks are coated with biotin labeling HPV16-E7/HLA-A*02:01 complex monomer or Biotin labeling P20 control peptides/HLA-A*02:01 monomer.For HPV16-E711-19 peptides/HLA-A*02:01 complex comes Indivedual phage clones in self enrichment phage display elutriation pond are cultivated in coating pan.The combination of phage clone is by HRP Conjugated anti-M13 antibody tests and use HRP Substrate developments.Absorptivity is read at 450nm.15 positive colonies are by from biting The ELISA of 135 phage clones of thalline elutriation enrichment is screened to identify.11 Unique clones are by 15 ELISA positives The DNA sequencing of phage clone is identified.By flow cytometry (facs analysis), loaded using HPV16-E7 11-19 peptides The HLA-A*02 that T2 cells living are further tested on specific and unique clone and liver cell surface:The combination of 01/ peptide complex. Control includes not loading the horse anti-mouse IgG (only secondary antibody) that the T2 cells (only cell) of peptide and R-PE are conjugated.It is in short, negative The T2 cells scFv phage clones dyeing purified first of HPV16-E711-19 peptides or P20 peptides pond is loaded with, then using mouse Anti- M13mAb carries out second and dyed, and the horse anti-mouse IgG being conjugated using the R-PE from Vector Labs carries out third time Dyeing.Each staining procedure is in progress 30-60 minutes on ice and cell washes twice between staining procedure.In 10 unique grams In grand test, T2 cells (the SEQ ID NOs of 8 specific recognition HPV16-E711-19 loads:223-230).This 8 phagocytosis Body clone-specific is bound to the T2 cells of HPV16-E7 11-19 loads and in HLA-A*02:Nonrecognition loads in the case of 01 There are the T2 cells in P20 peptides pond, or do not load the T2 cells of peptide.

The sign of bi-specific antibody molecule of the embodiment 11. based on anti-HPV16-E7 affinity maturation variants

The generation of bispecific antibody

Bispecific antibody (BsAb) uses the affinity maturation separated in embodiment 10 using the methods described of embodiment 4 HPV16-E7/HLA-A*02:ScFv sequences (the SEQ ID NO of 01 specific bacteriophage clone：223-232) produce.Gained Single chain bispecific antibody includes the HPV16-E7/HLA-A*02 at N- ends:The scFv sequences and C- of 01 specific bacteriophage clone The anti-human CD3 ε mouse monoclonals scFv at end.

Bispecific antibody and HPV16-E7/HLA-A*02:7 kinds of HPV16-E7 BsAb of measure of 01 binding affinity Antibody (is derived from clone 7-1,7-3,7-6,7-7,7-8 and 7-9 of affinity maturation, corresponds respectively to SEQ ID NO:223、225 And 227-230, and parental clone US-7) with recombinating HPV16-E7/HLA-A*02:The binding affinity of 01 complex is by table Surface plasma resonance (BiaCore) measures.HPV16-E7 BsAb and HPV16-E7/HLA-A*02:Knot between 01 complex Parameter is closed according to the scheme for multi-cycle kinetic measurement of manufacturer, uses Biotin CAPture kits (GE Healthcare, catalog number (Cat.No.) 28920233) measured on Biacore X100 (GE Healthcare).For all of measure Protein is diluted using HBS-EP electrophoretic buffers.By 5 μ g/mL biotinylation HPV16-E7 11-19/HLA-A*02:01/β2M Complex by make solution with 5 μ L/min flow through fluid cell be fixed within 75 seconds using streptavidin (capture~3, 800RU streptavidins) pre- functionalization sensor wafer CAP on (MHC for each circulating capture~120RU is compound Body).Analyzed towards being incorporated under 150nM, 75nM, 37.5nM, 18.8nM and 9.4nM for HPV16-E7 BsAb, each run by Combine within 2 minutes under 30 μ L/min and dissociation in 10 minutes forms.At the end of circulation, surface uses and comes from Biotin CAPture The regeneration buffer regeneration of kit.Data assess software using BiaCore X-100 and use 1:1 binding site pattern analysis. Then calculations incorporated parameter (association rate constant k_a, dissociation constant k_dAnd equilibrium dissociation constant K_d), and be shown in Table 9.With from Parental clone US-7 BsAb is compared, and the BsAb from clone 7-1,7-3,7-6,7-7 and 7-9 shows that increased combination is affine Power, and the BsAb from clone 7-8 shows the binding affinity of reduction.

Table 9

HPV16-E7 bispecific antibodies are directed to the cross reactivity and binding affinity of a variety of HLA-A02 allele

As described in example 5 above, the different hypotypes of HLA-A02 groups are quite conservative, and are directed to a variety of HLA- The cross reactivity of A02 hypotypes is highly desirable.Therefore, tested to determine by parental clone US-7 and affinity maturation Variant generation bispecific antibody (BsAb) whether the non-HLA-A*02 with HLA-A02 groups:01 hypotype cross reaction.Especially Ground, the HPV16-E7 11-19/MHC I class complexs of the various hypotypes with HLA-A02 allele are produced, and use utilization The Pall ForteBio LLC's (Menlo Park, CA) of Biolayer Interferometry (BLI) technology QKe system measurements its with HPV16-E7 11-19/HLA-A*02:The binding affinity of 01 specific antibody.The BsAb bags of test Include parental clone US-7 and affinity maturation variant clone 7-1,7-3,7-6,7-7,7-8 and 7-9.There to be different HLA-A02 5 μ g/mL biotinylations HPV16-E711-19 peptides/HLA-A02 complexs of hypotype are loaded to avidin chain enzyme bio-sensing On device.After excessive antigen (HPV16-E7 peptides/HLA-A02 complexs) is washed off, BsAb tests association and dissociation under 10 μ g/mL Dynamics.Use 1：1 binding site, partial fitting model calculations incorporated parameter.BLI measurement results are shown in table 10 below.

Table 10.HLA-A02 hypotype incorporating parametrics

"-", is indicated without detectable specific binding

As a result show that the antibody cloning each tested specifically combines at least four in seven kinds of HLA-A02 hypotypes.It is several Kind affinity maturation variant antibody combines more HLA-A02 hypotypes than parental clone, and many affinity maturation variants resist For body with the affinity combination HPV16-E7 11-19/HLA-A02 higher than parental clone, it passes through lower K_DValue represents.

Peptide binding specificity determines

In order to confirm affinity maturation variant antibody identification peptide specificity, be loaded with HPV16-E7 11-19 peptides, P20 peptides pond or the T2 cells progress facs analysis without peptide.As a result show, the BsAb of all tests, including parental clone US-7 and Affinity maturation variant 7-1,7-3,7-6,7-7,7-8 and 7-9, specifically it is bound to and is loaded with HPV16-E7 11-19 peptides T2 cells, but the T2 cells for being loaded with P20 peptides pond or not loading any peptide are not bound to.FACS data are shown in table herein 12。

By the epitope mapping of Alanine-scanning

Clone the #US-7's and its HPV16-E7 11-19 of affinity maturation variant identification by BsAb to accurately study Sensitive residue, design and synthesize a series of mutant peptides make it have nine with alanine substitute residue in each (table 11 Shown in).

Table 11.HPV16-E7 11-19 wild types and mutant peptide

Then by facs analysis test b sAb clone be loaded with the T2 cells of the peptide from table 11, be loaded with feminine gender it is right T2 cells according to P20 peptides pond or the combination with the T2 cells without peptide load.With reference to the MHC on T2 cells peptide by BB7.2 Mouse antibodies dyeing is assessed.Compared with the control T2 cells without peptide, except HPV16-E7 11-19mut8 all peptides show Higher BB7.2 antibody bindings (data are not shown) are shown, representing can be into except HPV16-E7 11-19mut8 all peptides Combine the MHC on T2 cells work(.Analyzed for BsAb, cell is dyed with 10 μ g/mL BsAb, is then carried out the anti-His of APC and is resisted 20 times of dilutions of body (R＆D System#IC050A).FACS average fluorescent strengths (MFI) value of each FACS measure is shown in table In 12.The C-terminal part of the scFv combination HPV16-E7 peptides of parent US-7 and affinity maturation is predicted, because at the 5-8 of position Alanine substitution generally reduce with peptide load T2 cells combination.Single FACS combination mensurations result such as Figure 10 A and 10B Shown in.

Table 12.HPV16-E7 11-19 Alanine-scanning (FACS, average fluorescent strength)

*：Sensitive position：<50%MFI (T2 for being loaded with wild type HPV16-E7 11-19)

**：The combination of reduction：50-75%MFI (T2 for being loaded with wild type HPV16-E7 11-19)

The Chimeric antigen receptor presentation T that embodiment 12. produces the scFvs variants with anti-HPV16-E7 affinity maturations is thin Born of the same parents (CAR-T)

Construction expression affinity maturation HPV16-E7scFv Chimeric antigen receptor is simultaneously transduceed into T cell.Pass through The scFv of the affinity maturation separated in embodiment 10 (7-1,7-3,7-6,7-7,7-8 and 7-9, is corresponded respectively into SEQ ID NO:223rd, 225 and 227-230) or parent US-7scFv and there is CD28 and CD3 ζ signal transductions domain (SEQ ID NO: 256) polypeptide has 4-1BB and CD3 ζ signal transductions domain (SEQ ID NO:257) peptide fusion, build for producing Raw coding HPV16-E7/HLA-A*02:The CAR carriers of 01 specific C AR slow virus.

Contain HPV16-E7/HLA-A*02:The slow virus of 01 specific chimeric antigen receptor is transfected by using CAR carriers 293T cells produce.Human T cells be used for using CD3/CD28 pearls (Invitrogen 1) is stimulated Transduceed after it in the presence of the 100U/ml white element -2 of Jie.The slow virus of concentration is wrapped in Retronectin (Takara) T cell is applied in 6 porose discs of quilt 72 hours.Five days after transduction, HPV16-E7 CAR-T cells and simulation transducer cell Use biotinylation HPV16-E7 11-19 peptides/HLA-A*02:01 tetramer and CD4 or CD8 antibody dye and pass through streaming altogether Cytometry.Figure 11 A-11C show the flow cytometry results of HPV16-E7 CAR-T cells and simulation transducer cell, as a result It is summarized in table 13.Each in the test CAR of scFv with affinity maturation is with higher than the CAR's with parent scFv Transduction efficiency.

Table 13

Clone	% tetramers⁺
		Simulation	0.12
US-7 4-1BB/CD3ζ	39.8
		US-7CD28/CD3ζ	43.0
7-1 4-1BB/CD3ζ	69.6
		7-1CD28/CD3ζ	67.9
7-3 4-1BB/CD3ζ	74.4
		7-3CD28/CD3ζ	70.5
7-6 4-1BB/CD3ζ	64.3
		7-6CD28/CD3ζ	68.3
7-7 4-1BB/CD3ζ	68.4
		7-7CD28/CD3ζ	62.9
7-8 4-1BB/CD3ζ	71.2
		7-8CD28/CD3ζ	63.6
7-9 4-1BB/CD3ζ	77.3
		7-9CD28/CD3ζ	66.1

Embodiment 13. is surveyed with the CAR-T with anti-HPV16-E7 affinity maturation variants of cancerous cell line T cell killing It is fixed

The vitro cytotoxicity research of HPV16-E7 CAR-T cells

By LDH CTAs (Promega) measure expression with affinity maturation scFv (7-1,7-3,7-6, 7-7,7-8 and 7-9, correspond respectively to SEQ ID NO:223rd, 225 and 227-230) or parent US-7scFv CAR T cell Cytotoxicity.According to manufacturer's scheme activation purchase from AllCells human T-cell, then transduce and make using CAR Expanded with CD3/CD28 Dynabead (Invitrogen).Activation CAR-T cells are adding 100U/ml IL-2 with 10%FBS RPMI1640 culture mediums in culture and keep, and the 7-14 days use.The CAR-T cells (effector cell) and target cancer of activation Cell is with 5:1 ratio co-cultures 16 hours.Then cytotoxicity is determined by measuring LDH activity in culture supernatants.

The target cancer cells of test include cervical cancer tumer line CaSki (ATCC CRL-1550；HLA-A2⁺, HPV16⁺) and palace Neck cancer cell line MS-751 (ATCC HTB-34；HLA-A2⁺,HPV16^-), and lymphoma mantle cell cell line JeKo-1 (ATCC CRL-3006；HLA-A2⁺,HPV16^-).As shown in figure 12, compared with parental clone US-7, clone (7-1,7- of affinity maturation 3rd, 7-6,7-7,7-8 and 7-9) confirm the killing activities of notable elevated CD28/CD3 ζ and 4-1BB/CD3 ζ forms.

Sequence table

SEQ ID NO:1, HPV16 E7 albumen

MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCV QSTHVDIRTLEDLLMGTLGIVCPICSQKP

SEQ ID NO:2, HPV16 E7 CDS

ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTAC TGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAG AGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAG ACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA

SEQ ID NO:3, HPV16 E7 7-15

TLHEYMLDL

SEQ ID NO:4, HPV16 E7 11-19

YMLDLQPET

SEQ ID NO:5, HPV16 E7 16-25

QPETTDLYCY

SEQ ID NO:6, HPV16-E7 44-52

QAEPDRAHY

SEQ ID NO:7, HPV16-E7 46-55

EPDRAHYNIV

SEQ ID NO:8, HPV16-E7 49-57

RAHYNIVTF

SEQ ID NO:9, HPV16 E7 82-90

LLMGTLGIV

SEQ ID NO:10, HPV16 E7 86-93

TLGIVCPI

SEQ ID NO:The variants of 11, HPV16 E7 11-19

YMLDVQPET

SEQ ID NO:The A1 of 12, HPV16 E7 11-19

AMLDLQPET

SEQ ID NO:The A5 of 13, HPV16 E7 11-19

YMLDAQPET

SEQ ID NO:The A8 of 14, HPV16 E7 11-19

YMLDLQPAT

SEQ ID NO:15, HCVR 1

QVQLVQSGAEVKKPGASVKVSCKASGYTFIDYYIHWVRQAPGQGLEWMGWINPKSGGTKYAQKFQGRVT MTRDTSITTAYMELSSLTSDDTAVYYCARSYYYGGVKDYWGQGTLVTVSS

SEQ ID NO:16, HCVR 2

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVT ITADESTSTAYMELSSLRSEDTAVYYCARGVYGYSGSAPYDEWGQGTLVTVSS

SEQ ID NO:17, HCVR 3

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSMAAISWVRQAPGQGLEWMGRIIPMIGIPDYAQEFQGRVT FTADRSTSTAYMELSSLRSDDTAVYYCARGPLYWWHSDSWGQGTLVTVSS

SEQ ID NO:18, HCVR 4

QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGTNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARMHGSYGSYFDKWGQGTLVTVSS

SEQ ID NO:19, HCVR 5

QVQLVQSGAEVKKPGSSVKVSCKASGGTFRSYAVSWVRQAPGQGLEWMGRIIPIFGTPDYAQKFQGRVT ITADASTSTAHMELSGLRSEDTAVYYCARSSMYQYSHDDWGQGTLVTVSS

SEQ ID NO:20, HCVR 8

EVQLVQSGAEVKKPGASVKVSCKASGYTFANYGINWVRQAPGQGLEWMGWISASNGNTNYAQKLQGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSYYAGYYMFMDFWGQGTLVTVSS

SEQ ID NO:21, HCVR 9

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINQDGSEKYYVDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARYSVWGMMDEWGQGTLVTVSS

SEQ ID NO:22, HCVR 10

QVQLVQSGGGVVQPGRSQRLSCAASGFPFSSYSMNWVRRAPGKGLEWVSYISSSSSTIYYADSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQNYYADYDYWGQGTLVTVSS

SEQ ID NO:23, HCVR 11

QVQLVQSGAEVKKPGASVKVSCKTSGYTFSDYGISWVRQAPGQGLEWLGWISTYNGNTDLAQKVRGRVT LTTDTSTSTAYMELRSLRSEDTAVYYCARGYTGSYYATGDEWGQGTLVTVSS

SEQ ID NO:24, HCVR 13

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISPYNGNTNYAQKLQGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARGYKDYWGQGTLVTVSS

SEQ ID NO:25, HCVR 17

QVQLVQSGAEVRKPGSSVKVSCKASGDIFRKFAVSWVRQAPGQGPEWMGRIIPNLDITDKPQKFQGRVT LSADKSTSTVYLELSSLRSEDTAVYYCARGSIYQWSGWDSWGQGTLVTVSS

SEQ ID NO:26, HCVR 22

EVQLVESGGGLVKPGGSLRLSCAASGFNFSDYYMTWIRQAPGKGLEWLSYISPGGTTYYAASVEGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARGHSFSYGEDYWGQGTLVTVSS

SEQ ID NO:27, HCVR 26

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVT MTRDTSISTAYMELSRLRSDDTAVYYCARGWHQYGIMDYWGQGTLVTVSS

SEQ ID NO:28, HCVR 27

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARRSVWSAYDSWGQGTLVTVSS

SEQ ID NO:29, HCVR 31

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVGNIKEDGSVKYYVDSVKGRFT ISRDNAKNSVYLEMNNLRAEDTAVYYCARRGVWGHIDFWGQGTLVTVSS

SEQ ID NO:30, HCVR 32

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVT ITADKSTSTAYMELSSLRSEDTAVYYCARGTYTWGSDTWGQGTLVTVSS

SEQ ID NO:31, HCVR 39

QVQLVESGGGLVQPGGSLRLSCAASGFSFSRHAMNWVRQAPGKGLEWVSAISGSGGNTYYSDSVEGRFT MSRDNSKNTLYLQMNSLRAEDTAVYYCARYWGSYDKWGQGTLVTVSS

SEQ ID NO:32, HCVR 40

EVQLVQSGAEVKKPGASVKVSCTASGYSFSDYYIYWLRQGPGQGLEWMGWINPNTGVTHYAQKYQGRVT MTRDTSINTVYMELSRLRSDDTAVYYCARNVYRYSMMHDSWGQGTLVTVSS

SEQ ID NO:33, HCVR 41

QAQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVRGRFT ISRDNAKNSLFLQMNSLRAEDTAVYYCARRGVWGYFDSWGQGTLVTVSS

SEQ ID NO:34, HCVR US-5

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVT ITADESTRTAYMELSSLRSEDTAVYYCARVYGSYFYDDWGQGTLVTVSS

SEQ ID NO:35, HCVR US-7

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:36, LCVR 1

QAVLTQPPSASGTPGQRVTISCSGSSSNIGTNPVTWYQHLPGTAPKLLIYGNYQRPSGVPDRFSGSKSG TSASLAISGLQSEDEADYYCAAWDDSLTGYVFGTGTKVTVLG

SEQ ID NO:37, LCVR 2

QSVLTQPPSLSVAPGKTASITCGGNNIGMKSVHWYQQRPGQAPVLVIYYDSDRPSGIPGRFSGSNSGNT ATLTISRVEAGDEADYYCQVWDTTGDHPGYVFGTGTKVTVLG

SEQ ID NO:38, LCVR 3

QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQHFPGAAPKLLIYDNYKRPSGIPDRFSGSKSG TSATLGITGLQTGDEAEYYCGTWDSSLSAYVFGTGTKVTVLG

SEQ ID NO:39, LCVR 4

SYELTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYNSDRPSGIPERFSGSNSGNT ATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVLG

SEQ ID NO:40, LCVR 5

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKR

SEQ ID NO:41, LCVR 8

QSVLTQPPSASGTPGQRVTISCSGHNSNIETNPVSWYQQVPGTTPKLLIYSYYYRPSGVPDRFSGSKSG TSASLAISGLQSEDEADYFCASWDDSVQGYVFGSGTKVTVLG

SEQ ID NO:42, LCVR 9

SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNT ASLTITGAQAEDEADYYCNSRDSSGNHQVFGTGTKVTVLG

SEQ ID NO:43, LCVR 10

QSVLTQPPSVSVAPGKTARITCGGNNIGSKNVHWYQQKPGQAPVLVVYDDHDGPSGIPERFSGSNSGNT ATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG

SEQ ID NO:44, LCVR 11

QAVLTQPPSASGTPGQRVTISCSGSSSNIGINTVNWYQQLPGTAPKLLIYGNSHRPSGVPDRFSGSKSG ASASLAITGLQADDEGAYFCQSYDTSLTVIFGGGTKLTVLG

SEQ ID NO:45, LCVR 13

QTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGYYPNWFQQKPGQAPRALIYSTNNKHSWTPARFSGSLL GGKAALTLSGVQPEDEAEYYCLLYYGGAQLWVFGGGTKLTVLG

SEQ ID NO:46, LCVR 17

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGQAPNLLIYGASSFQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTKVDIKR

SEQ ID NO:47, LCVR 22

QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNSVNWYQQVPGTAPKLLIYSNNQRPSGVPDRFSGSKSG TSASLAITKLQSEDEADYYCAAWDDSLNGYVFGTGTKVTVLG

SEQ ID NO:48, LCVR 26

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYESNNRPSGVPDRFSGSKS GTSASLAITGLQAEDEADYYCQSYDTSLKGVFGPGTKVTVLG

SEQ ID NO:49, LCVR 27

SSELTQDPAVSVALGQTVTITCQGDILRNYYATWYQQKPGQAPSLVLYGQNNRPSGIPDRFSGSTSGNT ASLTITGAQAEDEGDYYCDSRDSSGNHWVFGGGTKLTVLG

SEQ ID NO:50, LCVR 31

SSELTQDPALSVALGQTVTITCRGDNLRNSYASWYQQKPGQAPVVVLYGKNRRPSGIPDRFSGSSSGNT ASLTITGAQADDEADYYCNSRDSSGFPPYVFGPGTKVTVLG

SEQ ID NO:51, LCVR 32

SYVLTQPPSVSVSPGQTASITCSGDKLGEKYVYWYQQRPGQSPVLVIFQNDKRPSGIPERLSGSRSGNT ATLTISGTQAMDEADYYCQSWASSSVYVFGTGTKLTVLG

SEQ ID NO:52, LCVR 39

SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPVLVLYGKDNRPSGIPDRFSGSSSGNT ASLTITGAQAEDEADYYCNSRDSSGNHRVFGGGTKLTVLG

SEQ ID NO:53, LCVR 40

QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNHVNWYQQLPGTAPKLLISNNNQRPSGVPDRFSGSKSG TSASLAISGLQSEDEADYYCAAWDDSLNVPVFGGGTQLTVLS

SEQ ID NO:54, LCVR 41

SSELTQDPAVSVALGQTVRITCQGDTLRSFYASWYQQKPGQAPVLVFYGKNNRPSGIPERFSGSNSENT ASLTIAGAQAEDEADYYCNSRDSGGDPVVFGGGTKLTVLG

SEQ ID NO:55, LCVR US-5

QAVVTQEPSLTVSPGGTVTLTCASSTGAVTSSYYPNWFQQKPGQAPRALIYSTSNTHSWTPARFSGSLL GGKAALTVSGVQPEDEAEYYCLLYYGGARVFGGGTKLTVLG

SEQ ID NO：56, LCVR US-7

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLG

SEQ ID NO：57, HC-CDR1 1

GYTFIDYY

SEQ ID NO：58, HC-CDR1 2

GGTFSSYA

SEQ ID NO：59, HC-CDR1 3

GGTFSMAA

SEQ ID NO：60, HC-CDR1 4

GFTFSSYA

SEQ ID NO：61, HC-CDR1 5

GGTFRSYA

SEQ ID NO：62, HC-CDR1 8

GYTFANYG

SEQ ID NO：63, HC-CDR1 9

GFTFSSYW

SEQ ID NO：64, HC-CDR1 10

GFPFSSYS

SEQ ID NO：65, HC-CDR1 11

GYTFSDYG

SEQ ID NO：66, HC-CDR1 13

GYTFTSYG

SEQ ID NO：67, HC-CDR1 17

GDIFRKFA

SEQ ID NO：68, HC-CDR1 22

GFNFSDYY

SEQ ID NO：69, HC-CDR1 26

GYTFTGYY

SEQ ID NO：70, HC-CDR1 27

GFTFSSYA

SEQ ID NO：71, HC-CDR1 31

GFTFSSYW

SEQ ID NO：72, HC-CDR1 32

GGTFSSYA

SEQ ID NO：73, HC-CDR1 39

GFSFSRHA

SEQ ID NO：74, HC-CDR1 40

GYSFSDYY

SEQ ID NO：75, HC-CDR1 41

GFTFSSYS

SEQ ID NO：76, HC-CDR1 US-5

GGTFSSYG

SEQ ID NO：77, HC-CDR1 US-7

GGSFSGYY

SEQ ID NO：78, HC-CDR2 1

INPKSGGT

SEQ ID NO：79, HC-CDR2 2

IIPIFGTA

SEQ ID NO：80, HC-CDR2 3

IIPMIGIP

SEQ ID NO：81, HC-CDR2 4

ISYDGTNK

SEQ ID NO：82, HC-CDR2 5

IIPIFGTP

SEQ ID NO：83, HC-CDR2 8

ISASNGNT

SEQ ID NO：84, HC-CDR2 9

INQDGSEK

SEQ ID NO：85, HC-CDR2 10

ISSSSSTI

SEQ ID NO：86, HC-CDR2 11

ISTYNGNT

SEQ ID NO：87, HC-CDR2 13

ISPYNGNT

SEQ ID NO：88, HC-CDR2 17

IIPNLDIT

SEQ ID NO：89, HC-CDR2 22

ISPGGTT

SEQ ID NO：90, HC-CDR2 26

INPNSGGT

SEQ ID NO：91, HC-CDR2 27

ISGSGGST

SEQ ID NO：92, HC-CDR2 31

IKEDGSVK

SEQ ID NO：93, HC-CDR2 32

IIPILGIA

SEQ ID NO：94, HC-CDR2 39

ISGSGGNT

SEQ ID NO：95, HC-CDR2 40

INPNTGVT

SEQ ID NO：96, HC-CDR2 41

ISSSSSYI

SEQ ID NO：97, HC-CDR2 US-5

IIPIFGTT

SEQ ID NO：98, HC-CDR2 US-7

INHSGST

SEQ ID NO：99, HC-CDR3 1

ARSYYYGGVKDY

SEQ ID NO：100, HC-CDR3 2

ARGVYGYSGSAPYDE

SEQ ID NO：101, HC-CDR3 3

ARGPLYWWHSDS

SEQ ID NO：102, HC-CDR3 4

ARMHGSYGSYFDK

SEQ ID NO：103, HC-CDR3 5

ARSSMYQYSHDD

SEQ ID NO：104, HC-CDR3 8

ARSYYAGYYMFMDF

SEQ ID NO：105, HC-CDR3 9

ARYSVWGMMDE

SEQ ID NO：106, HC-CDR3 10

ARQNYYADYDY

SEQ ID NO：107, HC-CDR3 11

ARGYTGSYYATGDE

SEQ ID NO：108, HC-CDR3 13

ARGYKDY

SEQ ID NO：109, HC-CDR3 17

ARGSIYQWSGWDS

SEQ ID NO：110, HC-CDR3 22

ARGHSFSYGEDY

SEQ ID NO：111, HC-CDR3 26

ARGWHQYGIMDY

SEQ ID NO：112, HC-CDR3 27

ARRSVWSAYDS

SEQ ID NO：113, HC-CDR3 31

ARRGVWGHIDF

SEQ ID NO：114, HC-CDR3 32

ARGTYTWGSDT

SEQ ID NO：115, HC-CDR3 39

ARYWGSYDK

SEQ ID NO：116, HC-CDR3 40

ARNVYRYSMMHDS

SEQ ID NO：117, HC-CDR3 41

ARRGVWGYFDS

SEQ ID NO：118, HC-CDR3 US-5

ARVYGSYFYDD

SEQ ID NO：119, HC-CDR3 US-7

ARAPQSWYVGDV

SEQ ID NO：120, LC-CDR1 1

SSNIGTNP

SEQ ID NO：121, LC-CDR1 2

NIGMKS

SEQ ID NO：122, LC-CDR1 3

SSNIGNNY

SEQ ID NO：123, LC-CDR1 4

NIGSKS

SEQ ID NO：124, LC-CDR1 5

QGISSW

SEQ ID NO：125, LC-CDR1 8

NSNIETNP

SEQ ID NO：126, LC-CDR1 9

SLRSYY

SEQ ID NO：127, LC-CDR1 10NIGSKN

SEQ ID NO：128, LC-CDR1 11

SSNIGINT

SEQ ID NO：129, LC-CDR1 13

TGAVTSGYY

SEQ ID NO：130, LC-CDR1 17

QSISSY

SEQ ID NO：131, LC-CDR1 22

SSNIGSNS

SEQ ID NO：132, LC-CDR1 26

SSNIGAGYD

SEQ ID NO：133, LC-CDR1 27

ILRNYY

SEQ ID NO：134, LC-CDR1 31

NLRNSY

SEQ ID NO：135, LC-CDR1 32

KLGEKY

SEQ ID NO：136, LC-CDR1 39

SLRNYY

SEQ ID NO：137, LC-CDR1 40

SSNIGSNH

SEQ ID NO：138, LC-CDR1 41

TLRSFY

SEQ ID NO：139, LC-CDR1 US-5

TGAVTSSYY

SEQ ID NO：140, LC-CDR1 US-7

SSNIGTRM

SEQ ID NO：141, LC-CDR2 1

GNY

SEQ ID NO：142, LC-CDR2 2

YDS

SEQ ID NO：143, LC-CDR2 3

DNY

SEQ ID NO：144, LC-CDR2 4

YNS

SEQ ID NO：145, LC-CDR2 5

AAS

SEQ ID NO：146, LC-CDR2 8

SYY

SEQ ID NO：147, LC-CDR2 9

GKN

SEQ ID NO：148, LC-CDR2 10

DDH

SEQ ID NO：149, LC-CDR2 11

GNS

SEQ ID NO：150, LC-CDR2 13

STN

SEQ ID NO：151, LC-CDR2 17

GAS

SEQ ID NO：152, LC-CDR2 22

SNN

SEQ ID NO：153, LC-CDR2 26

ESN

SEQ ID NO：154, LC-CDR2 27

GQN

SEQ ID NO：155, LC-CDR2 31

GKN

SEQ ID NO：156, LC-CDR2 32

QND

SEQ ID NO：157, LC-CDR2 39

GKD

SEQ ID NO：158, LC-CDR2 40

NNN

SEQ ID NO：159, LC-CDR2 41

GKN

SEQ ID NO：160, LC-CDR2 US-5

STS

SEQ ID NO：161, LC-CDR2 US-7

NNN

SEQ ID NO：162, LC-CDR3 1

AAWDDSLTGYV

SEQ ID NO：163, LC-CDR3 2

QVWDTTGDHPGYV

SEQ ID NO：164, LC-CDR3 3

GTWDSSLSAYV

SEQ ID NO：165, LC-CDR3 4

QVWDSSSDHWV

SEQ ID NO：166, LC-CDR3 5

QQANSFPLT

SEQ ID NO：167, LC-CDR3 8

ASWDDSVQGYV

SEQ ID NO：168, LC-CDR3 9

NSRDSSGNHQV

SEQ ID NO：169, LC-CDR3 10

QVWDSSSDHVV

SEQ ID NO：170, LC-CDR3 11

QSYDTSLTVI

SEQ ID NO：171, LC-CDR3 13

LLYYGGAQLWV

SEQ ID NO：172, LC-CDR3 17

QQSYSAPLT

SEQ ID NO：173, LC-CDR3 22

AAWDDSLNGYV

SEQ ID NO：174, LC-CDR3 26

QSYDTSLKGV

SEQ ID NO：175, LC-CDR3 27

DSRDSSGNHWV

SEQ ID NO：176, LC-CDR3 31

NSRDSSGFPPYV

SEQ ID NO：177, LC-CDR3 32

QSWASSSVYV

SEQ ID NO：178, LC-CDR3 39

NSRDSSGNHRV

SEQ ID NO：179, LC-CDR3 40

AAWDDSLNVPV

SEQ ID NO：180, LC-CDR3 41

NSRDSGGDPVV

SEQ ID NO：181, LC-CDR3 US-5

LLYYGGARV

SEQ ID NO:182, LC-CDR3 US-7

AAWDDNLKSYV

SEQ ID NO:183, HC-CDR1 share

G-F/G/Y-S/T-F-S/T-S-Y-A/G

SEQ ID NO:184, HC-CDR2 share 1

I-N/I-P-X-X-G-G/T/I-T/A/P

SEQ ID NO:185, HC-CDR2 share 2

I-S-X-S/D-G/N-G/S-N-T/I/K

SEQ ID NO:186, HC-CDR3 share 1

A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D

SEQ ID NO:187, HC-CDR3 share 2

A-R-G-X-X-X-Y-Y/G/S

SEQ ID NO:188, HC-CDR3 share 3

A-R-G-X-X-Y-Q/W-W-S-X-D-D

SEQ ID NO:189, LC-CDR1 share 1

N-I-G-S-N/K

SEQ ID NO:190, LC-CDR1 share 2

L-R-S/N-X-Y

SEQ ID NO:191, LC-CDR3 share

A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V

SEQ ID NO:192, AFP158 control peptides

FMNKFIYEI

SEQ ID NO:193, C3 control peptides (A2E-7)

YLLPAIVHI

SEQ ID NO:194, A2E-1

LLDVPTAAV

SEQ ID NO:195, A2E-2

TLWVDPYEV

SEQ ID NO：196, A2E-3

FLLDHLKRV

SEQ ID NO：197, A2E-4

LLLDVPTAAV

SEQ ID NO：198, A2E-5

VLFRGGPRGLLAV

SEQ ID NO：199, A2E-6

SLLPAIVEL

SEQ ID NO：200, A2E-8

FLLPTGAEA

SEQ ID NO：201, A2E-9

LLDPKLCYLL

SEQ ID NO：202, A2E-11

MLLSVPLLLG

SEQ ID NO：203, A2E-17

MVDGTLLLL

SEQ ID NO：204, DMTN controls

SLPHFHHPET

SEQ ID NO：205, PIM1 controls

LLYDMVCGDIP

SEQ ID NO：206, IFI30 controls

LLLDVPTAAVQ

SEQ ID NO：207, IFI30 controls

LLLDVPTAAVQA

SEQ ID NO：208, SSR1 controls

VLFRGGPRGLLAVA

SEQ ID NO：209, RPS6KB1 controls

YMAPEILMRS

SEQ ID NO:210, CSF2RA controls

FIYNADLMNC

SEQ ID NO:211, IL7 controls

KQYESVLMVSI

SEQ ID NO:212, β balls represent control

KVNVDEVGGE

SEQ ID NO:213, clone 7-1 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCGTGGTACCGAGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:214, clone 7-2 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTTTGC GCGCGCCGCAGTCCTGGTACGTTGGTGATGTATGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:215, clone 7-3 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCATGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCCCCGCAGTCTTGGTACCGTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:216, clone 7-5 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATGGGATGAAAACCCGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:217, clone 7-6 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATGGGAGGACAACCGGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:218, clone 7-7 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTGTGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTACGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:219, clone 7-8 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATGGGATGACAACGTCAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:220, clone 7-9 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGATAACCCGAAAAGTTATCAC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:221, clone 7-10 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCATGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGA CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATGTCAGT AGACACGTCCAAGAGGCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:222, clone 7-11 CDS

TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCCTGTTCT GGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTACCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCAT CTTTAATAACAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGG CCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAACCTGAAAAGTTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGATGGTGG TGGATCCCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCC TCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCTCCCAGGGAAGGGGCTG GAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTACCTCAAGAGTCGAGTCACCATATCAGT AGACACGTCCAAGAGGCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGC GCGCTCCGCAGTCTTGGTACGTTGGTGATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO:223, clone 7-1 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYRGDVWGQGTLVTVSS

SEQ ID NO:224, clone 7-2 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCLRAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:225, clone 7-3 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETMSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYRGDVWGQGTLVTVSS

SEQ ID NO:226, clone 7-5 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWDENPKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:227, clone 7-6 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWEDNRKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:228, clone 7-7 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRVVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:229, clone 7-8 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWDDNVKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:230, clone 7-9 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNPKSYHFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:231, clone 7-10 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLTIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTMSVDTSKRQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:232, clone 7-11 albumen

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGGGGSGGGGSDGGGSLEMAQVQLQQWGAGLLKP SETLSLTCAVYGGSFSGYYWSWIRQLPGKGLEWIGEINHSGSTNYNPYLKSRVTISVDTSKRQFSLKLSSVTAADTA VYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:233, HCVR 7-1

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARAPQSWYRGDVWGQGTLVTVSS

SEQ ID NO:234, HCVR 7-2

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCLRAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:235, HCVR 7-3

QVQLQQWGAGLLKPSETMSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARAPQSWYRGDVWGQGTLVTVSS

SEQ ID NO:236, HCVR 7-10

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTM SVDTSKRQFSLKLSSVTAADTAVYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:237, HCVR 7-11

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQLPGKGLEWIGEINHSGSTNYNPYLKSRVTI SVDTSKRQFSLKLSSVTAADTAVYYCARAPQSWYVGDVWGQGTLVTVSS

SEQ ID NO:238, LCVR 7-5

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWDENPKSYVFGTGTKVTVLG

SEQ ID NO:239, LCVR 7-6

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWEDNRKSYVFGTGTKVTVLG

SEQ ID NO:240, LCVR 7-7

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRVVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLG

SEQ ID NO:241, LCVR 7-8

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCATWDDNVKSYVFGTGTKVTVLG

SEQ ID NO:242, LCVR 7-9

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLAIIGLQSDDEADYYCAAWDDNPKSYHFGTGTKVTVLG

SEQ ID NO:243, LCVR 7-10

SYELTQPPSVSGTPGQRVAISCSGSSSNIGTRMVTWYQHVPGTAPKLLIFNNNQRPSGVPDRFSASKSG TSASLTIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLG

SEQ ID NO:244, HC-CDR3 7-1

ARAPQSWYRGDV

SEQ ID NO:245, HC-CDR3 7-2

LRAPQSWYVGDV

SEQ ID NO:246, LC-CDR1 7-7

SSNIGTRV

SEQ ID NO:247, LC-CDR3 7-5

ATWDENPKSYV

SEQ ID NO:248, LC-CDR3 7-6

ATWEDNRKSYV

SEQ ID NO:249, LC-CDR3 7-8

ATWDDNVKSYV

SEQ ID NO:250, LC-CDR3 7-9

AAWDDNPKSYH

SEQ ID NO:251, HC-FR1 7-3

VQLQQWGAGLLKPSETMSLTCAVY

SEQ ID NO:252, HC-FR2 7-11

WSWIRQLPGKGLEWIGE

SEQ ID NO:253, HC-FR3 7-10

NYNPSLKSRVTMSVDTSKRQFSLKLSSVTAADTAVYYC

SEQ ID NO:254, HC-FR3 7-11

NYNPYLKSRVTISVDTSKRQFSLKLSSVTAADTAVYYC

SEQ ID NO:255, LC-FR3 7-10

QRPSGVPDRFSASKSGTSASLTIIGLQSDDEADYYC

SEQ ID NO:256, CD28/CD3 ζ

AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:257,4-1BB/CD3 ζ

TGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR

SEQ ID NO:The A2 of 258, HPV16 E7 11-19

YALDLQPET

SEQ ID NO:The A3 of 259, HPV16 E7 11-19

YMADLQPET

SEQ ID NO:The A4 of 260, HPV16 E7 11-19

YMLALQPET

SEQ ID NO:The A6 of 261, HPV16 E7 11-19

YMLDLAPET

SEQ ID NO:The A7 of 262, HPV16 E7 11-19

YMLDLQAET

SEQ ID NO:The A9 of 263, HPV16 E7 11-19

YMLDLQPEA

Sequence table

<110>Biotech company of You Rui sections (EUREKA THERAPEUTICS, INC.)

<120>Target construct of HPV16-E7 peptides/MHC complexs and application thereof

<130> 750042000240

<140>Not yet specify

<141>Enclose simultaneously

<150> US 62/158,735

<151> 2015-05-08

<150> US 62/197,480

<151> 2015-07-27

<160> 263

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 98

<212> PRT

<213>Artificial sequence

<220>

<223>HPV16 E7 albumen

<400> 1

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser

20 25 30

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

50 55 60

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

65 70 75 80

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

85 90 95

Lys Pro

<210> 2

<211> 297

<212> DNA

<213>Artificial sequence

<220>

<223> HPV16 E7 CDS

<400> 2

atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact 60

gatctctact gttatgagca attaaatgac agctcagagg aggaggatga aatagatggt 120

ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt ttgttgcaag 180

tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg tactttggaa 240

gacctgttaa tgggcacact aggaattgtg tgccccatct gttctcagaa accataa 297

<210> 3

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 7-15

<400> 3

Thr Leu His Glu Tyr Met Leu Asp Leu

1 5

<210> 4

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19

<400> 4

Tyr Met Leu Asp Leu Gln Pro Glu Thr

1 5

<210> 5

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 16-25

<400> 5

Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr

1 5 10

<210> 6

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16-E7 44-52

<400> 6

Gln Ala Glu Pro Asp Arg Ala His Tyr

1 5

<210> 7

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16-E7 46-55

<400> 7

Glu Pro Asp Arg Ala His Tyr Asn Ile Val

1 5 10

<210> 8

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16-E7 49-57

<400> 8

Arg Ala His Tyr Asn Ile Val Thr Phe

1 5

<210> 9

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 82-90

<400> 9

Leu Leu Met Gly Thr Leu Gly Ile Val

1 5

<210> 10

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 86-93

<400> 10

Thr Leu Gly Ile Val Cys Pro Ile

1 5

<210> 11

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 variant

<400> 11

Tyr Met Leu Asp Val Gln Pro Glu Thr

1 5

<210> 12

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A1

<400> 12

Ala Met Leu Asp Leu Gln Pro Glu Thr

1 5

<210> 13

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A5

<400> 13

Tyr Met Leu Asp Ala Gln Pro Glu Thr

1 5

<210> 14

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A8

<400> 14

Tyr Met Leu Asp Leu Gln Pro Ala Thr

1 5

<210> 15

<211> 119

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 1

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro Lys Ser Gly Gly Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Thr Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Tyr Tyr Tyr Gly Gly Val Lys Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 16

<211> 122

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 2

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Val Tyr Gly Tyr Ser Gly Ser Ala Pro Tyr Asp Glu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 119

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 3

<400> 17

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Met Ala

20 25 30

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

35 40 45

Gly Arg Ile Ile Pro Met Ile Gly Ile Pro Asp Tyr Ala Gln Glu Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Pro Leu Tyr Trp Trp His Ser Asp Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 18

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 4

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

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

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Met His Gly Ser Tyr Gly Ser Tyr Phe Asp Lys Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 119

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 5

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr Ser Thr Ala His

65 70 75 80

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

85 90 95

Ala Arg Ser Ser Met Tyr Gln Tyr Ser His Asp Asp Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 20

<211> 121

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 8

<400> 20

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

1 5 10 15

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

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Ser Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Tyr Tyr Ala Gly Tyr Tyr Met Phe Met Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 9

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asn Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Ser Val Trp Gly Met Met Asp Glu Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 22

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 10

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Asn Tyr Tyr Ala Asp Tyr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 23

<211> 121

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 11

<400> 23

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

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

35 40 45

Gly Trp Ile Ser Thr Tyr Asn Gly Asn Thr Asp Leu Ala Gln Lys Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Tyr Thr Gly Ser Tyr Tyr Ala Thr Gly Asp Glu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 13

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Ser Ser

<210> 25

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 17

<400> 25

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ile Phe Arg Lys Phe

20 25 30

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

35 40 45

Gly Arg Ile Ile Pro Asn Leu Asp Ile Thr Asp Lys Pro Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Ile Tyr Gln Trp Ser Gly Trp Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 26

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 22

<400> 26

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly His Ser Phe Ser Tyr Gly Glu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 27

<211> 119

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 26

<400> 27

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Trp His Gln Tyr Gly Ile Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 28

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 27

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 29

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 31

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Asn Ile Lys Glu Asp Gly Ser Val Lys Tyr Tyr Val Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Gly Val Trp Gly His Ile Asp Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 30

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 32

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 31

<211> 116

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 39

<400> 31

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Arg His

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Glu Gly Arg Phe Thr Met Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 32

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 40

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Val Tyr Arg Tyr Ser Met Met His Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 33

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 41

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 34

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR US-5

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Arg Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 35

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR US-7

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 36

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 1

<400> 36

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Asn

20 25 30

Pro Val Thr Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Thr Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 37

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 2

<400> 37

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Met Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Gly Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Thr Gly Asp His

85 90 95

Pro Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 38

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 3

<400> 38

Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln His Phe Pro Gly Ala Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Tyr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Glu Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

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

100 105 110

<210> 39

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 4

<400> 39

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

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 40

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 5

<400> 40

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

1 5 10 15

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

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 41

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 8

<400> 41

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly His Asn Ser Asn Ile Glu Thr Asn

20 25 30

Pro Val Ser Trp Tyr Gln Gln Val Pro Gly Thr Thr Pro Lys Leu Leu

35 40 45

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

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Trp Asp Asp Ser Val

85 90 95

Gln Gly Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 42

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 9

<400> 42

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

1 5 10 15

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

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Gln Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

100 105

<210> 43

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 10

<400> 43

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

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp His Asp Gly Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 44

<211> 110

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 11

<400> 44

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Asp Asp Glu Gly Ala Tyr Phe Cys Gln Ser Tyr Asp Thr Ser Leu

85 90 95

Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 45

<211> 112

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 13

<400> 45

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

1 5 10 15

Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly

20 25 30

Tyr Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala

35 40 45

Leu Ile Tyr Ser Thr Asn Asn Lys His Ser Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Tyr Tyr Gly Gly

85 90 95

Ala Gln Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 46

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 17

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Ala Ser Ser Phe Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg

100 105

<210> 47

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 22

<400> 47

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

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

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Lys Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 48

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 26

<400> 48

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

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Glu Ser Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

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

85 90 95

Leu Lys Gly Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 49

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 27

<400> 49

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

1 5 10 15

Thr Val Thr Ile Thr Cys Gln Gly Asp Ile Leu Arg Asn Tyr Tyr Ala

20 25 30

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

35 40 45

Gly Gln Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Gly Asp Tyr Tyr Cys Asp Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 50

<211> 110

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 31

<400> 50

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Lys Asn Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Phe Pro

85 90 95

Pro Tyr Val Phe Gly Pro Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 51

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 32

<400> 51

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Phe

35 40 45

Gln Asn Asp Lys Arg Pro Ser Gly Ile Pro Glu Arg Leu Ser Gly Ser

50 55 60

Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Ala Ser Ser Ser Val Tyr

85 90 95

Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 52

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 39

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Lys Asp Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 53

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 40

<400> 53

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

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

35 40 45

Ile Ser Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

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

100 105 110

<210> 54

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 41

<400> 54

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

1 5 10 15

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

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Phe Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Glu Asn Thr Ala Ser Leu Thr Ile Ala Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Gly Gly Asp Pro

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 55

<211> 110

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR US-5

<400> 55

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

1 5 10 15

Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Ser

20 25 30

Tyr Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala

35 40 45

Leu Ile Tyr Ser Thr Ser Asn Thr His Ser Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Val Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Tyr Tyr Gly Gly

85 90 95

Ala Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 56

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR US-7

<400> 56

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

<210> 57

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 1

<400> 57

Gly Tyr Thr Phe Ile Asp Tyr Tyr

1 5

<210> 58

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 2

<400> 58

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

<210> 59

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 3

<400> 59

Gly Gly Thr Phe Ser Met Ala Ala

1 5

<210> 60

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 4

<400> 60

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 61

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 5

<400> 61

Gly Gly Thr Phe Arg Ser Tyr Ala

1 5

<210> 62

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 8

<400> 62

Gly Tyr Thr Phe Ala Asn Tyr Gly

1 5

<210> 63

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 9

<400> 63

Gly Phe Thr Phe Ser Ser Tyr Trp

1 5

<210> 64

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 10

<400> 64

Gly Phe Pro Phe Ser Ser Tyr Ser

1 5

<210> 65

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 11

<400> 65

Gly Tyr Thr Phe Ser Asp Tyr Gly

1 5

<210> 66

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 13

<400> 66

Gly Tyr Thr Phe Thr Ser Tyr Gly

1 5

<210> 67

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 17

<400> 67

Gly Asp Ile Phe Arg Lys Phe Ala

1 5

<210> 68

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 22

<400> 68

Gly Phe Asn Phe Ser Asp Tyr Tyr

1 5

<210> 69

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 26

<400> 69

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 70

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 27

<400> 70

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 71

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 31

<400> 71

Gly Phe Thr Phe Ser Ser Tyr Trp

1 5

<210> 72

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 32

<400> 72

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

<210> 73

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 39

<400> 73

Gly Phe Ser Phe Ser Arg His Ala

1 5

<210> 74

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 40

<400> 74

Gly Tyr Ser Phe Ser Asp Tyr Tyr

1 5

<210> 75

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 41

<400> 75

Gly Phe Thr Phe Ser Ser Tyr Ser

1 5

<210> 76

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 US-5

<400> 76

Gly Gly Thr Phe Ser Ser Tyr Gly

1 5

<210> 77

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR1 US-7

<400> 77

Gly Gly Ser Phe Ser Gly Tyr Tyr

1 5

<210> 78

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 1

<400> 78

Ile Asn Pro Lys Ser Gly Gly Thr

1 5

<210> 79

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 2

<400> 79

Ile Ile Pro Ile Phe Gly Thr Ala

1 5

<210> 80

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 3

<400> 80

Ile Ile Pro Met Ile Gly Ile Pro

1 5

<210> 81

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 4

<400> 81

Ile Ser Tyr Asp Gly Thr Asn Lys

1 5

<210> 82

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 5

<400> 82

Ile Ile Pro Ile Phe Gly Thr Pro

1 5

<210> 83

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 8

<400> 83

Ile Ser Ala Ser Asn Gly Asn Thr

1 5

<210> 84

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 9

<400> 84

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210> 85

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 10

<400> 85

Ile Ser Ser Ser Ser Ser Thr Ile

1 5

<210> 86

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 11

<400> 86

Ile Ser Thr Tyr Asn Gly Asn Thr

1 5

<210> 87

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 13

<400> 87

Ile Ser Pro Tyr Asn Gly Asn Thr

1 5

<210> 88

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 17

<400> 88

Ile Ile Pro Asn Leu Asp Ile Thr

1 5

<210> 89

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 22

<400> 89

Ile Ser Pro Gly Gly Thr Thr

1 5

<210> 90

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 26

<400> 90

Ile Asn Pro Asn Ser Gly Gly Thr

1 5

<210> 91

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 27

<400> 91

Ile Ser Gly Ser Gly Gly Ser Thr

1 5

<210> 92

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 31

<400> 92

Ile Lys Glu Asp Gly Ser Val Lys

1 5

<210> 93

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 32

<400> 93

Ile Ile Pro Ile Leu Gly Ile Ala

1 5

<210> 94

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 39

<400> 94

Ile Ser Gly Ser Gly Gly Asn Thr

1 5

<210> 95

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 40

<400> 95

Ile Asn Pro Asn Thr Gly Val Thr

1 5

<210> 96

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 41

<400> 96

Ile Ser Ser Ser Ser Ser Tyr Ile

1 5

<210> 97

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 US-5

<400> 97

Ile Ile Pro Ile Phe Gly Thr Thr

1 5

<210> 98

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR2 US-7

<400> 98

Ile Asn His Ser Gly Ser Thr

1 5

<210> 99

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 1

<400> 99

Ala Arg Ser Tyr Tyr Tyr Gly Gly Val Lys Asp Tyr

1 5 10

<210> 100

<211> 15

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 2

<400> 100

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

1 5 10 15

<210> 101

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 3

<400> 101

Ala Arg Gly Pro Leu Tyr Trp Trp His Ser Asp Ser

1 5 10

<210> 102

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 4

<400> 102

Ala Arg Met His Gly Ser Tyr Gly Ser Tyr Phe Asp Lys

1 5 10

<210> 103

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 5

<400> 103

Ala Arg Ser Ser Met Tyr Gln Tyr Ser His Asp Asp

1 5 10

<210> 104

<211> 14

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 8

<400> 104

Ala Arg Ser Tyr Tyr Ala Gly Tyr Tyr Met Phe Met Asp Phe

1 5 10

<210> 105

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 9

<400> 105

Ala Arg Tyr Ser Val Trp Gly Met Met Asp Glu

1 5 10

<210> 106

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 10

<400> 106

Ala Arg Gln Asn Tyr Tyr Ala Asp Tyr Asp Tyr

1 5 10

<210> 107

<211> 14

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 11

<400> 107

Ala Arg Gly Tyr Thr Gly Ser Tyr Tyr Ala Thr Gly Asp Glu

1 5 10

<210> 108

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 13

<400> 108

Ala Arg Gly Tyr Lys Asp Tyr

1 5

<210> 109

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 17

<400> 109

Ala Arg Gly Ser Ile Tyr Gln Trp Ser Gly Trp Asp Ser

1 5 10

<210> 110

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 22

<400> 110

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

1 5 10

<210> 111

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 26

<400> 111

Ala Arg Gly Trp His Gln Tyr Gly Ile Met Asp Tyr

1 5 10

<210> 112

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 27

<400> 112

Ala Arg Arg Ser Val Trp Ser Ala Tyr Asp Ser

1 5 10

<210> 113

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 31

<400> 113

Ala Arg Arg Gly Val Trp Gly His Ile Asp Phe

1 5 10

<210> 114

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 32

<400> 114

Ala Arg Gly Thr Tyr Thr Trp Gly Ser Asp Thr

1 5 10

<210> 115

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 39

<400> 115

Ala Arg Tyr Trp Gly Ser Tyr Asp Lys

1 5

<210> 116

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 40

<400> 116

Ala Arg Asn Val Tyr Arg Tyr Ser Met Met His Asp Ser

1 5 10

<210> 117

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 41

<400> 117

Ala Arg Arg Gly Val Trp Gly Tyr Phe Asp Ser

1 5 10

<210> 118

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 US-5

<400> 118

Ala Arg Val Tyr Gly Ser Tyr Phe Tyr Asp Asp

1 5 10

<210> 119

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 US-7

<400> 119

Ala Arg Ala Pro Gln Ser Trp Tyr Val Gly Asp Val

1 5 10

<210> 120

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 1

<400> 120

Ser Ser Asn Ile Gly Thr Asn Pro

1 5

<210> 121

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 2

<400> 121

Asn Ile Gly Met Lys Ser

1 5

<210> 122

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 3

<400> 122

Ser Ser Asn Ile Gly Asn Asn Tyr

1 5

<210> 123

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 4

<400> 123

Asn Ile Gly Ser Lys Ser

1 5

<210> 124

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 5

<400> 124

Gln Gly Ile Ser Ser Trp

1 5

<210> 125

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 8

<400> 125

Asn Ser Asn Ile Glu Thr Asn Pro

1 5

<210> 126

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 9

<400> 126

Ser Leu Arg Ser Tyr Tyr

1 5

<210> 127

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 10

<400> 127

Asn Ile Gly Ser Lys Asn

1 5

<210> 128

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 11

<400> 128

Ser Ser Asn Ile Gly Ile Asn Thr

1 5

<210> 129

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 13

<400> 129

Thr Gly Ala Val Thr Ser Gly Tyr Tyr

1 5

<210> 130

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 17

<400> 130

Gln Ser Ile Ser Ser Tyr

1 5

<210> 131

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 22

<400> 131

Ser Ser Asn Ile Gly Ser Asn Ser

1 5

<210> 132

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 26

<400> 132

Ser Ser Asn Ile Gly Ala Gly Tyr Asp

1 5

<210> 133

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 27

<400> 133

Ile Leu Arg Asn Tyr Tyr

1 5

<210> 134

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 31

<400> 134

Asn Leu Arg Asn Ser Tyr

1 5

<210> 135

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 32

<400> 135

Lys Leu Gly Glu Lys Tyr

1 5

<210> 136

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 39

<400> 136

Ser Leu Arg Asn Tyr Tyr

1 5

<210> 137

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 40

<400> 137

Ser Ser Asn Ile Gly Ser Asn His

1 5

<210> 138

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 41

<400> 138

Thr Leu Arg Ser Phe Tyr

1 5

<210> 139

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 US-5

<400> 139

Thr Gly Ala Val Thr Ser Ser Tyr Tyr

1 5

<210> 140

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 US-7

<400> 140

Ser Ser Asn Ile Gly Thr Arg Met

1 5

<210> 141

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 1

<400> 141

Gly Asn Tyr

1

<210> 142

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 2

<400> 142

Tyr Asp Ser

1

<210> 143

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 3

<400> 143

Asp Asn Tyr

1

<210> 144

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 4

<400> 144

Tyr Asn Ser

1

<210> 145

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 5

<400> 145

Ala Ala Ser

1

<210> 146

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 8

<400> 146

Ser Tyr Tyr

1

<210> 147

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 9

<400> 147

Gly Lys Asn

1

<210> 148

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 10

<400> 148

Asp Asp His

1

<210> 149

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 11

<400> 149

Gly Asn Ser

1

<210> 150

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 13

<400> 150

Ser Thr Asn

1

<210> 151

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 17

<400> 151

Gly Ala Ser

1

<210> 152

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 22

<400> 152

Ser Asn Asn

1

<210> 153

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 26

<400> 153

Glu Ser Asn

1

<210> 154

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 27

<400> 154

Gly Gln Asn

1

<210> 155

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 31

<400> 155

Gly Lys Asn

1

<210> 156

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 32

<400> 156

Gln Asn Asp

1

<210> 157

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 39

<400> 157

Gly Lys Asp

1

<210> 158

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 40

<400> 158

Asn Asn Asn

1

<210> 159

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 41

<400> 159

Gly Lys Asn

1

<210> 160

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 US-5

<400> 160

Ser Thr Ser

1

<210> 161

<211> 3

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR2 US-7

<400> 161

Asn Asn Asn

1

<210> 162

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 1

<400> 162

Ala Ala Trp Asp Asp Ser Leu Thr Gly Tyr Val

1 5 10

<210> 163

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 2

<400> 163

Gln Val Trp Asp Thr Thr Gly Asp His Pro Gly Tyr Val

1 5 10

<210> 164

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 3

<400> 164

Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr Val

1 5 10

<210> 165

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 4

<400> 165

Gln Val Trp Asp Ser Ser Ser Asp His Trp Val

1 5 10

<210> 166

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 5

<400> 166

Gln Gln Ala Asn Ser Phe Pro Leu Thr

1 5

<210> 167

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 8

<400> 167

Ala Ser Trp Asp Asp Ser Val Gln Gly Tyr Val

1 5 10

<210> 168

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 9

<400> 168

Asn Ser Arg Asp Ser Ser Gly Asn His Gln Val

1 5 10

<210> 169

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 10

<400> 169

Gln Val Trp Asp Ser Ser Ser Asp His Val Val

1 5 10

<210> 170

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 11

<400> 170

Gln Ser Tyr Asp Thr Ser Leu Thr Val Ile

1 5 10

<210> 171

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 13

<400> 171

Leu Leu Tyr Tyr Gly Gly Ala Gln Leu Trp Val

1 5 10

<210> 172

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 17

<400> 172

Gln Gln Ser Tyr Ser Ala Pro Leu Thr

1 5

<210> 173

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 22

<400> 173

Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val

1 5 10

<210> 174

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 26

<400> 174

Gln Ser Tyr Asp Thr Ser Leu Lys Gly Val

1 5 10

<210> 175

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 27

<400> 175

Asp Ser Arg Asp Ser Ser Gly Asn His Trp Val

1 5 10

<210> 176

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 31

<400> 176

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

1 5 10

<210> 177

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 32

<400> 177

Gln Ser Trp Ala Ser Ser Ser Val Tyr Val

1 5 10

<210> 178

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 39

<400> 178

Asn Ser Arg Asp Ser Ser Gly Asn His Arg Val

1 5 10

<210> 179

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 40

<400> 179

Ala Ala Trp Asp Asp Ser Leu Asn Val Pro Val

1 5 10

<210> 180

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 41

<400> 180

Asn Ser Arg Asp Ser Gly Gly Asp Pro Val Val

1 5 10

<210> 181

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 US-5

<400> 181

Leu Leu Tyr Tyr Gly Gly Ala Arg Val

1 5

<210> 182

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 US-7

<400> 182

Ala Ala Trp Asp Asp Asn Leu Lys Ser Tyr Val

1 5 10

<210> 183

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR1 is shared

<220>

<221> VARIANT

<222> 2

<223>Xaa=F or G or Y

<220>

<221> VARIANT

<222> 3

<223>Xaa=S or T

<220>

<221> VARIANT

<222> 5

<223>Xaa=S or T

<220>

<221> VARIANT

<222> 8

<223>Xaa=A or G

<400> 183

Gly Xaa Xaa Phe Xaa Ser Tyr Xaa

1 5

<210> 184

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR2 shares 1

<220>

<221> VARIANT

<222> 2

<223>Xaa=N or I

<220>

<221> VARIANT

<222> 4, 5

<223>Xaa=any amino acid

<220>

<221> VARIANT

<222> 7

<223>Xaa=G or T or I

<220>

<221> VARIANT

<222> 8

<223>Xaa=T or A or P

<400> 184

Ile Xaa Pro Xaa Xaa Gly Xaa Xaa

1 5

<210> 185

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR2 shares 2

<220>

<221> VARIANT

<222> 3

<223>Xaa=any amino acid

<220>

<221> VARIANT

<222> 4

<223>Xaa=S or D

<220>

<221> VARIANT

<222> 5

<223>Xaa=G or N

<220>

<221> VARIANT

<222> 6

<223>Xaa=G or S

<220>

<221> VARIANT

<222> 8

<223>Xaa=T or I or K

<400> 185

Ile Ser Xaa Xaa Xaa Xaa Asn Xaa

1 5

<210> 186

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR3 shares 1

<220>

<221> VARIANT

<222> 3

<223>Xaa=S or R

<220>

<221> VARIANT

<222> 4

<223>Xaa=Y or S or G

<220>

<221> VARIANT

<222> 5

<223>Xaa=Y or V

<220>

<221> VARIANT

<222> 6

<223>Xaa=Y or W

<220>

<221> VARIANT

<222> 8

<223>Xaa=any amino acid

<400> 186

Ala Arg Xaa Xaa Xaa Xaa Gly Xaa Tyr Asp

1 5 10

<210> 187

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR3 shares 2

<220>

<221> VARIANT

<222> 4, 5, 6

<223>Xaa=any amino acid

<220>

<221> VARIANT

<222> 8

<223>Xaa=Y or G or S

<400> 187

Ala Arg Gly Xaa Xaa Xaa Tyr Xaa

1 5

<210> 188

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223>HC-CDR3 shares 3

<220>

<221> VARIANT

<222> 4, 5, 10

<223>Xaa=any amino acid

<220>

<221> VARIANT

<222> 7

<223>Xaa=Q or W

<400> 188

Ala Arg Gly Xaa Xaa Tyr Xaa Trp Ser Xaa Asp Asp

1 5 10

<210> 189

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>LC-CDR1 shares 1

<220>

<221> VARIANT

<222> 5

<223>Xaa=N or K

<400> 189

Asn Ile Gly Ser Xaa

1 5

<210> 190

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>LC-CDR1 shares 2

<220>

<221> VARIANT

<222> 3

<223>Xaa=S or N

<220>

<221> VARIANT

<222> 4

<223>Xaa=any amino acid

<400> 190

Leu Arg Xaa Xaa Tyr

1 5

<210> 191

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>LC-CDR3 is shared

<220>

<221> VARIANT

<222> 1

<223>Xaa=A or Q or N

<220>

<221> VARIANT

<222> 2

<223>Xaa=S or A or V

<220>

<221> VARIANT

<222> 3

<223>Xaa=W or Y or R

<220>

<221> VARIANT

<222> 5

<223>Xaa=S or D

<220>

<221> VARIANT

<222> 7

<223>Xaa=L or S or G

<220>

<221> VARIANT

<222> 8, 9, 10

<223>Xaa=any amino acid

<400> 191

Xaa Xaa Xaa Asp Xaa Ser Xaa Xaa Xaa Xaa Val

1 5 10

<210> 192

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>AFP158 control peptides

<400> 192

Phe Met Asn Lys Phe Ile Tyr Glu Ile

1 5

<210> 193

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>C3 control peptides A2E-7

<400> 193

Tyr Leu Leu Pro Ala Ile Val His Ile

1 5

<210> 194

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-1

<400> 194

Leu Leu Asp Val Pro Thr Ala Ala Val

1 5

<210> 195

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-2

<400> 195

Thr Leu Trp Val Asp Pro Tyr Glu Val

1 5

<210> 196

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-3

<400> 196

Phe Leu Leu Asp His Leu Lys Arg Val

1 5

<210> 197

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-4

<400> 197

Leu Leu Leu Asp Val Pro Thr Ala Ala Val

1 5 10

<210> 198

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-5

<400> 198

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

1 5 10

<210> 199

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-6

<400> 199

Ser Leu Leu Pro Ala Ile Val Glu Leu

1 5

<210> 200

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-8

<400> 200

Phe Leu Leu Pro Thr Gly Ala Glu Ala

1 5

<210> 201

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-9

<400> 201

Leu Leu Asp Pro Lys Leu Cys Tyr Leu Leu

1 5 10

<210> 202

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-11

<400> 202

Met Leu Leu Ser Val Pro Leu Leu Leu Gly

1 5 10

<210> 203

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> A2E-17

<400> 203

Met Val Asp Gly Thr Leu Leu Leu Leu

1 5

<210> 204

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>DMTN is compareed

<400> 204

Ser Leu Pro His Phe His His Pro Glu Thr

1 5 10

<210> 205

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>PIM1 is compareed

<400> 205

Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro

1 5 10

<210> 206

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>IFI30 is compareed

<400> 206

Leu Leu Leu Asp Val Pro Thr Ala Ala Val Gln

1 5 10

<210> 207

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223>IFI30 is compareed

<400> 207

Leu Leu Leu Asp Val Pro Thr Ala Ala Val Gln Ala

1 5 10

<210> 208

<211> 14

<212> PRT

<213>Artificial sequence

<220>

<223>SSR1 is compareed

<400> 208

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

1 5 10

<210> 209

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>RPS6KB1 is compareed

<400> 209

Tyr Met Ala Pro Glu Ile Leu Met Arg Ser

1 5 10

<210> 210

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>CSF2RA is compareed

<400> 210

Phe Ile Tyr Asn Ala Asp Leu Met Asn Cys

1 5 10

<210> 211

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>IL7 is compareed

<400> 211

Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile

1 5 10

<210> 212

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223>Beta Globulin compares

<400> 212

Lys Val Asn Val Asp Glu Val Gly Gly Glu

1 5 10

<210> 213

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-1 CDS

<400> 213

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagctctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtcgt ggtaccgagg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 214

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-2 CDS

<400> 214

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagctctgt gaccgccgcg 660

gacacggccg tgtattactg tttgcgcgcg ccgcagtcct ggtacgttgg tgatgtatgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 215

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-3 CDS

<400> 215

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccatg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagctctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgcc ccgcagtctt ggtaccgtgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 216

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-5 CDS

<400> 216

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca acatgggatg aaaacccgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 217

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-6 CDS

<400> 217

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca acatgggagg acaaccggaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 218

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-7 CDS

<400> 218

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtgtgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttacgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 219

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-8 CDS

<400> 219

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca acatgggatg acaacgtcaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 220

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-9 CDS

<400> 220

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg ataacccgaa aagttatcac 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaaccagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 221

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-10 CDS

<400> 221

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcatgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tgaccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg gtggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagcccccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt ccctcaagag tcgagtcacc 600

atgtcagtag acacgtccaa gaggcagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 222

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Clone 7-11 CDS

<400> 222

tcctatgagc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcgccatc 60

tcctgttctg gaagcagctc caacatcggg actcgtatgg tgacctggta ccagcatgtc 120

ccagggacgg cccccaaact cctcatcttt aataacaatc agcggccctc aggggtccct 180

gaccgattct ctgcctccaa gtctggcacc tcagcctccc tggccatcat tgggctccag 240

tctgacgatg aggctgatta ttactgtgca gcatgggatg acaacctgaa aagttatgtc 300

ttcggaactg ggaccaaggt caccgtccta ggttctagag gtggtggtgg tagcggcggc 360

ggcggctctg atggtggtgg atccctcgag atggcccagg tgcagctaca gcagtggggc 420

gcaggactgt tgaagccttc ggagaccctg tccctcacct gcgctgtcta tggtgggtcc 480

ttcagtggtt actactggag ctggatccgc cagctcccag ggaaggggct ggagtggatt 540

ggggaaatca atcatagtgg aagcaccaac tacaacccgt acctcaagag tcgagtcacc 600

atatcagtag acacgtccaa gaggcagttc tccctgaagc tgagttctgt gaccgccgcg 660

gacacggccg tgtattactg tgcgcgcgct ccgcagtctt ggtacgttgg tgatgtttgg 720

ggtcaaggta ctctggtgac cgtctcctca 750

<210> 223

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-1 protein

<400> 223

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 224

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-2 protein

<400> 224

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 225

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-3 protein

<400> 225

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

Lys Pro Ser Glu Thr Met Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 226

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-5 protein

<400> 226

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Glu Asn Pro

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 227

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-6 protein

<400> 227

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Glu Asp Asn Arg

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 228

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-7 protein

<400> 228

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Val Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 229

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-8 protein

<400> 229

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Asn Val

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 230

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-9 protein

<400> 230

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Pro

85 90 95

Lys Ser Tyr His Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Ser

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 231

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-10 protein

<400> 231

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Arg

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 232

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>Clone 7-11 protein

<400> 232

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly Gly Gly Ser

115 120 125

Leu Glu Met Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

130 135 140

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

145 150 155 160

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

165 170 175

Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn

180 185 190

Pro Tyr Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Arg

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 233

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 7-1

<400> 233

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 234

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 7-2

<400> 234

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 235

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 7-3

<400> 235

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 236

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 7-10

<400> 236

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 237

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223> HCVR 7-11

<400> 237

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Tyr Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 238

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-5

<400> 238

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Glu Asn Pro

85 90 95

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

100 105 110

<210> 239

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-6

<400> 239

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Glu Asp Asn Arg

85 90 95

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

100 105 110

<210> 240

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-7

<400> 240

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Val Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

<210> 241

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-8

<400> 241

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Asn Val

85 90 95

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

100 105 110

<210> 242

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-9

<400> 242

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Pro

85 90 95

Lys Ser Tyr His Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

100 105 110

<210> 243

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223> LCVR 7-10

<400> 243

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

1 5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Arg

20 25 30

Met Val Thr Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Phe Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Thr Ile Ile Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Asn Leu

85 90 95

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

100 105 110

<210> 244

<211> 12

<212> PRT

<213> HC-CDR3 7-1

<220>

<223>Synthesis

<400> 244

Ala Arg Ala Pro Gln Ser Trp Tyr Arg Gly Asp Val

1 5 10

<210> 245

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> HC-CDR3 7-2

<400> 245

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

1 5 10

<210> 246

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR1 7-7

<400> 246

Ser Ser Asn Ile Gly Thr Arg Val

1 5

<210> 247

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 7-5

<400> 247

Ala Thr Trp Asp Glu Asn Pro Lys Ser Tyr Val

1 5 10

<210> 248

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 7-6

<400> 248

Ala Thr Trp Glu Asp Asn Arg Lys Ser Tyr Val

1 5 10

<210> 249

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 7-8

<400> 249

Ala Thr Trp Asp Asp Asn Val Lys Ser Tyr Val

1 5 10

<210> 250

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> LC-CDR3 7-9

<400> 250

Ala Ala Trp Asp Asp Asn Pro Lys Ser Tyr His

1 5 10

<210> 251

<211> 24

<212> PRT

<213>Artificial sequence

<220>

<223> HC-FR1 7-3

<400> 251

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

1 5 10 15

Met Ser Leu Thr Cys Ala Val Tyr

20

<210> 252

<211> 17

<212> PRT

<213>Artificial sequence

<220>

<223> HC-FR2 7-11

<400> 252

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

1 5 10 15

Glu

<210> 253

<211> 38

<212> PRT

<213>Artificial sequence

<220>

<223> HC-FR3 7-10

<400> 253

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 254

<211> 38

<212> PRT

<213>Artificial sequence

<220>

<223> HC-FR3 7-11

<400> 254

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 255

<211> 36

<212> PRT

<213>Artificial sequence

<220>

<223> LC-FR3 7-10

<400> 255

Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Ala Ser Lys Ser Gly

1 5 10 15

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

20 25 30

Asp Tyr Tyr Cys

35

<210> 256

<211> 222

<212> PRT

<213>Artificial sequence

<220>

<223> CD28/CD3zeta

<400> 256

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val

35 40 45

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

50 55 60

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

65 70 75 80

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

85 90 95

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

100 105 110

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

115 120 125

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

130 135 140

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

145 150 155 160

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

165 170 175

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

180 185 190

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

195 200 205

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 257

<211> 225

<212> PRT

<213>Artificial sequence

<220>

<223> 4-1BB/CD3zeta

<400> 257

Thr Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

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

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

65 70 75 80

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

85 90 95

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

100 105 110

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

115 120 125

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

130 135 140

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

145 150 155 160

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

165 170 175

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

180 185 190

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

195 200 205

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

210 215 220

Arg

225

<210> 258

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A2

<400> 258

Tyr Ala Leu Asp Leu Gln Pro Glu Thr

1 5

<210> 259

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A3

<400> 259

Tyr Met Ala Asp Leu Gln Pro Glu Thr

1 5

<210> 260

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A4

<400> 260

Tyr Met Leu Ala Leu Gln Pro Glu Thr

1 5

<210> 261

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A6

<400> 261

Tyr Met Leu Asp Leu Ala Pro Glu Thr

1 5

<210> 262

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A7

<400> 262

Tyr Met Leu Asp Leu Gln Ala Glu Thr

1 5

<210> 263

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> HPV16 E7 11-19 A9

<400> 263

Tyr Met Leu Asp Leu Gln Pro Glu Ala

1 5

Claims

1. a kind of anti-E7MC constructs of separation, it includes antibody moiety, and antibody moiety specific binding includes HPV16-E7 Complex (the HPV16-E7/ of (HPV16) peptide of human papilloma virus hypotype 16 and ajor histocompatibility (MHC) I proteinoid MHC I class complexs, or E7MC).

2. the anti-E7MC constructs of the separation of claim 1, wherein, the MHC I proteinoid is selected from the group：HLA-A*02: 01、HLA-A*02:02、HLA-A*02:06、HLA-A*02:07 and HLA-A*02:11..

3. the anti-E7MC constructs of the separation of claim 2, wherein, the MHC I proteinoid is HLA-A*02:01.

4. the anti-E7MC constructs of the separation of any one of claims 1 to 3, wherein, the HPV16-E7 peptides, which have, to be selected from by SEQ ID NO:The amino acid sequence of the group of 3-14 compositions.

5. the anti-E7MC constructs of the separation of claim 4, wherein, the HPV16-E7 peptides have YMLDLQPET (SEQ ID NO:4) amino acid sequence.

6. the anti-E7MC constructs of the separation of any one of claim 1 to 5, wherein, the antibody moiety be full length antibody, Fab, Fab', (Fab') 2, Fv or scFv (scFv).

7. the anti-E7MC constructs of the separation of any one of claim 1 to 5, wherein, the anti-E7MC constructs of the separation with About 0.1pM to about 500nM K_dIt is bound to the HPV16-E7/MHC I class complexs.

8. the anti-E7MC constructs of the separation of any one of claim 1 to 7, wherein, the antibody moiety includes：

I) weight chain variable district, it is included：Complementary determining region of heavy chain (HC-CDR) 1, the HC-CDR1 include amino acid sequence G-F/ G/Y-S/T-F-S/T-S-Y-A/G(SEQ ID NO:183), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；HC- CDR2, the HC-CDR2 include amino acid sequence I-N/I-P-X-X-G-G/T/I-T/A/P or I-S-X-S/D-G/N-G/S- N-T/I/K(SEQ ID NO:184 or 185), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And HC-CDR3, it is described HC-CDR3 includes amino acid sequence A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D, A-R-G-X-X-X-Y-Y/G/S or A-R- G-X-X-Y-Q/W-W-S-X-D-D(SEQ ID NOs:186-188), or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors； And

Ii) light chain variable district, it is included：Complementary determining region of light chain (LC-CDR) 1, the LC-CDR1 include amino acid sequence N- I-G-S-N/K or L-R-S/N-X-Y(SEQ ID NO:189 or 190), or it includes the change of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors Body；And LC-CDR3, the LC-CDR3 include amino acid sequence A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V (SEQ ID NO:191), or it includes any one in variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and wherein X can be any ammonia Base acid.

9. the anti-E7MC constructs of the separation of any one of claim 1 to 7, wherein the antibody moiety includes：

I) weight chain variable district, it is included：HC-CDR1, the HC-CDR1 include SEQ ID NO:Any one of 57-77 ammonia Base acid sequence, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；HC-CDR2, the HC-CDR2 include SEQ ID NO: Any one of 78-98 amino acid sequence, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And HC-CDR3, it is described HC-CDR3 includes SEQ ID NO:The amino acid sequence of any one of 99-119,244 and 245, or its include at most about 5 The variant of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor；And

Ii) light chain variable district, it is included：LC-CDR1, the LC-CDR1 include SEQ ID NO:Appointing in 120-140 and 246 The amino acid sequence of one, or it includes the variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；LC-CDR2, the LC-CDR2 include SEQ ID NO:Any one of 141-161 amino acid sequence, or it includes the variant of at most about 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors；And LC- CDR3, the LC-CDR3 include SEQ ID NO:Any one of 162-182 and 247-250 amino acid sequence, or it includes The variant of at most about 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

10. the anti-E7MC constructs of the separation of claim 9, wherein, the antibody moiety includes：A) weight chain variable district, it is wrapped The NO of ID containing SEQ:Any one of 15-35 and 233-237 amino acid sequence, or itself and SEQ ID NO:15-35 and 233- Any one of 237 have the variant of at least about 95% sequence identity；And b) light chain variable district, it includes SEQ ID NO: Any one of 36-56 and 238-243 amino acid sequence, or itself and SEQ ID NO:Any one of 36-56 and 238-243 Variant with least about 95% sequence identity.

11. the anti-E7MC constructs of the separation of any one of claim 1 to 10, wherein, the anti-E7MC constructs of the separation For polyspecific.

12. the anti-E7MC constructs of the separation of claim 11, wherein, the anti-E7MC constructs of the separation are series connection scFv, It is anti-that bifunctional antibody (Db), Single-chain bifunctional antibody (scDb), double affinity target (DART) antibody, Double variable regions (DVD) again Body, pestle-mortar (knob-into-hole；KiH) antibody, depressed place lock (dock and lock；DNL) antibody, chemical crosslinking antibody, different Multimer antibody or different conjugate antibody.

13. the anti-E7MC constructs of the separation of claim 12, wherein, the anti-E7MC constructs of the separation be comprising two by By the scFv of peptide linker connection series connection scFv.

14. the anti-E7MC constructs of the separation of any one of claim 11 to 13, wherein, the anti-E7MC constructs of the separation Further include the secondary antibody part of the second antigen of specific binding.

15. the anti-E7MC constructs of the separation of claim 14, wherein, second antigen is selected from the group：CD3γ、CD3δ、 CD3 ε, CD3 ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.

16. the anti-E7MC constructs of the separation of claim 14, wherein, second antigen is CD3 ε, and wherein described separation Anti- E7MC constructs be comprising have to the HPV16-E7/MHC I class complexs specific N-terminal scFv and to CD3 ε have There is specific C-terminal scFv series connection scFv.

17. the anti-E7MC constructs of the separation of any one of claim 1 to 10, wherein, the anti-E7MC constructs of the separation For Chimeric antigen receptor, it includes the extracellular containing the antibody moiety, membrane-spanning domain and includes CD3 ζ intracellular signal transduction sequences The intracellular signal transduction domain of row and CD28 intracellular signal transduction sequences.

18. the anti-E7MC constructs of the separation of any one of claim 1 to 10, wherein, the anti-E7MC constructs of the separation To include the immunoconjugates of the antibody moiety and effector molecule, wherein the effector molecule is selected from the group consisted of Therapeutic agent：Medicine, toxin, radio isotope, protein, peptide and nucleic acid.

19. the anti-E7MC constructs of the separation of any one of claim 1 to 10, wherein, the anti-E7MC constructs of the separation To include the immunoconjugates of the antibody moiety and mark.

20. a kind of pharmaceutical composition, it includes the anti-E7MC constructs of the separation of any one of claim 1 to 18.

21. a kind of host cell, it expresses the anti-E7MC constructs of the separation of any one of claim 1 to 19.

22. a kind of nucleic acid, it encodes the polypeptide fractions of the anti-E7MC constructs of the separation of any one of claim 1 to 19.

23. a kind of effector cell, it expresses the anti-E7MC constructs of the separation of claim 17.

24. the effector cell of claim 23, wherein, the effector cell is T cell.

A kind of 25. side for detecting the cell for presenting the complex comprising HPV16-E7 peptides and MHC I proteinoid in its surface Method, it includes making the cell contact and detect the mark on the cell with the anti-E7MC constructs of the separation of claim 19 Presence.

26. a kind of treat the individual method with HPV16-E7 positive diseases, it includes applying to the individual：

A) pharmaceutical composition of the claim 20 of effective dose, or

B) effector cell of the claim 23 or 24 of effective dose.

27. a kind of diagnose the individual method with HPV16-E7 positive diseases, it includes：

A) the anti-E7MC constructs of the separation of the claim 19 of effective dose are applied to the individual；And

B) level of the mark in the individual is determined, wherein the level of the mark shows that the individual is suffered from higher than threshold level There are the HPV16-E7 positive diseases.

28. a kind of diagnose the individual method with HPV16-E7 positive diseases, it includes：

A) contact the anti-E7MC constructs of the separation from individual sample with the claim 19；And

B) quantity of the cell combined in the sample with the anti-E7MC constructs separated is determined, wherein being separated with described Anti- E7MC constructs with reference to the quantitative value of cell show that the individual suffers from the HPV16-E7 positives disease higher than threshold level Disease.

29. the method for any one of claim 26 to 28, wherein, the HPV16-E7 positive diseases are cancer.

30. the method for claim 29, wherein, the HPV16-E7 positive cancers are squamous cell carcinoma.

31. the method for claim 29, wherein, the HPV16-E7 positive cancers are cervical carcinoma, anogenital cancer, head and neck cancer Or throat cancer.