JP2018516879A - Constructs targeting the HPV16-E7 peptide / MHC complex and uses thereof - Google Patents

Abstract

The application provides a construct comprising an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein. Methods for making and using these constructs are also provided. The present invention specifically binds to a complex comprising, for example, human papillomavirus subtype 16 (HPV16) E7 peptide and a major histocompatibility complex (MHC) class I protein (HPV16-E7 / MHC class I complex, or E7MC). An isolated anti-E7MC construct comprising an antibody moiety is provided. [Selection] Figure 8A

Description

This application is related to US Provisional Application No. 62 / 158,735 filed May 8, 2015 and US Provisional Application No. 62 / 197,480 filed July 27, 2015. All of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION The present invention relates to antibody constructs that specifically bind MHC molecules complexed with HPV16-E7 peptides, and their uses including treating and diagnosing diseases.

Submitting Sequence Listings in ASCII Text Files The contents of the following submissions in ASCII text files are hereby incorporated by reference in their entirety: Sequence Listing in computer readable form (CRF) (file name: 750042000240SEQLIST.txt, Data recording: May 6, 2016, size: 125 KB).

  Human papillomavirus (HPV) is a small non-enveloped DNA virus that causes warts or benign papillomas when infected with epithelial cells. Persistent infection with high-risk HPV types can result in more severe cytological abnormalities or lesions that can progress to cancer if not treated. The annual incidence of HPV-related cancer exceeds 26,000 in the United States (by CDC) and over 600,000 worldwide (Forman D. et al., Vaccine, 30: Addendum 5: pages 12-23, 2012) Year). Various types of HPV are responsible for causing almost all cases of cervical cancer, the third most common cause of cancer-related deaths in women worldwide (Doorbar J., Clin. Sci., 110: 525-541, 2006). In addition to cervical cancer, HPV is also associated with anogenital cancer (anus, penis, vagina and vulva) and head and neck cancer (oropharynx: the base of the tongue and the back of the throat, including the tonsils). In a meta-analysis of 5,046 head and neck squamous cell carcinoma (HNSCC) cancer specimens from 60 studies, the overall prevalence of HPV was found to be 25.9%. Of the HPV positive HNSCCs, HPV-16 was found in 86.7% of HPV positive oropharyngeal SCC, 68.2% of oral SCC, and 69.2% of laryngeal SCC (Kreimer AR et al. , Cancer Epidemiol Biomarkers Prev. 14: 467-475, 2005). A more recent study categorizing HPV status by the presence of more than 1,000 mapped RNA sequencing (RNA-Seq) readings of HPV E6 and E7 proteins (The Cancer Genome Atlas Network, Nature 517: 576-582 Page (2015), 36 (12.9%) of 279 were found to be HPV positive.

Cancer development during persistent infection with high-risk HPV subtypes has been reported to promote genomic instability and cellular transformation by degrading p53 and Rb in a proteasome-dependent manner It is mainly due to the expression of genes E6 and E7 (Doorbar, supra). HPV E6 and E7 oncoproteins are continuously expressed in lesions, but tumors occur only years after the first cell immortalization event. Indeed, continued expression of E6 and E7 is required for maintenance of the transformed phenotype and for prevention of cell growth arrest and / or apoptosis (McLaughlin-Drubin ME and Muenger K., Virology, 384: 335-344, 2009). Since established lesions do not express L1, there is a need for treatment of HPV ⁺ cancer despite the availability of approved prophylactic vaccines for HPV-6, 11, 16, and 18 subtypes of L1 protein. Not satisfied.

Proteins expressed by oncogenic viruses such as HPV are excellent targets for immunotherapy because they are not expressed in normal cells. E6 and E7 are intracellular proteins that have not been targeted by conventional drug development attempts using low molecular weight compounds or antibody approaches to cell surface proteins. Recent studies on HPV16 positive high-grade vulvar intraepithelial neoplasia have demonstrated T cell responses and clinical responses in 50% of patients treated with vaccination approaches utilizing E6 / E7 peptides (Kenter G. et al. G. et al., N. Engl. J. Med. 361: 1838-1847, 2009; Welters M. J. et al., N. Engl. J. Med. S. et al., Br. J. Cancer 102: 1129-1136, 2010). Of the 13 distinct predicted HLA-A ^* 02 ^: 01 binding E7 peptides, only E7 _11-19 (YMLDLQPET) was naturally processed in vitro and HPV-16 transformed HLA-A ^* It was found to be displayed on the 02:01 expressing cell line (Riemer AB et al., J. Biol. Chem. 285 (38): 29608-29622, 2010). This peptide is conserved in 16 of 17 HPV-16 variants in the HPV database (Zhang GL et al., Database, 1-12, 2014).

Recent advances in using phage display to generate mAbs have made it possible to select drugs from the large antibody repertoire with superior specificity for defined epitopes. Several such mAbs specific for solid tumor antigens have been successfully selected from phage display libraries in the context of HLA-A01 and HLA-A02 (Noy et al., Expert Rev. Anticancer Ther. 5 ( 3): 523-536, 2005; Chames et al., Proc. Natl. Acad. Sci. USA 97: 7969-7974, 2000; Held et al., Eur. J. Immunol.34: 2919-2929. Lev et al., Cancer Res. 62: 3184-3194, 2002; Klechsky et al., Cancer Res. 68 (15): 6360-6367, 2008). More recently, a well-described T cell epitope, a human mAb specific for the human WT1 / HLA-A02 complex, has been shown to induce multiple cancers via Fc-mediated effector cell function in cellular assays and in vivo models. It has been shown to inhibit cell lines and primary cancer cells (Dao et al., Sci. Transl. Med. 5: 176ra33, 2013; Veomett et al., Clin. Cancer Res. Doi: 10.15158 / 1078 -0432, 2014).
The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated by reference in their entirety.

Forman D. Vaccine, 30: Addendum 5: F12-23, 2012 Doorbar J. et al. Clin. Sci. 110: 525-541, 2006 Kreimer A.M. R. Et al., Cancer Epidemiol Biomarkers Prev. 14: 467-475, 2005 The Cancer Genome Atlas Network, Nature 517: 576-582, 2015 McLaughlin-Drubin M.M. E. And Muenger K.M. Virology, 384: 335-344, 2009 Kenter G. G. Et al. Engl. J. et al. Med. 361: 1838-1847, 2009 Welters M.M. J. et al. Et al. Engl. J. et al. Med. 361: 1838-1847, 2010 Dayana S. Et al., Br. J. et al. Cancer 102: 1129-1136, 2010 Riemer A. B. Et al. Biol. Chem. 285 (38): 29608-29622, 2010 Zhang G. L. Et al., Database, 1-12, 2014 Noy et al., Expert Rev. Anticancer Ther. Volume 5 (3): 523-536, 2005 Chames et al., Proc. Natl. Acad. Sci. USA 97: 7969-7974, 2000 Held et al., Eur. J. et al. Immunol. 34: 2919-2929, 2004 Lev et al., Cancer Res. 62: 3184-3194, 2002 Klechevsky et al., Cancer Res. Volume 68 (No. 15): 6360-6367, 2008 Dao et al., Sci. Transl. Med. Volume 5: 176ra33, 2013 Veomett et al., Clin. Cancer Res. doi: 10.1158 / 1078-0432, 2014

  The application, in one aspect, comprises a construct that binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein (referred to herein as an “HPV16-E7 / MHC class I complex” or “E7MC”) (eg, Isolated constructs). In some embodiments, the construct (“anti-E7MC construct”) comprises an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein (referred to herein as an “anti-E7MC antibody moiety”). Call).

  Accordingly, in some embodiments, an anti-E7MC construct (eg, an isolated anti-E7MC construct) comprising an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein is provided. . In some embodiments, the HPV16-E7 / MHC class I complex is present on the cell surface. In some embodiments, the HPV16-E7 / MHC class I complex is present on the surface of a cancer cell.

In some embodiments, the anti-E7MC construct comprises an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is the HLA-A ^* 02 ^: 01 subtype of the HLA-A02 allele.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. This antibody portion cross-reacts with a complex comprising an HPV16-E7 peptide and a second MHC class I protein having an HLA allele different from the MHC class I protein. In some embodiments, the antibody portion cross-reacts with a complex comprising a variant of HPV16-E7 peptide comprising one amino acid substitution (eg, a conservative amino acid substitution) and an MHC class I protein.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. The HPV16-E7 peptide is from about 8 to about 12 (eg, any of about 8, 9, 10, 11 or 12) amino acids in length. In some embodiments, the HPV16-E7 peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-14. In some embodiments, the HPV16-E7 peptide has the amino acid sequence YMLDLQPET (SEQ ID NO: 4).

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. The antibody portion is a full length antibody, Fab, Fab ′, (Fab ′) 2, Fv or single chain Fv (scFv). In some embodiments, the antibody portion is fully human, semi-synthetic with a human antibody framework region, or humanized.

In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. Wherein the antibody moiety is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM Bind to the HPV16-E7 / MHC class I complex with an equilibrium dissociation constant (K _d ) of 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM). In some embodiments, the isolated anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, It binds to the HPV16-E7 / MHC class I complex with a _Kd of 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM).

  In some embodiments, the anti-E7MC construct comprises an antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, the antibody portion comprising i) GF / G / Y A heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of S / TFS / TSYA / G (SEQ ID NO: 183), or up to about 3 (eg, about Any of 1, 2 or 3) variants thereof comprising amino acid substitutions, I / N / I-X-X-G-G / T / IT-A / P or I-S-X-S HC-CDR2 comprising the amino acid sequence of / DG / NG / SNT / I / K (SEQ ID NO: 184 or 185), or up to about 3 (eg, any of about 1, 2 or 3) Or) variants thereof containing amino acid substitutions, and ARS / RY 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- HC-CDR3 comprising any one amino acid sequence of YQ / WWSXDD (SEQ ID NO: 186-188), or up to about 3 (eg, about 1, 2 or 3 Any) a heavy chain variable domain comprising a variant thereof containing amino acid substitutions; and ii) NIGGSN / K or LRS / NXY (SEQ ID NO: 189 or 190) A light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and A / Q / N -S / A / VW / Y / RDS / DSSL / S / GXXXXV (SEQ ID NO: 91) or a light chain variable domain comprising a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, wherein X is It can be any amino acid.

  In some embodiments, the anti-E7MC construct comprises an antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, the antibody portion comprising i) any of SEQ ID NOs: 57-77. HC-CDR1 comprising one amino acid sequence (or consisting thereof) or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2 comprising (consisting of in some embodiments) any one of the amino acid sequences of SEQ ID NOs: 78-98, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Its variants, including amino acid substitutions, and the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245 (in some embodiments, HC-CDR3, or a heavy chain variable domain comprising a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and ii) SEQ ID NOs 120-140 LC-CDR1 comprising (and in some embodiments consisting of) any one amino acid sequence, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions LC-CDR2 comprising (consisting in some embodiments) an amino acid sequence of any one of SEQ ID NOs: 141-161, or up to about 3 (eg, any of about 1, 2, or 3) ) Including variants thereof including amino acid substitutions, and amino acid sequences of any one of SEQ ID NOs: 162-182 and 247-250 (some embodiments) In consists) LC-CDR3 or at most about 5, (e.g., a light chain variable domain comprising a variant thereof comprising substitution of one) amino acids from about 1, 2, 3, 4 or 5.

  In some embodiments, the anti-E7MC construct comprises an antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, the antibody portion comprising i) any of SEQ ID NOs: 57-77. HC-CDR1 comprising any one amino acid sequence, HC-CDR2 comprising (in some embodiments consisting of) any one amino acid sequence of SEQ ID NOs: 78-98, and SEQ ID NOs: 99-119, 244 and 245 A heavy chain variable domain comprising (in some embodiments consisting of) HC-CDR3 comprising any one amino acid sequence; or up to about 5 in the HC-CDR region; (Eg, any of about 1, 2, 3, 4 or 5) variants thereof comprising amino acid substitutions; and ii) SEQ ID NOs 120- LC-CDR1 comprising any one amino acid sequence of 40 and 246 (consisting in some embodiments), comprising any one amino acid sequence of SEQ ID NOs: 141-161 (consisting in some embodiments) A light chain variable domain comprising LC-CDR2, and LC-CDR3 comprising (consisting of in some embodiments) any one of the amino acid sequences of SEQ ID NOs: 162-182 and 247-250; or in the LC-CDR region It includes variants thereof containing up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC construct comprises an antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, the antibody portion comprising a) SEQ ID NOs: 15-35 and 233. A heavy chain variable domain comprising any one of the amino acid sequences of ~ 237 or consisting of any one of SEQ ID NOs: 15-35 and 233-237 (e.g., Including variants thereof having at least about 95%, 96%, 97%, 98% or 99% sequence identity; and b) any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243 A light chain variable domain (consisting of in some embodiments) or any one of SEQ ID NOs: 36-56 and 238-243 At least about 95% (e.g., at least about 95%, 96%, 97%, 98% or 99% or) a variant thereof having a sequence identity. In some embodiments, the antibody portion comprises a heavy chain variable domain (consisting in some embodiments) of any one of SEQ ID NOs: 15-35 and 233-237 and SEQ ID NOs: 36-56 and A light chain variable domain comprising (consisting of in some embodiments) an amino acid sequence of any one of 238-243.

  In some embodiments, the anti-E7MC construct comprises a first antibody portion that competes with a second antibody portion according to any of the above antibody portions for binding to a target HPV16-E7 / MHC class I complex. In some embodiments, the first antibody portion binds to the same or substantially the same epitope as the second antibody portion. In some embodiments, binding of the first antibody moiety to the target HPV16-E7 / MHC class I complex comprises at least binding of the second antibody moiety to the target HPV16-E7 / MHC class I complex. Inhibits about 70% (eg, at least about 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa. In some embodiments, the first antibody portion and the second antibody portion cross-compete for binding to the target HPV16-E7 / MHC class I complex, ie, each of the first and second antibody portions. Competes with the other for binding to the target HPV16-E7 / MHC class I complex.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC construct), the isolated anti-E7MC construct is a full-length antibody. In some embodiments, the isolated anti-E7MC construct is monospecific. In some embodiments, the isolated anti-E7MC construct is multispecific. In some embodiments, the isolated anti-E7MC construct is bispecific. In some embodiments, the isolated anti-E7MC molecule is a tandem scFv, diabody (Db), single chain diabody (scDb), dual-affinity retargeting ( DART) antibody, double variable domain (DVD) antibody, knob-into-hole (KiH) antibody, dock and lock (DNL) antibody, chemically cross-linked antibody, heteromultimeric antibody Or a heteroconjugate antibody. In some embodiments, the isolated anti-E7MC construct is a tandem scFv comprising two scFvs linked by a peptide linker. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. The isolated anti-E7MC construct further comprises a second antigen-binding moiety that specifically binds to a second antigen. In some embodiments, the second antigen binding portion is an antibody portion. In some embodiments, the second antigen is an antigen on the surface of a T cell. In some embodiments, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, and natural killer T cells. In some embodiments, the second antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM. In some embodiments, the second antigen is CD3ε and the isolated anti-E7MC construct is an N-terminal scFv specific for HPV16-E7 / MHC class I complex and a C-terminal scFv specific for CD3ε. Is a tandem scFv. In some embodiments, the second antigen is an antigen on the surface of natural killer cells, neutrophils, monocytes, macrophages or dendritic cells.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. This isolated anti-E7MC construct comprising an antibody moiety that binds to is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor comprises an extracellular domain comprising an antibody portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling sequence and a costimulatory signaling sequence. In some embodiments, the costimulatory signaling sequence is a CD28 intracellular signaling sequence.

  In some embodiments, according to any of the anti-E7MC constructs (eg, isolated anti-E7MC constructs), the anti-E7MC construct is specific for a complex comprising an HPV16-E7 peptide and an MHC class I protein. The isolated anti-E7MC construct is an immunoconjugate comprising an antibody moiety and an effector molecule. In some embodiments, the effector molecule is a therapeutic agent selected from the group consisting of drugs, toxins, radioisotopes, proteins, peptides and nucleic acids. In some embodiments, the therapeutic agent is a drug or toxin. In some embodiments, the effector molecule is a label.

  In still other embodiments, pharmaceutical compositions comprising an anti-E7MC construct (eg, an isolated anti-E7MC construct) according to any of the above embodiments are provided. In some embodiments, the pharmaceutical composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, host cells that express or are associated with an anti-E7MC construct or polypeptide component thereof are provided. In some embodiments, a nucleic acid encoding an anti-E7MC construct or a polypeptide component thereof is provided. In some embodiments, a vector comprising the nucleic acid is provided. In some embodiments, effector cells that express or are associated with an anti-E7MC construct are provided. In some embodiments, the effector cell is a T cell.

  In some embodiments, a method of detecting a cell presenting on its surface a complex comprising an HPV16-E7 peptide and an MHC class I protein comprising the steps of: a) HPV16-E7 peptide and MHC class I protein Contacting an anti-E7MC construct (eg, an isolated anti-E7MC construct) according to any of the above embodiments comprising an antibody moiety that specifically binds to a complex comprising: b) a label, and a label on the cell A method is provided that includes detecting the presence of.

  In some embodiments, a method of treating an individual having an HPV16-E7 positive disease, wherein the individual is treated with an effective amount of an anti-E7MC construct according to any of the above embodiments (eg, an isolated anti-E7MC construct). A method comprising administering a pharmaceutical composition comprising In some embodiments, the pharmaceutical composition further comprises cells (eg, effector cells) associated with the isolated anti-E7MC construct. In some embodiments, a method of treating an individual having an HPV16-E7 positive disease, comprising administering to the individual an effective amount of an effector cell expressing any of the anti-E7MC CARs. Provided. In some embodiments, the effector cell is a T cell. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer or oropharyngeal cancer. In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma (SCC).

  In some embodiments, a method of diagnosing an individual having an HPV16-E7 positive disease, wherein a) an isolated amount of an isolated anti-E7MC construct comprising a label according to any of the above embodiments is administered to the individual. And b) determining the level of labeling in the individual, wherein a level of labeling above the threshold level indicates that the individual has an HPV16-E7 positive disease. In some embodiments, a method of diagnosing an individual having an HPV16-E7 positive disease, wherein a) a sample from the individual is isolated with an isolated anti-E7MC construct comprising a label according to any of the above embodiments And b) determining the number of cells bound to the isolated anti-E7MC construct in the sample, the number of cells bound to the isolated anti-E7MC construct above a threshold level. A value of is provided that includes the step of indicating that the individual has an HPV16-E7 positive disease. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer or oropharyngeal cancer. In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma (SCC).

  Also provided are methods of making any of the constructs described herein, articles of manufacture and kits suitable for the methods described herein.

FIG. 1 shows a size exclusion chromatography (SEC) chromatogram of the HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex after concentration by ultrafiltration. Properly folded peptide / MHC complex monomer: 212 mL; misfolded aggregate: 111 mL; free β2M: 267 mL.

FIG. 2 shows the results of a phage clone ELISA for specific binding of biotinylated HPV16-E7 11-19 peptide / HLA-A ^* 02: 01 vs. biotinylated C3 control peptide / HLA-A ^* 02 ^: 01 FIG.

FIG. 3 shows the results of a phage clone FACS binding assay for binding of T2 cells loaded with HPV16-E7 11-19 peptide versus T2 cells loaded with C3 control peptide. 1: Negative control of cells only; 2: Control of 2 ° antibody only; 3: HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 specific antibody phage clone.

FIG. 4 shows the results of phage clone number 11 FACS binding assay for T2 cells loaded with HPV16-E7 11-19 peptide having a single alanine substitution at position 1, 5 or 8. 1: Negative control with cells only; 2: Control with secondary antibodies only; 3: HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 specific antibody phage clone number 11.

FIG. 5 shows the results of a phage clone FACS binding assay for binding of T2 cells loaded with HPV16-E7 11-19 peptide versus T2 cells loaded with a peptide derived from a normally expressed endogenous protein. For each phage clone, the loaded peptides are HPV16-E7 11-19, A2E1, A2E2, A2E3, A2E4, A2E5, A2E6, A2E7, A2E8, A2E9, A2E11 and A2E17 from left to right.

FIG. 6 shows SDS-PAGE analysis to determine the purity of anti-HPV16-E7 11-19 / MHC bispecific antibody.

FIG. 7 shows HPV16-E7 mediated by 1 μg / ml and 0.2 μg / ml anti-HPV16-E7 11-19 / HLA-A ^* 02 ^: 01 bispecific antibody prepared from various phage clones. It is a figure which shows the killing by the T cell of T2 cell which loaded 11-19 peptide. Negative controls include T2 cells loaded with AFP158 peptide and a bispecific antibody specific for AFP158 / HLA-A ^* 02 ^: 01. For each phage clone, the loaded peptides were from left to right 1 μg / ml HPV16-E7 11-19, 1 μg / ml AFP158, 0.2 μg / ml HPV16-E7 11-19 and 0.2 μg / ml AFP158. Negative control peptide: AFP158 peptide; negative control antibody: anti-AFP158 / HLA-A ^* 02: 01 bispecific antibody.

FIG. 8A shows T cell killing of two cancer cell lines (CaSki and MS-751) mediated by anti-HPV16-E7 11-19 / MHC bispecific antibody (BsAb).

FIG. 8B shows T cell killing of two cancer cell lines (CaSki and MS-751) mediated by various anti-HPV16-E7 11-19 / MHC bispecific antibody (BsAb) concentrations of BsAb. It is a figure which shows the dose dependence of.

FIG. 9 shows a schematic of the chimeric antigen receptor construct.

FIGS. 10A and 10B show the results of a BsAb FACS binding assay for T2 cells loaded with a WT sequence or HPV16-E7 11-19 peptide with a single alanine substitution at positions 1-9. FIG. 10A shows the results for BsAbs based on clones US-7, 7-1, 7-3 and 7-6. FIG. 10B shows the results for BsAbs based on clones 7-7 and 7-8. FIGS. 10A and 10B show the results of a BsAb FACS binding assay for T2 cells loaded with a WT sequence or HPV16-E7 11-19 peptide with a single alanine substitution at positions 1-9. FIG. 10A shows the results for BsAbs based on clones US-7, 7-1, 7-3 and 7-6. FIG. 10B shows the results for BsAbs based on clones 7-7 and 7-8.

FIGS. 11A-11C show flow cytometric analysis of T cells transduced with various CARs with anti-HPV16-E7 affinity matured scFv or parental scFv; cells were treated with HPV16-E7 11-19 peptide / Stained with HLA-A ^* 02 ^: 01 tetramer and co-stained with anti-CD4 and anti-CD8 antibodies. FIG. 11A shows US-7 4-1BB, 7-1 4-1BB, 7-3 4-1BB, 7-6 4-1BB, 7-7 4-1BB and 7-8 4-1BB CAR-T cells. Flow cytometric analysis of is shown. FIG. 11B shows flow cytometric analysis for 7-9 4-1BB, US-7 CD28, 7-1 CD28, 7-3 CD28, 7-6 CD28 and 7-7 CD28 CAR-T cells. FIG. 11C shows flow cytometric analysis for 7-8 CD28 and 7-9 CD28 CAR-T cells, as well as mock-transduced T cells. FIGS. 11A-11C show flow cytometric analysis of T cells transduced with various CARs with anti-HPV16-E7 affinity matured scFv or parental scFv; cells were treated with HPV16-E7 11-19 peptide / Stained with HLA-A ^* 02 ^: 01 tetramer and co-stained with anti-CD4 and anti-CD8 antibodies. FIG. 11A shows US-7 4-1BB, 7-1 4-1BB, 7-3 4-1BB, 7-6 4-1BB, 7-7 4-1BB and 7-8 4-1BB CAR-T cells. Flow cytometric analysis of is shown. FIG. 11B shows flow cytometric analysis for 7-9 4-1BB, US-7 CD28, 7-1 CD28, 7-3 CD28, 7-6 CD28 and 7-7 CD28 CAR-T cells. FIG. 11C shows flow cytometric analysis for 7-8 CD28 and 7-9 CD28 CAR-T cells, as well as mock-transduced T cells. FIGS. 11A-11C show flow cytometric analysis of T cells transduced with various CARs with anti-HPV16-E7 affinity matured scFv or parental scFv; cells were treated with HPV16-E7 11-19 peptide / Stained with HLA-A ^* 02 ^: 01 tetramer and co-stained with anti-CD4 and anti-CD8 antibodies. FIG. 11A shows US-7 4-1BB, 7-1 4-1BB, 7-3 4-1BB, 7-6 4-1BB, 7-7 4-1BB and 7-8 4-1BB CAR-T cells. Flow cytometric analysis of is shown. FIG. 11B shows flow cytometric analysis for 7-9 4-1BB, US-7 CD28, 7-1 CD28, 7-3 CD28, 7-6 CD28 and 7-7 CD28 CAR-T cells. FIG. 11C shows flow cytometric analysis for 7-8 CD28 and 7-9 CD28 CAR-T cells, as well as mock-transduced T cells.

FIG. 12 shows a cancer cell line positive for HLA-A ^* 02 ^: 01 mediated by T cells expressing anti-HPV16-E7 / HLA-A ^* 02 ^: 01 CAR with affinity matured scFv or parental scFv , And killing of either cancer cell lines positive or negative for HPV16-E7. Mock-transduced cells were included as a control.

The present application relates to an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein (referred to herein as an “HPV16-E7 / MHC class I complex” or “E7MC”). Provided is an isolated construct (referred to herein as an “anti-E7MC construct”) comprising an “anti-E7MC antibody portion”). The anti-E7MC construct specifically recognizes an HPV16-E7 / MHC class I complex, eg, an MHC-presenting HPV16-E7 peptide, on the surface of a cell expressing HPV16-E7. The anti-E7MC construct can specifically bind to the N-terminal part, C-terminal part or central part of the HPV16-E7 peptide in the complex and / or comprises HPV16-E7 peptide and different subtypes of MHC class I proteins Can cross-react with at least one complex (eg, the anti-E7MC construct is HPV16-E7 peptide / HLA-A ^* 02:01 complex and HPV16-E7 peptide / HLA-A ^* Binds both to the 02:02 complex). Anti-E7MC constructs allow specific targeting of E7MC-presenting cells (ie, cells presenting HPV16-E7 peptides bound to MHC molecules on their surface), eg, disease cells expressing HPV16-E7 To do. This strategy offers significant technical advantages over the use of antibodies against HPV16-E7 protein that cannot specifically target E7MC presenting cells. Furthermore, the anti-E7MC antibody portion, when fused to a detectable moiety, is more sensitive to changes in the number and distribution of E7MC presenting cells, a measure of disease progression that is potentially more relevant than circulating HPV16-E7 levels. Allows diagnosis and prognosis of HPV16-E7 positive disease or disorder.

Using phage display technology, we generated multiple monoclonal antibodies that were specific and high affinity for the HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex. Flow cytometry and T cell mediated cytotoxicity assays demonstrated that the antibody recognizes HPV16-E7 peptide pulsed T2 cells in an HPV16-E7 and HLA-A ^* 02 ^: 01 restricted manner. . When the antibody was enhanced as an anti-CD3 bispecific antibody, it redirected human T cells to kill HPV16-E7 positive and HLA-A ^* 02 ^: 01 positive target cells. The data presented herein demonstrates that antibodies against HPV16-E7 peptides in the context of HLA complexes can be effective therapeutic agents for cancer indications such as solid tumor indications.

  Accordingly, the application provides a construct (eg, an isolated construct) that includes an antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein. The construct can be, for example, a full-length anti-E7MC antibody, a multispecific anti-E7MC molecule (eg, a bispecific anti-E7MC antibody), an anti-E7MC chimeric antigen receptor (“CAR”) or an anti-E7MC immunoconjugate.

  In another aspect, nucleic acids encoding anti-E7MC constructs or portions of the anti-E7MC antibody portion of these constructs are provided.

  In another aspect, a composition comprising an anti-E7MC construct comprising an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein is provided. The composition can be a pharmaceutical composition comprising an anti-E7MC construct or an effector cell that expresses or is associated with an anti-E7MC construct (eg, a T cell that expresses an anti-E7MC CAR).

  Also provided are methods of making and using anti-E7MC constructs (or cells expressing or associated with anti-E7MC constructs) for treatment or diagnostic purposes, and kits and articles of manufacture useful for such methods.

Definitions As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms resulting from a disease, the extent of the disease Weakening, stabilizing the disease (eg preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (eg metastasis), preventing or delaying the recurrence of the disease, Delay or slow down progression, alleviate disease state, provide disease remission (partial or total), dose of one or more other drug therapies needed to treat the disease Reducing disease, delaying disease progression, increasing or improving quality of life, increasing weight gain, and / or extending survival Be. Reduction of cancer pathological consequences (eg, tumor volume, etc.) is also encompassed by “treatment”. The methods of the invention contemplate any one or more of these aspects of treatment.

  The terms “recurrence”, “relapse” or “relapse” refer to the return of cancer or disease after clinical assessment of disease disappearance. Diagnosis of distant metastasis or local recurrence can be considered relapse.

  The term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment.

  “Activation” as used herein in reference to T cells refers to a state of a T cell that is sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function.

  The term “antibody portion” includes full length antibodies and antigen binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally comprise three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2 and LC-CDR3). , Heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2 and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein can be defined or identified by Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989). Year; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are sandwiched between adjacent stretches known as framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops. The heavy and light chain constant regions are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, respectively, characterized by the presence of α, δ, ε, γ and μ heavy chains. Some major antibody classes are IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain). And so on.

  As used herein, the term “antigen-binding fragment” includes, for example, diabody, Fab, Fab ′, F (ab ′) 2, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2, two From a portion of an antibody comprising a bispecific dsFv (dsFv-dsFv ′), a disulfide stabilized diabody (ds diabody), a single chain antibody molecule (scFv), an scFv dimer (bivalent diabody), one or more CDRs Multispecific antibody formed, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds antigen but does not contain complete antibody structure An antibody fragment comprising The antigen-binding fragment can bind to the same antigen to which the parent antibody or parent antibody fragment (eg, parent scFv) binds. In some embodiments, the antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

  The term “epitope” as used herein refers to a particular group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions can bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

  As used herein, the first antibody moiety has at least about 50% (eg, at least about 55%) target E7MC binding of the second antibody moiety in the presence of an equimolar concentration of the first antibody moiety. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) if any, the first antibody moiety is directed against the target E7MC. “Compete” with the second antibody moiety for binding, or vice versa. A high-throughput process for “binning” antibodies based on their cross-competition is described in PCT Publication No. WO 03/48731.

  As used herein, the term “specifically binds” or “specific for” determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. , Refers to measurable and reproducible interactions, such as binding between a target and an antibody or antibody portion. For example, an antibody or antibody portion that specifically binds to a target (which may be an epitope) has a higher affinity, avidity, easier and / or longer persistence than its binding to other targets The antibody or antibody portion that binds this target in time. In some embodiments, an antibody or antibody portion that specifically binds an antigen has an antigen (eg, HPV16-E7 peptide / MHC) with a binding affinity that is at least about 10 times its binding affinity for other targets. Reacts with one or more antigenic determinants of a class I protein complex).

  As used herein, an “isolated” anti-E7MC construct is (1) not associated with a protein found in nature, (2) free of other proteins from the same source, (3) different species Refers to an anti-E7MC construct that is expressed by the cell from which it is derived, or (4) is not naturally occurring.

  As used herein, the term “isolated nucleic acid” refers to an “isolated nucleic acid”, depending on its origin, (1) all or part of a polynucleotide in which the “isolated nucleic acid” is found in nature. (2) a nucleic acid of genomic, cDNA or synthetic origin or some combination thereof that is (2) operably linked to a non-naturally relevant polynucleotide, or (3) does not naturally occur as part of a larger sequence. Intended to mean.

As used herein, the term “CDR” or “complementarity determining region” refers to a non-contiguous antigen combining site found in the variable region of both heavy and light chain polypeptides. ) Is meant to mean. These particular regions are described in Kabat et al. Biol. Chem. 252: 6609-6616 (1977); Kabat et al. S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Biol. Mol. Biol. 196: 901-917 (1987); and MacCallum et al., J. MoI. Mol. Biol. 262: 732-745 (1996), where the definition includes duplication or subset of amino acid residues when compared against each other. Nevertheless, the application of either definition to refer to an antibody or grafted antibody CDR or variant thereof is intended to be within the scope of the terms defined and used herein. The amino acid residues encompassing the CDRs defined by each of the references cited above are shown in Table 1 below for comparison.

  The term “chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is from a particular species or belongs to a particular antibody class or subclass as long as they exhibit the biological activity of the invention. Same or homologous to the corresponding sequence, but the remainder of the chain (s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass and fragments of such an antibody (See US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

  With respect to an antibody or antibody portion, the term “semi-synthetic” means that the antibody or antibody portion has one or more naturally occurring sequences and one or more non-naturally occurring (ie, synthetic) sequences. .

  “Fv” is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from heavy and light chains) that contribute to amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for the antigen) has the ability to recognize and bind to the antigen, albeit with a lower affinity than the entire binding site. .

“Single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment comprising V _H and V _L antibody domains linked to a single polypeptide chain. In some embodiments, scFv polypeptides, the desired structure for antigen binding so that scFv can be formed, further comprises a polypeptide linker between the V _H and V _L domains. For reviews of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, 269-315 (1994).

The term “diabody” refers to a V _H domain and a V V domain such that interchain pairing is achieved rather than intrachain pairing of the V domain, resulting in a bivalent fragment, ie, a fragment having two antigen binding sites. Refers to small antibody fragments prepared by constructing scFv fragments (see previous paragraph) that typically have a short linker (eg, about 5 to about 10 residues) between the _L domain. A bispecific diabody is a heterodimer of two “crossover” scFv fragments in which the V _H and _VL domains of the two antibodies reside on different polypeptide chains. Diabodies are described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), which is more fully described.

“Humanized” forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are non-residues such as mice, rats, rabbits or non-human primates whose residues from the recipient's hypervariable region (HVR) have the desired antibody specificity, affinity and ability. Human immunoglobulin (recipient antibody) replaced by residues from the hypervariable region of a human species (donor antibody). In some examples, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody comprises substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin. However, all or substantially all of the FR is of human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin Fc. For further details, see 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).

  With respect to polypeptide and antibody sequences identified herein, “percent (%) amino acid sequence identity” or “homology” refers to a sequence that considers any conservative substitutions as part of the sequence identity. After alignment, it is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptides being compared. Alignments aimed at determining percent amino acid sequence identity can be performed using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. It can be achieved in various ways within the technical scope. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research Volume 32 (No. 5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5 (1): 113, 2004).

  The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcRs of the present invention are those that bind IgG antibodies (γ receptors), including allelic variants and alternatively spliced forms of these receptors, FcγRI, FcγRII and FcγRIII Contains subclass receptors. FcγRII receptors include FcγRIIA (“activated receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed M. in Daeron, Annu. Rev. Immunol. 15: 203-2: 203-2. (See 1997)). The term includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcR is described 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. Biol. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term “FcR” herein. This term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976)) and Kim et al., J. Immunol. 24: 249. (1994)).

  The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2-microglobulin. Multiple functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18 pages 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and in the regulation of serum IgG levels. FcRn can act as a salvage receptor that binds pinocytosed IgG, transports it in an intact form both intracellularly and across the cell, and rescues them from the default degradation pathway.

  The “CH1 domain” (also referred to as “C1” of the “H1” domain) of a human IgG Fc region usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

  The “hinge region” is generally defined as a stretch of human IgG1 from Glu216 to Pro230 (Burton, Molec. Immunol. 22: 161-206 (1985)). The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form an interheavy chain SS bond at the same position.

  The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of the “H2” domain) typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it does not pair closely with another domain. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains of an intact native IgG molecule. It has been speculated that carbohydrates may provide an alternative to domain-domain pairing and may help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985).

  The “CH3 domain” (also referred to as the “H3” domain (or) “C2”) is a stretch of residues C-terminal to the CH2 domain in the Fc region (ie, from about amino acid residue 341 of the antibody sequence). , Typically up to the C-terminus at amino acid residue 446 or 447 of IgG).

  A “functional Fc fragment” has an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, , B cell receptor; downregulation of BCR) and the like. Such effector functions generally require an Fc region to combine with a binding domain (eg, an antibody variable domain) and can be assessed using various assays known in the art.

An antibody having a variant IgG Fc with “altered” FcR binding affinity or ADCC activity is either enhanced or attenuated compared to a parent polypeptide or a polypeptide comprising a native sequence Fc region. An antibody having FcR binding activity (eg, FcγR or FcRn) and / or ADCC activity. A variant Fc that “increases binding” to an FcR binds at least one FcR with a higher affinity (eg, a lower apparent K _d or IC ₅₀ value) than the parent polypeptide or native sequence IgG Fc. . According to some embodiments, the improvement in binding compared to the parent polypeptide is about 3-fold, eg, about 5, 10, 25, 50, 60, 100, 150, 200 or up to 500-fold in binding. Or an improvement of about 25% to 1000%. A polypeptide variant that “shows reduced binding” to FcR binds at least one FcR with a lower affinity (eg, higher apparent K _d or higher IC ₅₀ value) than the parent polypeptide. The reduction in binding compared to the parent polypeptide can be about 40% or more reduction in binding.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to Fc receptors (FcR) present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils and macrophages. Of the cytotoxic effector cells that specifically bind to antigen-bearing target cells and subsequently allow the target cells to be killed using a cytotoxin. Refers to the form. The antibody “arms” the cytotoxic cells and is absolutely required for such killing. NK cells, the main cell for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), summarized in Table 3 on page 464. In vitro ADCC assays such as those described in US Pat. Nos. 5,500,362 or 5,821,337 can be performed to assess ADCC activity of the molecule of interest. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest can be assessed in vivo in animal models such as those disclosed, for example, in Clynes et al. PNAS (USA) 95: 652-656 (1998).

Variant Fc regions that exhibit “increased ADCC” or mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells more effectively than a polypeptide having a wild-type IgG Fc or a parent polypeptide A polypeptide comprising a variant Fc region in the assay and in vitro or in if the amount of polypeptide (or parent polypeptide) having a wild-type Fc region is essentially the same.
A polypeptide that is substantially more effective in mediating ADCC in vivo. In general, such variants are identified using any in vitro ADCC assay known in the art, such as in an animal model, for example, an assay or method for determining ADCC activity. In some embodiments, the variant is about 5-fold to about 100-fold, eg, about 25-about 50-fold more effective in mediating ADCC than the wild-type Fc (or parent polypeptide).

  “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigens. To assess complement activation, see, for example, Gazzano-Santoro et al. Immunol. Methods 202: 163 (1996) can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in US Pat. No. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are specifically incorporated herein by reference. Idusogie et al. Immunol. 164: 4178-4184 (2000).

  Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, as long as the nucleotide sequence encoding the protein can include intron (s) in some versions.

  The term “operably linked” refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the latter expression. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in functional association with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In general, operably linked DNA sequences are contiguous and, if necessary, join two protein coding regions in the same reading frame.

  “Homology” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, these molecules are Homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by 100 divided by the number of positions compared. For example, if 6 out of 10 positions in two sequences are identical or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. In general, a comparison is made when two sequences are aligned to give maximum homology.

  An “effective amount” of an anti-E7MC construct or composition disclosed herein is an amount sufficient to achieve a specifically stated purpose. An “effective amount” can be determined experimentally and by known methods relating to the stated purpose.

  The term “therapeutically effective amount” refers to an amount of an anti-E7MC construct or composition disclosed herein that is effective to “treat” a disease or disorder in an individual. In the case of cancer, a therapeutically effective amount of an anti-E7MC construct or composition disclosed herein can reduce the number of cancer cells; it can reduce the size or weight of the tumor; Can inhibit tumor cell invasion (ie slow down to some extent, preferably stop); inhibit tumor metastasis (ie slow down to some extent, preferably stop); inhibit tumor growth to some extent; And / or one or more of the symptoms associated with cancer may be reduced to some extent. The anti-E7MC constructs or compositions disclosed herein can be cytostatic and / or cytotoxic as long as they can prevent growth and / or kill existing cancer cells. In some embodiments, the therapeutically effective amount is a growth inhibiting amount. In some embodiments, a therapeutically effective amount is an amount that prolongs patient survival. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of the patient.

  As used herein, “pharmaceutically acceptable” or “pharmacologically compatible” means a material that is not biologically or otherwise undesirable, for example, a material Is a pharmaceutical composition that is administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Can be incorporated into. A pharmaceutically acceptable carrier or excipient is preferably included on the Inactive Ingredient Guide that meets the required standards for toxicological and manufacturing testing and / or created by the US Food and Drug Administration.

  The term “label” as used herein refers to a detectable compound or composition that can be conjugated directly or indirectly to an anti-E7MC antibody moiety. The label can be detectable by itself (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical alteration of the detectable substrate compound or composition.

  It is understood that the embodiments of the invention described herein include embodiment “consisting of” and / or embodiment “consisting essentially of”.

  Reference to “about” a value or parameter herein includes (and describes) variations to that value or parameter itself. For example, description referring to “about X” includes description of “X”.

  As used herein, reference to a value or parameter “not” generally means and describes the value or parameter “other than”. For example, a method not being used to treat a type X cancer means that the method is used to treat a type of cancer other than X.

  As used herein and in the appended claims, the singular forms “a, a,” or “or,” and “the, the, the,” the. Includes multiple referents unless the context clearly specifies otherwise.

Anti-E7MC Constructs In one aspect, the invention provides an antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein (“HPV16-E7 / MHC class I complex” or “E7MC”). An HPV16-E7 / MHC class I complex specific construct (anti-E7MC construct) is provided. The specificity of the anti-E7MC construct is derived from the anti-E7MC antibody portion that specifically binds to E7MC, eg, a full-length antibody or antigen-binding fragment thereof. In some embodiments, reference to a moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein (eg, an antibody moiety) includes: a) full-length HPV16-E7, free HPV16-E7 peptide, At least about 10 of its binding affinity for each of MHC class I protein not bound to peptide and MHC class I protein bound to non-HPV16-E7 peptide (eg, at least about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more of affinity); or b) full length HPV16-E7, free HPV16-E7 peptide, not bound to peptide For MHC class I proteins and non-HPV16-E7 peptides Combined about 1/10 or less of its _{K d} for the binding to each of the MHC class I protein (e.g., about 1 / 10,1 / 20,1 / 30,1 / 40,1 / 50,1 / 75 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less), with a K _d that the portion binds to E7MC means. Binding affinity can be determined by methods known in the art, for example, ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation assay (RIA). K _d can be determined by methods known in the art, such as a surface plasmon resonance (SPR) assay utilizing a Biacore instrument or a kinetic exclusion assay (KinExA) utilizing, for example, a Sapidyne instrument. .

  Contemplated anti-E7MC constructs include, for example, full-length anti-E7MC antibodies, multispecific (eg, bispecific) anti-E7MC molecules, anti-E7MC chimeric antigen receptor (CAR) and anti-E7MC immunoconjugates.

For example, in some embodiments, an anti-E7MC construct (eg, an isolated anti-E7MC construct) comprising an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein is provided. Is done. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02: 01 (GenBank accession number: AAO20853). In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100pM, 500pM, 1nM, 10nM, 50nM, with a _{K d} of any) of 100nM or 500 nM, couples E7MC. In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, or 2) variant of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

In some embodiments, an anti-E7MC construct comprising an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01 is provided. . In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100pM, 500pM, 1nM, 10nM, 50nM, with a _{K d} of any) of 100nM or 500 nM, couples E7MC. In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, or 2) variant of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

In some embodiments, an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or a maximum And variants thereof containing substitutions of about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1, 2, Or any of 3) variants thereof comprising amino acid substitutions, and HC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOS: 186-188; or up to about 3 (eg, any of about 1, 2, or 3) A) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) an sequence of SEQ ID NO: 189 or 190 LC-CDR1 comprising a non-acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to An anti-E7MC construct comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising a substitution of about 3 (eg, any of about 1, 2 or 3) amino acids is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100pM, 500pM, 1nM, 10nM, 50nM, with a _{K d} of any) of 100nM or 500 nM, couples E7MC. In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, or 2) variant of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

  In some embodiments, an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising i) an amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 (consisting of in some embodiments); or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; SEQ ID NOs: 78-98 HC-CDR2 comprising (consisting of in some embodiments) any one amino acid sequence; or that comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids A HC-CDR3 comprising (consisting of in some embodiments) the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245; Or a heavy chain variable domain sequence comprising a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and ii) any of SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising (in some embodiments) comprising one amino acid sequence; or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids LC-CDR2 comprising (consisting of in some embodiments) any one amino acid sequence of SEQ ID NOs: 141-161; or substitution of up to about 3 (eg, about 1, 2 or 3) amino acids And any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250 (consisting of it in some embodiments) C-CDR3; or an anti-E7MC comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence comprising a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids A construct is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, E7MC is bound with a Kd of 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM). In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

  In some embodiments, an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising i) an amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 (consisting of in some embodiments); HC-CDR2 (consisting of in some embodiments) comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NOs: 99-119, 244 And a heavy chain variable domain sequence comprising HC-CDR3 comprising (in some embodiments consisting of) any one amino acid sequence; or HC-CDR sequences up to about 5 (eg, about 1, 2 Any of 3, 4, or 5) variants thereof containing amino acid substitutions; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence (consisting of it in some embodiments); LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161 (consisting in some embodiments); And a light chain variable domain sequence comprising LC-CDR3 comprising (consisting of in some embodiments) comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or at most about in the LC-CDR sequence An anti-E7MC construct comprising an anti-E7MC antibody portion comprising a variant thereof comprising substitution of 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, E7MC is bound with a Kd of 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM). In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

  In some embodiments, an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 Or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98% or 99%) sequence identity A light chain variable domain comprising, or consisting of, in some embodiments) any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about 96%, 97% , 98% or 99%) of the anti-E7MC antibody portion comprising a variant thereof with sequence identity No anti E7MC construct. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the anti-E7MC construct is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM, E7MC is bound with a Kd of 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM). In some embodiments, the anti-E7MC construct comprises at least one (eg, at least 2, 3, Cross-react with any of 4, 5, or 6) complexes. In some embodiments, the anti-E7MC construct crosses at least one (eg, at least 2, 3, 4 or 5) complex comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. react.

  In some embodiments, a first competing with a second anti-E7MC antibody portion according to any of the anti-E7MC antibody portions described herein for binding to a target HPV16-E7 / MHC class I complex. An anti-E7MC construct comprising an anti-E7MC antibody portion is provided. In some embodiments, the first anti-E7MC antibody portion binds to the same or substantially the same epitope as the second anti-E7MC antibody portion. In some embodiments, the binding of the first anti-E7MC antibody moiety to the target HPV16-E7 / MHC class I complex comprises the second anti-E7MC antibody moiety to the target HPV16-E7 / MHC class I complex. Inhibition of binding is at least about 70% (eg, at least about 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa. In some embodiments, the first anti-E7MC antibody portion and the second anti-E7MC antibody portion cross-compete for binding to the target HPV16-E7 / MHC class I complex, ie, the first and second Each of the antibody moieties competes with the other for binding to the target HPV16-E7 / MHC class I complex.

  For example, in some embodiments, for binding to a target HPV16-E7 / MHC class I complex, i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or up to about 3 (eg, about 1, 2 Or any of 3) variants thereof comprising amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids Including variants thereof, and HC-CDR3 comprising any one amino acid sequence of SEQ ID NO: 186-188; or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids A heavy chain variable domain sequence; and ii) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or And variants thereof comprising substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or up to about 3 (eg, about 1, 2, or 3) An anti-E7MC construct comprising an anti-E7MC antibody portion that competes with an antibody portion comprising a light chain variable domain comprising a variant thereof comprising amino acid substitutions is provided.

  In some embodiments, for binding to the target HPV16-E7 / MHC class I complex, i) an HC- comprising (in some embodiments consisting of) an amino acid sequence of any one of SEQ ID NOs: 57-77. CDR1; or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; comprising any one amino acid sequence of SEQ ID NOs: 78-98 (some HC-CDR2 comprising: in embodiments); or variants thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions; and SEQ ID NOs: 99-119, 244 and 245 HC-CDR3 comprising (in some embodiments) any one of the amino acid sequences; or up to about 5 (eg, about 1, 2, 3, 4 or 5 Any) a heavy chain variable domain sequence comprising a variant thereof comprising amino acid substitutions; and ii) an LC-CDR1 comprising (in some embodiments consisting of) an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 Or variants thereof containing substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; comprising any one amino acid sequence of SEQ ID NOs: 141-161 (some implementations) LC-CDR2 comprising thereof in the form; or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and any one of SEQ ID NOs: 162-182 and 247-250 LC-CDR3 comprising (consisting of in some embodiments) an amino acid sequence; or up to about 5 (eg, about 1, , Anti E7MC constructs comprising anti E7MC antibody portion that competes with the antibody portion that comprises a light chain variable domain sequence comprising the variant comprises substitution of one) amino acids of the 3, 4 or 5 is provided.

  In some embodiments, for binding to the target HPV16-E7 / MHC class I complex, i) an HC- comprising (in some embodiments consisting of) an amino acid sequence of any one of SEQ ID NOs: 57-77. CDR1; HC-CDR2 comprising (consisting of in some embodiments) any one amino acid sequence of SEQ ID NOs: 78-98; and any one amino acid sequence of SEQ ID NOs: 99-119, 244, and 245 ( A heavy chain variable domain sequence comprising HC-CDR3 (in some embodiments); or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; Variants thereof comprising substitutions; and ii) comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 (in some embodiments) LC-CDR1 comprising: an amino acid sequence of any one of SEQ ID NOs: 141-161 (consisting in some embodiments); and any one of SEQ ID NOs: 162-182 and 247-250 A light chain variable domain sequence comprising LC-CDR3 comprising (consisting of in some embodiments) one amino acid sequence; or up to about 5 (eg, about 1, 2, 3, 4 or 5 in the LC-CDR sequence) Any) An anti-E7MC construct is provided that comprises an anti-E7MC antibody portion that competes with an antibody portion comprising that variant containing amino acid substitutions.

  In some embodiments, the binding to a target HPV16-E7 / MHC class I complex comprises the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 (in some embodiments, consists thereof). A heavy chain variable domain, or variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and SEQ ID NOs: 36-56 and 238-243, respectively. A light chain variable domain comprising any one amino acid sequence (consisting of it in some embodiments), or at least about 95% (eg, at least about 96%, 97%, 98% or 99% of any) sequence Provided is an anti-E7MC construct comprising an anti-E7MC antibody portion that competes with an antibody portion comprising that variant having identity.

  Different aspects are discussed in further detail in the various sections below.

Anti-E7MC antibody portion The anti-E7MC construct comprises an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein.

  In some embodiments, the anti-E7MC antibody portion specifically binds to E7MC present on the surface of the cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is in a solid tumor. In some embodiments, the cancer cell is a metastatic cancer cell.

  In some embodiments, the HPV16-E7 peptide is an MHC class I restricted peptide. In some embodiments, the HPV16-E7 peptide is about 8 to about 12 (eg, any of about 8, 9, 10, 11 or 12) amino acids in length.

  In some embodiments, the HPV16-E7 peptide comprises amino acids 7-15 of HPV16-E7 (TLHEYMLDL, SEQ ID NO: 3), amino acids 11-19 of HPV16-E7 (YMLDLQPET, SEQ ID NO: 4, herein referred to as “HPV16 -E7 11-19 "), amino acids 16-25 of HPV16-E7 (QPETTDLYCY, SEQ ID NO: 5), amino acids 44-52 of HPV16-E7 (QAEPDRAHY, SEQ ID NO: 6), amino acids 46-55 of HPV16-E7 (EPDRAHYNIV, SEQ ID NO: 7), amino acids 49-57 of HPV16-E7 (RAHYNIVTF, SEQ ID NO: 8), amino acids 82-90 of HPV16-E7 (LLMGTLGIV, SEQ ID NO: 9), or amino acids 86-93 of HPV16-E7 ( TLGIVCPI It comprises the sequence of SEQ ID NO: 10) (made of it in some embodiments).

In some embodiments, the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, HLA-A is HLA-A02. In some embodiments, HLA-A02 is HLA-A ^* 02 ^: 01.

  In some embodiments, the anti-E7MC antibody portion is a full length antibody. In some embodiments, the anti-E7MC antibody portion comprises antigen binding fragments, such as Fab, Fab ′, F (ab ′) 2, Fv fragments, disulfide stabilized Fv fragments (dsFv) and single chain antibody molecules (scFv). An antigen-binding fragment selected from the group consisting of In some embodiments, the anti-E7MC antibody portion is an scFv. In some embodiments, the anti-E7MC antibody portion is human, humanized or semi-synthetic.

  In some embodiments, the anti-E7MC antibody portion specifically binds to the N-terminal portion of the HPV16-E7 peptide in the complex. In some embodiments, the anti-E7MC antibody portion specifically binds to the C-terminal portion of the HPV16-E7 peptide in the complex. In some embodiments, the anti-E7MC antibody portion specifically binds to the central portion of the HPV16-E7 peptide in the complex.

In some embodiments, the anti-E7MC antibody portion (or an anti-E7MC construct comprising an anti-E7MC antibody portion) comprises full length HPV16-E7, free HPV16-E7 peptide, MHC class I protein not bound to peptide and non-HPV16- At least about 10 of its binding affinity for each of the MHC class I proteins bound to the E7 peptide (eg, at least about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 It binds to complexes containing HPV16-E7 peptides and MHC class I proteins with a double affinity (including any). In some embodiments, the anti-E7MC antibody portion (or an anti-E7MC construct comprising an anti-E7MC antibody portion) comprises full length HPV16-E7, free HPV16-E7 peptide, MHC class I protein not bound to peptide and non-HPV16- About 1/10 or less of its K _d for binding to each of the MHC class I proteins bound to the E7 peptide (eg, about 1/10, 1/20, 1/30, 1/40, 1/50, HPV16-E7 peptide and MHC with a _Kd of 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) Binds to complexes containing class I proteins.

In some embodiments, the anti-E7MC antibody portion (or an anti-E7MC construct comprising an anti-E7MC antibody portion) is between about 0.1 pM and about 500 nM (eg, including any range between these values; Binds to complexes comprising HPV16-E7 peptides and MHC class I proteins with a _Kd of about 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM) To do. In some embodiments, the anti-E7MC antibody portion (or an anti-E7MC construct comprising an anti-E7MC antibody portion) is between about 1 pM and about 250 pM (eg, including any range between these values, about 1 , with a _{K d} of any) of 10,25,50,75,100,150,200 or 250 pM, it binds to a complex containing HPV16-E7 peptides and MHC class I proteins. In some embodiments, the anti-E7MC antibody portion (or an anti-E7MC construct comprising an anti-E7MC antibody portion) is between about 1 nM and about 500 nM (eg, including any range between these values, about 1 Binds to complexes comprising HPV16-E7 peptides and MHC class I proteins with a _Kd of 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nM) To do.

  In some embodiments, the anti-E7MC antibody portion specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, the anti-E7MC antibody portion comprising HPV16-E7 peptide and MHC class I protein. Cross-reacts with at least one complex containing an allelic variant. In some embodiments, the allelic variant has a maximum of about 10 (eg, any of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 when compared to MHC class I protein). Or) have amino acid substitutions. In some embodiments, the allelic variant is the same serotype as the MHC class I protein. In some embodiments, the allelic variant is a serotype that is different from the MHC class I protein. In some embodiments, the anti-E7MC antibody portion does not cross-react with any complex comprising HPV16-E7 peptides and allelic variants of MHC class I proteins. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

  In some embodiments, the anti-E7MC antibody portion specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, the anti-E7MC antibody portion comprising an MHC class I protein and one amino acid substitution ( For example, it cross-reacts with at least one complex comprising a variant of the HPV16-E7 peptide having conservative amino acid substitutions). In some embodiments, the anti-E7MC antibody portion does not cross-react with any complex comprising MHC class I protein and HPV16-E7 peptide variants.

For example, in some embodiments, the anti-E7MC antibody portion comprises HPV16-E7 11-19 (SEQ ID NO: 4) and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01). It binds specifically to the complex. In some embodiments, the anti-E7MC antibody portion comprises a complex comprising an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 12 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01). A complex comprising an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 13 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); and an alanine-substituted of SEQ ID NO: 14 Further binding to at least one of the complexes comprising HPV16-E7 peptide and MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01), including at least about 2 or 3 To do.

In some embodiments, the anti-E7MC antibody portion comprises a complex comprising the HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); A complex comprising 12 alanine-substituted HPV16-E7 peptides and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); and an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 13 And a complex comprising a MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01).

In some embodiments, the anti-E7MC antibody portion comprises a complex comprising the HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); A complex comprising 12 alanine-substituted HPV16-E7 peptides and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); and an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 14 And a complex comprising a MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01).

In some embodiments, the anti-E7MC antibody portion comprises a complex comprising the HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); It specifically binds to a complex comprising an alanine-substituted HPV16-E7 peptide of number 12 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01).

In some embodiments, the anti-E7MC antibody portion comprises a complex comprising the HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01); It specifically binds to a complex comprising an alanine substituted HPV16-E7 peptide of number 14 and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01).

In some embodiments, the anti-E7MC antibody portion specifically binds to a complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01. In some embodiments, the anti-E7MC antibody portion comprises a complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A ^* 02: 02 (GenBank accession number: AFL91480), HPV16-E7 11-19. (SEQ ID NO: 4) and HLA-A ^* 02: 03 (GenBank accession number: AAA03604), HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A ^* 02: 05 (GenBank accession number: AAA03603) ), HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A ^* 02: 06 (GenBank accession number: CCB78868), HPV16-E7 11-19 (SEQ ID NO: 4) and HLA -A ^* 02:07 (GenBank accession number: ACR55712 Complex comprising, and HPV16-E7 11-19 (SEQ ID NO: 4) and ^HLA-A * 02:11 (GenBank accession number: CAB56609) at least one complex comprising (at least about 2, 3, 4, Cross-react with 5 or 6).

In some embodiments, the anti-E7MC antibody portion comprises a complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01). And a complex comprising an HPV16-E7 11-19 variant having an amino acid sequence of YMLVQPPET (SEQ ID NO: 11) and an MHC class I protein (eg, HLA-A02, eg, HLA-A ^* 02 ^: 01) To join.

  In some embodiments, the anti-E7MC antibody portion is a semi-synthetic antibody portion comprising a fully human sequence and one or more synthetic regions. In some embodiments, the anti-E7MC antibody portion comprises a fully human light chain variable domain, and fully human FR1, HC-CDR1, FR2, HC-CDR2, FR3 and FR4 regions and a synthetic HC-CDR3. A semi-synthetic antibody portion comprising a semi-synthetic heavy chain variable domain. In some embodiments, the semi-synthetic heavy chain variable domain is about 5 to about 25 (eg, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Any of 18, 19, 20, 21, 22, 23, 24 or 25) comprising a fully synthetic HC-CDR3 having an amino acid long sequence. In some embodiments, the semi-synthetic heavy chain variable domain or synthetic HC-CDR3 is about 5 to about 25 (eg, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) a semi-synthetic library comprising a fully synthetic HC-CDR3 having a sequence of amino acid length (eg, semi-synthetic human live Each amino acid in this sequence is randomly selected from the standard human amino acids minus cysteine. In some embodiments, the synthetic HC-CDR3 is about 7 to about 15 (eg, any of about 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acids in length.

  The anti-E7MC antibody portion comprises, in some embodiments, a specific sequence or certain variants of such sequence. In some embodiments, the amino acid substitution in the variant sequence does not substantially reduce the ability of the anti-E7MC antibody portion to bind to E7MC. For example, changes can be made that do not substantially reduce E7MC binding affinity. Changes that substantially improve E7MC binding affinity or affect certain other properties such as specificity and / or cross-reactivity with related variants of E7MC are also contemplated.

  In some embodiments, the anti-E7MC antibody portion is i) HC-CDR3 comprising an amino acid sequence of any one of SEQ ID NOS: 186-188, or up to about 3 (eg, any of about 1, 2, or 3). A) a heavy chain variable domain comprising a variant thereof comprising amino acid substitutions; and ii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or up to about 3 (eg, any of about 1, 2, or 3) amino acids It includes a light chain variable domain containing that variant containing substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises i) a heavy chain variable domain comprising HC-CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 186-188; and ii) an amino acid sequence of SEQ ID NO: 191 It contains a light chain variable domain comprising LC-CDR3.

  In some embodiments, the anti-E7MC antibody portion comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or a substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids. A variant thereof, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and of SEQ ID NOs: 186-188 A heavy chain variable domain comprising HC-CDR3 comprising any one amino acid sequence, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and ii) SEQ ID NO: 189 Or LC-CDR1 comprising 190 amino acid sequences, or up to about 3 (eg, any of about 1, 2, or 3) amino acids A variant thereof comprising an acid substitution, and a light chain comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids Contains variable domains.

  In some embodiments, the anti-E7MC antibody portion comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or a substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids. A variant thereof, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and of SEQ ID NOs: 186-188 A heavy chain variable domain comprising HC-CDR3 comprising any one amino acid sequence; and ii) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or up to about 3 (eg, about 1, 2 or 3 Any) including variants thereof containing amino acid substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 It comprises a light chain variable domain.

  In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, HC-CDR2, comprising the amino acid sequence of SEQ ID NO: 184 or 185, and any of SEQ ID NOs: 186-188 A heavy chain variable domain comprising HC-CDR3 comprising one amino acid sequence; or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids in the HC-CDR sequence; and ii ) A light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191; or up to about 3 (eg, about 1 in the LC-CDR sequence) 2 or 3) including variants thereof containing amino acid substitutions.

In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, HC-CDR2, comprising the amino acid sequence of SEQ ID NO: 184 or 185, and any of SEQ ID NOs: 186-188 A heavy chain variable domain comprising HC-CDR3 comprising one amino acid sequence; and ii) a light chain variable comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 Includes domain. The sequences of the CDRs referred to herein are provided in Table 2 below.

  In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245, or up to about 5 (eg, about 1, 2, Either 3, 4 or 5) a heavy chain variable domain comprising a variant thereof comprising amino acid substitutions; and ii) an LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs 162-182 and 247-250, or a maximum A light chain variable domain comprising a variant thereof comprising substitution of about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids.

  In some embodiments, the anti-E7MC antibody portion comprises i) a heavy chain variable domain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244, and 245; and ii) SEQ ID NOs: 162- A light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250.

  In some embodiments, the anti-E7MC antibody portion is i) HC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 57-77, or up to about 5 (eg, about 1, 2, 3, 4, or 5) variants thereof containing amino acid substitutions, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98, or up to about 5 (eg, about 1, 2, 3, 4 or 5) Any) HC-CDR3 comprising a variant thereof comprising amino acid substitutions and an amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245, or up to about 5 (eg, about 1, 2, 3, 4 Or any of 5) a heavy chain variable domain comprising a variant thereof comprising amino acid substitutions; and ii) any one amino acid of SEQ ID NOs: 120-140 and 246 An amino acid sequence of any one of SEQ ID NOs: 141-161, comprising LC-CDR1 comprising a column, or variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids Including LC-CDR2, or a variant thereof containing substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250 It includes a light chain variable domain comprising LC-CDR3, or variants thereof containing up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion is i) HC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 57-77, or up to about 5 (eg, about 1, 2, 3, 4, or 5) variants thereof containing amino acid substitutions, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98, or up to about 5 (eg, about 1, 2, 3, 4 or 5) Any) a heavy chain variable domain comprising a variant thereof comprising amino acid substitutions, and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one of the amino acid sequences, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids Variants thereof comprising a substitution, LC-CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 141-161, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, And a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250.

  In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98. And a heavy chain variable domain sequence comprising HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245; or up to about 5 (eg, about 1, 2, Any of 3, 4, or 5) variants thereof comprising amino acid substitutions; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; any one of SEQ ID NOs: 141-161 LC-CDR2 containing the amino acid sequence; and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250 A light chain variable domain sequence comprising LC-CDR3 comprising: or a variant thereof comprising substitution of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the LC-CDR sequence .

  In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98. And a heavy chain variable domain sequence comprising HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245; or up to about 5 (eg, about 1, 2, 3, 4 or 5 Any) a variant thereof comprising amino acid substitutions, wherein these amino acid substitutions are in the HC-CDR1 or HC-CDR2, heavy chain variable domain sequences or variants thereof; and ii) SEQ ID NOs 120-140 and 246 LC-CDR1 containing any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 A light chain variable domain sequence comprising LC-CDR2 comprising a column; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs 162-182 and 247-250; or at most about 5 (eg, about 1, 2, Any of 3, 4 or 5) variants thereof comprising amino acid substitutions, wherein these amino acid substitutions comprise a light chain variable domain sequence or variant thereof in HC-CDR1 or HC-CDR2.

In some embodiments, the anti-E7MC antibody portion comprises i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98 And a heavy chain variable domain sequence comprising HC-CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245; and ii) comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 LC-CDR1; a light chain variable domain comprising LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250 Contains an array. The sequences of HC-CDRs referred to herein are provided in Table 3 below, and the sequences of LC-CDRs referred to herein are provided in Table 4 below.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97 A light chain variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243, or at least about 95 % Thereof (eg, including at least 96%, 97%, 98% or 99%) sequence identity thereof.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and any one of SEQ ID NOs: 36-56 and 238-243. Contains a light chain variable domain comprising an amino acid sequence.

  The heavy and light chain variable domains can be combined in various pairwise combinations to produce several anti-E7MC antibody portions.

  For example, in some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. Variants thereof comprising the substitution of: HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and A heavy chain variable domain comprising an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR1 comprising the amino acid sequence of number 120, or a barrier thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 141, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC comprising the amino acid sequence of SEQ ID NO: 162 -Comprises a light chain variable domain comprising CDR3, or variants thereof containing up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 120 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 120, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 162 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions; and SEQ ID NO: A heavy chain variable domain comprising a HC-CDR3 comprising 100 amino acid sequences, or variants thereof containing substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 121 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 142, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 163 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 100. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 121 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 142, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 163, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 100. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 121, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 142, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 163 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: HC-CDR3 comprising 101 amino acid sequences, or a heavy chain variable domain comprising a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 122 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 143, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 164 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 101. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 122 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 164, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 101. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 122, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 164 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 81, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising a HC-CDR3 comprising 102 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 123 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 144, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 165 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 123 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 144, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 165, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 123, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 144, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 165 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 103 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 124 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 145, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 166 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 103. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 124 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 145, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 166, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 103. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 145, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 166. Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 104 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 125 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 146, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 167 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 125 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 167, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 125, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 167 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, or variants thereof containing substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 105 amino acid sequences, or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 126 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 147, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 168 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 126 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 147, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 168, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 126; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 168. Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 106 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 127 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 148, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 169 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 106. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 127 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 148, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises: A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 127; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 148; Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 65, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 86, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising a HC-CDR3 comprising 107 amino acid sequences, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 128 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 149, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 170 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 65, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 128 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 170, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 65, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107. A heavy chain variable domain; and a light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 170 .

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 87, or variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 108 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 129 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 150, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 171 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 87, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 108. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 129 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 150, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 171, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 87, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 108. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 129, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 150, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 171 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 67, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, or variants thereof containing substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 109 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 130 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 151, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 172 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 67, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 109. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 130 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 151, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 172, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 67, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 109 A light chain variable domain comprising a heavy chain variable domain comprising: Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 89, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 110 amino acid sequences, or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 131 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 152, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 173 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 110. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 131 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 152, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 173, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 110. A light chain variable domain comprising: a heavy chain variable domain comprising; and LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 131, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 152, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 173 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 90, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising a HC-CDR3 comprising 111 amino acid sequences, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 132 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 153, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 174 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 111. A heavy chain variable domain comprising, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 132 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 174, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 111. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 132, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 174 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 70, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, or variants thereof containing substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 112 amino acid sequences, or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 133 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 154, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 175 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 70, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 112. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 133 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 175, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 70, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 112. A light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 133; an LC-CDR2 comprising an amino acid sequence of SEQ ID NO: 154; and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 175; Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 92, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: An HC-CDR3 comprising 113 amino acid sequences, or a heavy chain variable domain comprising a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 134 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 155, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 176 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 113. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 134 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 155, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 176, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises: A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 134; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 155; Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 114 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 135 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 156, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 177 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 114. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 135 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 156, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 177, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 114. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 135, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 156, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 177 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 73, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, or variants thereof containing substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 115 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 136 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 157, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 178 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 73, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 115. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 136 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 157, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 178, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 73, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 115. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 136; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 157; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 178. Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 95, or variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 116 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 137 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 158, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 179 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 116. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 137 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 158, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 179, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 74, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 116. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 137, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 158, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 179. Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 75, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 117 amino acid sequences, or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 138 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 159, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 180 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 75, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 117. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 138 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 159, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 180, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 75, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 117. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 138, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 159, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 180 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. A HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 97, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 118 amino acid sequences, or variants thereof comprising substitutions of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 139 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column no. 160, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 181 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 118. A heavy chain variable domain, or variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 139 LC-CDR1, a light chain variable domain comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 181 or up to about 5 in the LC-CDR sequence (eg, about 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises: A light chain variable domain comprising a heavy chain variable domain comprising: Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain, or variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; as well as the amino acid sequence of SEQ ID NO: 140 LC-CDR1, a light chain variable domain comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182, or up to about 5 in the LC-CDR sequence (eg, about 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 244 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 244. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 244. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 245 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 245. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 245. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 247 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 247, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 247 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 248 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 248, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 248 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 246 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain comprising, or a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 246 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 182, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119 A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 246; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161; Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 249 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 249, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A light chain variable domain comprising a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 249 Including.

  In some embodiments, the anti-E7MC antibody portion comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, or a substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids. HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: A heavy chain variable domain comprising HC-CDR3 comprising 119 amino acid sequences, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 140 LC-CDR1 comprising the amino acid sequence of or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; LC-CDR2 comprising the amino acid sequence of column number 161, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 250 Or a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A heavy chain variable domain comprising, or a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions in the HC-CDR sequence; and the amino acid sequence of SEQ ID NO: 140 A light chain variable domain comprising LC-CDR1, comprising LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 250, or up to about 5 in the LC-CDR sequence (eg, About 1, 2, 3, 4 or 5) including variants thereof containing amino acid substitutions.

  In some embodiments, the anti-E7MC antibody portion comprises HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 77, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119. A light chain variable domain comprising: a heavy chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 250 Including.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 36.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 38, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 38.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 18, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 39, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 39.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 19, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 40, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 40.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 20, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 41, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 20 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 41.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 21, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 42, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 42.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 22, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 43, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 43.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 23, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having any sequence identity, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 44, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Including variants thereof having sequence identity. In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 44.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having a sequence identity of (including any of In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 25, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 46, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 46.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 47, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 47.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 27, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 48, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 48.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 49, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 49.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 50, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 50.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 30, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 51, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having a sequence identity of (including any of In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 51.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 52, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 52.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 53, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 53.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 33, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 54, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 54.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 55, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having a sequence identity of (including any of In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 55.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 233, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having a sequence identity of (including any of In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 233 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 234, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 234 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 235, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 235 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 238, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 238.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 239, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 239.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 240, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 240.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 241, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having a sequence identity of (including any of In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 241.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 242, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 242.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 236, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% A variant thereof having a sequence identity of and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 243, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 236 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 243.

  In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 237, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Variants thereof having a sequence identity of and any light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56, or at least about 95% (eg, at least about 96%, 97%, 98% or 99) And variants thereof having sequence identity). In some embodiments, the anti-E7MC antibody portion comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 237 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.

  In some embodiments, the anti-E7MC antibody portion is a second anti-E7MC antibody portion according to any of the anti-E7MC antibody portions described herein for binding to the target HPV16-E7 / MHC class I complex. Conflict with. In some embodiments, the anti-E7MC antibody portion binds to the same or substantially the same epitope as the second anti-E7MC antibody portion. In some embodiments, binding of the anti-E7MC antibody moiety to the target HPV16-E7 / MHC class I complex comprises binding of the second anti-E7MC antibody moiety to the target HPV16-E7 / MHC class I complex, Inhibits at least about 70% (eg, at least about 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa. In some embodiments, the anti-E7MC antibody portion and the second anti-E7MC antibody portion cross-compete for binding to the target HPV16-E7 / MHC class I complex, ie, each of these antibody portions is a target Competes with the other for binding to HPV16-E7 / MHC class I complexes.

  For example, in some embodiments, the anti-E7MC antibody moiety is i) for binding to a target HPV16-E7 / MHC class I complex, i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or up to about 3 ( For example, a variant thereof comprising an amino acid substitution, either about 1, 2 or 3), an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, any of about 1, 2 or 3) Or) an HC-CDR3 comprising a variant thereof comprising amino acid substitutions, and an amino acid sequence of any one of SEQ ID NOS: 186-188; or substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids A heavy chain variable domain sequence comprising a variant thereof comprising: ii) an L comprising the amino acid sequence of SEQ ID NO: 189 or 190 -CDR1, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to about 3 (eg, Compete with an antibody portion comprising a light chain variable domain comprising a variant thereof containing amino acid substitutions (either approximately 1, 2 or 3).

  In some embodiments, the anti-E7MC antibody portion comprises i) an amino acid sequence of any one of SEQ ID NOs: 57-77 for binding to a target HPV16-E7 / MHC class I complex (some embodiments). HC-CDR1 comprising: or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; any one amino acid sequence of SEQ ID NOs: 78-98 HC-CDR2 comprising (consisting of in some embodiments); or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; and SEQ ID NO: 99 HC-CDR3 comprising (consisting of in some embodiments) any one of the amino acid sequences of ˜119, 244, and 245; or up to about 5 (eg, about Any of 2, 3, 4 or 5) a heavy chain variable domain sequence comprising a variant thereof comprising amino acid substitutions; and ii) comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 (some LC-CDR1 comprising: in embodiments); or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids; any one of SEQ ID NOs: 141-161 LC-CDR2 comprising (consisting of in some embodiments) an amino acid sequence; or variants thereof containing up to about 3 (eg, any of about 1, 2, or 3) amino acid substitutions; and SEQ ID NOs 162-182 And LC-CDR3 (consisting of in some embodiments) comprising any one amino acid sequence of and 247-250; About 5 (e.g., about any of 1, 2, 3, 4 or 5) that competes with antibody portion comprising a light chain variable domain sequence comprising the variant comprising a substitution of an amino acid.

  In some embodiments, the anti-E7MC antibody portion comprises i) an amino acid sequence of any one of SEQ ID NOs: 57-77 for binding to a target HPV16-E7 / MHC class I complex (some embodiments). HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 78-98 (consisting of in some embodiments); and any one of SEQ ID NOs: 99-119, 244 and 245 A heavy chain variable domain sequence comprising HC-CDR3 comprising (consisting of in some embodiments) one amino acid sequence; or up to about 5 (eg, about 1, 2, 3, 4 or 5 in the HC-CDR sequence) Any of) variants thereof comprising amino acid substitutions; and ii) comprising any one of the amino acid sequences of SEQ ID NOs: 120-140 and 246 LC-CDR1 (consisting of some embodiments); LC-CDR2 (consisting of in some embodiments) comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NOs: 162-182 and 247 A light chain variable domain sequence comprising LC-CDR3 comprising (consisting of in some embodiments) comprising any one amino acid sequence of ˜250; or up to about 5 (eg, about 1, 2 in the LC-CDR sequence) Compete with the antibody portion containing the variant, including amino acid substitutions (either 3, 4, or 5).

  In some embodiments, the anti-E7MC antibody portion comprises an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 for binding to a target HPV16-E7 / MHC class I complex (some Or a variant thereof having a sequence identity of at least about 95% (eg, at least about 96%, 97%, 98% or 99%, respectively), and SEQ ID NOs: 36- A light chain variable domain comprising, or consisting of, in some embodiments, any one of amino acids 56 and 238-243, or at least about 95% (eg, at least about 96%, 97%, 98% or 99% Compete with the antibody portion comprising that variant with sequence identity of

Full length anti-E7MC antibody The anti-E7MC construct is, in some embodiments, a full length antibody comprising an anti-E7MC antibody portion (also referred to herein as a “full length anti-E7MC antibody”). In some embodiments, the full length antibody is a monoclonal antibody.

  In some embodiments, the full-length anti-E7MC antibody comprises Fc sequences from immunoglobulins such as IgA, IgD, IgE, IgG and IgM. In some embodiments, the full length anti-E7MC antibody comprises an Fc sequence of IgG, such as any of IgG1, IgG2, IgG3 or IgG4. In some embodiments, the full length anti-E7MC antibody comprises a human immunoglobulin Fc sequence. In some embodiments, the full-length anti-E7MC antibody comprises a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-E7MC antibody has been altered or otherwise modified to have enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function. In which the Fc sequence is altered.

Thus, for example, in some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a full-length anti-E7MC antibody comprising an Fc region is provided. Is done. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) an Fc region A full length anti-E7MC antibody comprising is provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence. In some embodiments, the anti-E7MC antibody portion comprises at least one (eg, at least 2, 3 or more) variants of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with the complex of either 4, 5, or 6. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1 HC-CDR3 comprising any of its variants, including amino acid substitutions, and any one amino acid sequence of SEQ ID NOS: 186-188; or up to about 3 (eg, about 1, 2, or 3) Any) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) SEQ ID NO: 189 or 190 LC-CDR1 comprising the amino acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to Provided is an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, and b) a full-length anti-E7MC antibody comprising an Fc region. The In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, A heavy chain variable domain sequence comprising HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185 and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 186 to 188; and ii) amino acids of SEQ ID NO: 189 or 190 An anti-E7MC antibody portion comprising an LC-CDR1 comprising a sequence and a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191), and b) a full length anti-E7MC antibody comprising an Fc region are provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 An HC-CDR3 comprising a sequence, or a variant thereof comprising substitutions of up to about 5 (eg any of about 1, 2, 3, 4 or 5) amino acids A heavy chain variable domain; and ii) LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof containing amino acid substitutions, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids LC-CDR3 comprising the variant and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids A full-length anti-E7MC antibody comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof including a substitution is provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 LC-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or up to about 5 in the LC-CDR sequence (eg, about Any of 1, 2, 3, 4 or 5) an anti-E7MC antibody portion comprising a variant thereof containing an amino acid substitution; and b) a full length anti-E7MC antibody comprising an Fc region is provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 Anti E7MC antibody portion containing no light chain variable domain sequences; full length anti E7MC antibody comprising and b) Fc region is provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98% Or a variant thereof having a sequence identity of any one) and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about An anti-E7MC antibody that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein comprising a variant thereof having sequence identity of 96%, 97%, 98% or 99%) And b) a full length anti-E7MC antibody comprising an Fc region. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 An anti-E7MC antibody portion comprising a heavy chain variable domain comprising an amino acid sequence and a light chain variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243; and b) a full-length anti-E7MC antibody comprising an Fc region Provided. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

In some embodiments, the full length anti-E7MC antibody is between about 0.1 pM and about 500 nM (eg, including any range between these values, including about 0.1 pM, 1.0 pM, 10 pM, 50 pM). , 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM) with a K _d of binding to a complex comprising HPV16-E7 peptide and MHC class I protein. In some embodiments, the full length anti-E7MC antibody is between about 1 pM and about 250 pM (eg, including any range between these values, including about 1, 10, 25, 50, 75, 100, 150 , with a _{K d} of 200 or any 250 pM), binds to a complex containing HPV16-E7 peptides and MHC class I proteins.

Multispecific anti-E7MC molecule The anti-E7MC construct comprises, in some embodiments, a multispecific anti-E7MC molecule comprising an anti-E7MC antibody portion and a second binding portion (eg, a second antigen-binding portion). In some embodiments, the multispecific anti-E7MC molecule comprises an anti-E7MC antibody portion and a second antigen binding portion.

  A multispecific molecule is a molecule that has binding specificities for at least two different antigens or epitopes (eg, a bispecific antibody has binding specificities for two antigens or epitopes). Multispecific molecules having more than two valencies and / or specificities are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. Immunol. 147: 60 (1991). It should be understood that one skilled in the art can select appropriate characteristics of the individual multispecific molecules described herein to combine with each other to form the multispecific anti-E7MC molecules of the present invention.

  Thus, for example, in some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a second binding portion (eg, antigen binding) Multispecific (eg, bispecific) anti-E7MC molecules are provided. In some embodiments, the second binding moiety specifically binds to a complex comprising a different HPV16-E7 peptide bound to an MHC class I protein. In some embodiments, the second scFv specifically binds to a complex comprising an HPV16-E7 peptide bound to a different MHC class I protein. In some embodiments, the second binding moiety specifically binds to a different epitope on a complex comprising an MHC class I protein and an HPV16-E7 peptide. In some embodiments, the second binding moiety specifically binds to a different antigen. In some embodiments, the second binding moiety specifically binds to an antigen on the surface of a cell, such as a cytotoxic cell. In some embodiments, the second binding moiety specifically binds to an antigen on the surface of lymphocytes, eg, T cells, NK cells, neutrophils, monocytes, macrophages or dendritic cells. In some embodiments, the second binding moiety specifically binds to an effector T cell, eg, a cytotoxic T cell (also known as a cytotoxic T lymphocyte (CTL) or T killer cell).

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) a second antigen-binding portion that specifically binds CD3. A multispecific anti-E7MC molecule comprising is provided. In some embodiments, the second antigen binding moiety specifically binds to CD3ε. In some embodiments, the second antigen binding portion specifically binds to an agonist epitope of CD3ε. The term “agonist epitope” as used herein (a) upon binding of multispecific molecules, optionally upon binding of several multispecific molecules on the same cell. Consisting of an epitope that activates TCR signaling and induces T cell activation in the multispecific molecule, and / or (b) the amino acid residue of the epsilon chain of CD3, and its natural on T cells Epitopes that are accessible for binding by multispecific molecules when presented in the environment (ie, surrounded by TCR, CD3γ chain, etc.), and / or (c) upon binding of multispecific molecules, It means an epitope that does not result in stabilization of the spatial position of CD3ε as compared to CD3γ.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) for example, CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD16a, Multispecific anti-E7MC molecules comprising a second antigen binding moiety that specifically binds to an antigen on the surface of effector cells, including CD56, CD68 and GDS2D are provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a specific binding to a component of the complement system such as C1q. A multispecific anti-E7MC molecule comprising two antigen binding moieties is provided. C1q is a subunit of the C1 enzyme complex that activates the serum complement system.

  In some embodiments, the second antigen binding moiety specifically binds to an Fc receptor. In some embodiments, the second antigen binding moiety specifically binds to an Fcγ receptor (FcγR). FcγR is one of FcγRIII present on the surface of natural killer (NK) cells or FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2 and FcγRIIIB present on the surface of macrophages, monocytes, neutrophils and / or dendritic cells. Can be one. In some embodiments, the second antigen binding portion is an Fc region or a functional fragment thereof. “Functional fragment” as used in this context means that FcγR-bearing effector cells, in particular macrophages, monocytes, neutrophils and / or dendritic cells, kill target cells by cytotoxic lysis or phagocytosis. Refers to a fragment of an antibody Fc region that is still capable of binding to FcR, in particular FcγR, with sufficient specificity and affinity to allow. A functional Fc fragment can competitively inhibit binding of a portion of the original full-length Fc to an FcR, such as activated FcγRI. In some embodiments, the functional Fc fragment retains at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of its affinity for activated FcγR. In some embodiments, the Fc region or functional fragment thereof is an enhanced Fc region or functional fragment thereof. The term “enhanced Fc region” as used herein refers to Fc receptor-mediated effector functions, particularly antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC). And refers to Fc regions that have been modified to enhance antibody-mediated phagocytosis. This can be achieved, as known in the art, for example, increased affinity and / or inhibitory receptors (eg, FcγRIIIA (CD16A) expressed on natural killer (NK) cells) (eg, , FcγRIIB1 / B2 (CD32B)) can be achieved by altering the Fc region in a way that results in decreased binding. In yet other embodiments, the second antigen-binding moiety is an FcγR-bearing effector cell, particularly a macrophage, monocyte, neutrophil and / or dendritic cell, that targets the target cell by cytotoxic lysis or phagocytosis. An antibody or antigen-binding fragment thereof that specifically binds to FcR, in particular FcγR, with sufficient specificity and affinity to allow killing.

In some embodiments, the multispecific anti-E7MC molecule may allow killing of E7MC presenting target cells and / or effectively redirect CTL to lyse E7MC presenting target cells. In some embodiments, a multispecific (eg, bispecific) anti-E7MC molecule of the invention exhibits an in vitro EC ₅₀ in the range of 10-500 ng / ml, with about 1: 1 to about 50: 1. About 50% redirected lysis of target cells via CTL at a ratio of CTL to target cells (eg, about 1: 1 to about 15: 1 or about 2: 1 to about 10: 1) lysis) can be induced.

  In some embodiments, the multispecific (eg, bispecific) anti-E7MC molecule crosslinks stimulated or unstimulated CTL and target cells in such a way that the target cells are lysed. Is possible. This provides the advantage that generation of target-specific T cell clones or common antigen presentation by dendritic cells is not required for the multispecific anti-E7MC molecule to exert its desired activity. In some embodiments, the multispecific anti-E7MC molecules of the invention can redirect CTLs to lyse target cells in the absence of other activation signals. In some embodiments, the second antigen-binding portion of the multispecific anti-E7MC molecule specifically binds to CD3 (eg, specifically binds to CD3ε) and is mediated by CD28 and / or IL-2. No signaling is required to redirect the CTL to lyse the target cells.

Methods for measuring the preference of multispecific anti-E7MC molecules that bind simultaneously to two antigens (eg, antigens on two different cells) are within the normal ability of one skilled in the art. For example, if the second binding moiety specifically binds to CD3, the multispecific anti-E7MC molecule can be contacted with a mixture of CD3 ⁺ / HPV16-E7 ⁻ cells and CD3 ⁻ / HPV16-E7 ⁺ cells. The number of multispecific anti-E7MC molecule positive single cells and the number of cells cross-linked by the multispecific anti-E7MC molecule are then determined by microscopy or fluorescence activated cell sorting (FACS), as is known in the art. Can be evaluated by

For example, in some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a multispecific comprising a second antigen binding portion. Anti-E7MC molecules are provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the second antigen binding moiety specifically binds to a complex comprising different HPV16-E7 peptides bound to MHC class I proteins. In some embodiments, the second antigen binding moiety specifically binds to a complex comprising an HPV16-E7 peptide bound to a different MHC class I protein. In some embodiments, the second antigen binding portion specifically binds to a different epitope on a complex comprising an HPV16-E7 peptide and an MHC class I protein. In some embodiments, the second antigen binding moiety specifically binds to another antigen. In some embodiments, the second antigen binding moiety specifically binds an antigen on the surface of a cell, such as an E7MC presenting cell. In some embodiments, the second antigen binding moiety specifically binds to an antigen on the surface of a cell that does not express HPV16-E7. In some embodiments, the second antigen binding moiety specifically binds to an antigen on the surface of a cytotoxic cell. In some embodiments, the second antigen binding moiety specifically binds to an antigen on the surface of a lymphocyte, eg, a T cell, NK cell, neutrophil, monocyte, macrophage or dendritic cell. . In some embodiments, the second antigen-binding moiety specifically binds to an antigen on the surface of an effector T cell, eg, a cytotoxic T cell. In some embodiments, the second antigen binding moiety specifically binds to an antigen on the surface of an effector cell, including, for example, CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD16a, CD56, CD68 and GDS2D. To do. In some embodiments, the anti-E7MC antibody portion is human, humanized or semi-synthetic. In some embodiments, the second antigen binding portion is an antibody portion. In some embodiments, the second antigen binding moiety is a human, humanized or semi-synthetic antibody moiety. In some embodiments, the multispecific anti-E7MC molecule further comprises at least one (eg, at least about any of about 2, 3, 4, 5, or more) additional antigen binding moieties. In some embodiments, the anti-E7MC antibody portion comprises at least one (eg, at least 2, 3 or more) variants of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with the complex of either 4, 5, or 6. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) a second Multispecific anti-E7MC molecules comprising an antigen binding moiety are provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1 HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOS: 186-188, or up to about 3 (eg, about 1, 2 or 3) Any) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) SEQ ID NO: 189 or 190 LC-CDR1 comprising the amino acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to An anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, and b) a multispecific comprising a second antigen binding portion Anti-E7MC molecules are provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, A heavy chain variable domain sequence comprising HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 186 to 188; and ii) amino acids of SEQ ID NO: 189 or 190 An anti-E7MC antibody portion comprising an LC-CDR1 comprising a sequence and a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191; and b) a multispecific anti-E7MC molecule comprising a second antigen binding portion. Is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 An HC-CDR3 comprising a sequence, or a variant thereof comprising substitutions of up to about 5 (eg any of about 1, 2, 3, 4 or 5) amino acids A heavy chain variable domain; and ii) LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof containing amino acid substitutions, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids LC-CDR3 comprising the variant and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids An anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising a substitution; and b) a multispecific comprising a second antigen binding portion E7MC molecule is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 LC-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or up to about 5 (eg, about Any of 1, 2, 3, 4 or 5) an anti-E7MC antibody portion comprising a variant thereof containing an amino acid substitution; and b) a multispecific anti-E7MC molecule comprising a second antigen binding portion is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 Anti E7MC antibody portion containing no light chain variable domain sequence; and b) a multi-specific anti E7MC molecule comprising a second antigen binding portions are provided.

  In some embodiments, a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98% Or a variant thereof having a sequence identity of any one) and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about An anti-E7MC antibody portion comprising a variant thereof having sequence identity of 96%, 97%, 98% or 99%); and b) a multispecific anti-E7MC molecule comprising a second scFv is provided. The

  In some embodiments, a) a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and an amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243 An anti-E7MC antibody portion comprising a light chain variable domain; and b) a multispecific anti-E7MC molecule comprising a second antigen binding portion is provided.

  In some embodiments, the multispecific anti-E7MC molecule is, for example, a diabody (Db), a single chain diabody (scDb), a tandem scDb (Tandab), a linear dimer scDb (LD-scDb), a cyclic dimer scDb. (CD-scDb), di-diabody, tandem scFv, tandem di-scFv (eg, bispecific T cell engager), tandem tri-scFv, avian (a) body (tri (a) body), dual Specific Fab2, di-antibody, tetrabody, scFv-Fc-scFv fusion, dual affinity retargeting (DART) antibody, dual variable domain (DVD) antibody, IgG-scFab , ScFab-ds-scFv, Fv2-Fc, IgG-scFv fusion , Dock and lock (DNL) antibody, knob-into-hole (KiH) antibody (bispecific IgG prepared by KiH technology), DuoBody (bispecific IgG prepared by Duobody technology), heteromultimeric antibody Or a heteroconjugate antibody. In some embodiments, the multispecific anti-E7MC molecule is a tandem scFv (eg, tandem di-scFv, eg, a bispecific T cell engager).

Tandem scFv
A multispecific anti-E7MC molecule, in some embodiments, is a tandem scFv comprising an anti-E7MC antibody portion and a second scFv (also referred to herein as a “tandem scFv multispecific anti-E7MC antibody”). Call). In some embodiments, the tandem scFv multispecific anti-E7MC antibody further comprises at least one (eg, any of at least about 2, 3, 4, 5 or more) additional scFv.

In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a tandem scFv multispecific comprising a second scFv (eg, Bispecific) anti-E7MC antibodies are provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the second scFv specifically binds to a complex comprising a different HPV16-E7 peptide bound to an MHC class I protein. In some embodiments, the second scFv specifically binds to a complex comprising a different HPV16-E7 peptide and MHC class I protein. In some embodiments, the second scFv specifically binds to a different epitope on a complex comprising an HPV16-E7 peptide bound to an MHC class I protein. In some embodiments, the second scFv specifically binds to another antigen. In some embodiments, the second scFv specifically binds to an antigen on the surface of a cell, such as an E7MC presenting cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of a cell that does not express HPV16-E7. In some embodiments, the second scFv specifically binds to an antigen on the surface of the cytotoxic cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of lymphocytes, eg, T cells, NK cells, neutrophils, monocytes, macrophages or dendritic cells. In some embodiments, the second scFv specifically binds to an antigen on the surface of an effector T cell, eg, a cytotoxic T cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of effector cells, including, for example, CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD16a, CD56, CD68 and GDS2D. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic. In some embodiments, the tandem scFv multispecific anti-E7MC antibody further comprises at least one (eg, any of at least about 2, 3, 4, 5 or more) additional scFv. In some embodiments, the anti-E7MC antibody portion comprises at least one (eg, at least 2, 3 or more) variants of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with the complex of either 4, 5, or 6. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

In some embodiments, a) a first scFv that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) a second A tandem scFv multispecific (eg, bispecific) anti-E7MC antibody comprising an scFv is provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1 HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOS: 186-188, or up to about 3 (eg, about 1, 2 or 3) Any) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) a of SEQ ID NO: 189 or 190 LC-CDR1 comprising a non-acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to A first scFv comprising a light chain variable domain comprising a variant thereof comprising a substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, and b) a tandem scFv multispecific comprising a second scFv (Eg, bispecific) anti-E7MC antibodies are provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, A heavy chain variable domain sequence comprising HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185 and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 186 to 188; and ii) amino acids of SEQ ID NO: 189 or 190 A first scFv comprising an LC-CDR1 comprising a sequence, and a light chain variable domain comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191; and b) a tandem scFv comprising a second scFv multispecific (eg, Bispecific) anti-E7MC antibodies are provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) an amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 HC-CDR3 containing sequences, or variants thereof containing substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids A chain variable domain; and ii) LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids Or a variant thereof comprising a substitution of up to about 3 (e.g., about 1, 2 or 3) amino acids, including an amino acid sequence of any one of SEQ ID NOs: 141-161, or a variant thereof And LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids A first scFv comprising a light chain variable domain comprising the variant comprising; and b) a tandem scFv comprising a second scFv (E.g., bispecific) anti E7MC antibodies are provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) an amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one of the amino acid sequences; comprising any one amino acid sequence of SEQ ID NOs: 141 to 161 C-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 162-182 and 247-250; or up to about 5 in the LC-CDR sequence (eg, about 1 Any of 2, 3, 4 or 5) a first scFv comprising a variant thereof containing amino acid substitutions; and b) a tandem scFv comprising a second scFv multispecific (eg, bispecific) anti-E7MC An antibody is provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, i) an amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- Including LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 First scFv containing chain variable domain sequence; and b) a tandem scFv Multispecific includes a second scFv (e.g., bispecific) anti E7MC antibodies are provided.

  In some embodiments, a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98% Or a variant thereof having a sequence identity of any one) and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about A first scFv comprising a variant thereof having a sequence identity of (including 96%, 97%, 98% or 99%); and b) a tandem scFv multispecific comprising a second scFv (eg, two Bispecific) anti-E7MC antibodies are provided.

  In some embodiments, a) a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and an amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243 A tandem scFv multispecific (eg, bispecific) anti-E7MC antibody comprising a first scFv comprising a light chain variable domain; and b) a second scFv is provided.

  In some embodiments, a) a first scFv that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a tandem scFv multispecific comprising a second scFv (eg, A bispecific) anti-E7MC antibody is provided, the tandem scFv multispecific anti-E7MC antibody being tandem di-scFv or tandem tri-scFv. In some embodiments, the tandem scFv multispecific anti-E7MC antibody is tandem di-scFv. In some embodiments, the tandem scFv multispecific anti-E7MC antibody is a bispecific T cell engager.

For example, in some embodiments, a) a first scFv that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) specifically binds an antigen on the surface of a T cell. A tandem-scFv bispecific anti-E7MC antibody comprising a second scFv is provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the second scFv specifically binds to an antigen on the surface of an effector T cell, eg, a cytotoxic T cell. In some embodiments, the second scFv specifically binds to an antigen selected from the group consisting of, for example, CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM. . In some embodiments, the second scFv specifically binds to an agonist epitope on the antigen on the surface of the T cell, and binding of the second scFv to the antigen enhances T cell activation. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

In some embodiments, a) a first scFv that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) a T cell A tandem di-scFv bispecific anti-E7MC antibody comprising a second scFv that specifically binds to an antigen on the surface is provided.

  In some embodiments, a) i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or variants, sequences comprising up to about 3 (eg, any of about 1, 2, or 3) amino acid substitutions HC-CDR2 comprising the amino acid sequence of number 184 or 185, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and any one of SEQ ID NOs: 186-188 An HC-CDR3 comprising an amino acid sequence, or a heavy chain variable domain sequence comprising a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and ii) of SEQ ID NO: 189 or 190 LC-CDR1 containing the amino acid sequence, or containing up to about 3 (eg, any of about 1, 2 or 3) amino acid substitutions. HPV16 comprising a variant and a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids A tandem di-scFv duplex comprising a first scFv that specifically binds to a complex comprising an E7 peptide and an MHC class I protein, and b) a second scFv that specifically binds an antigen on the surface of a T cell. Specific anti-E7MC antibodies are provided.

  In some embodiments, a) i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an amino acid sequence of any one of SEQ ID NOs: 186-188 A heavy chain variable domain sequence comprising HC-CDR3 comprising: and ii) comprising a light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 A tandem di-scFv comprising a first scFv that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) a second scFv that specifically binds an antigen on the surface of a T cell. Bispecific anti-E7MC antibodies are provided.

  In some embodiments, a) i) HC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 57-77, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof comprising amino acid substitutions, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR3 comprising the variant thereof, and the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245, or up to about 5 (eg any of about 1, 2, 3, 4 or 5) Or) a heavy chain variable domain comprising a variant thereof containing amino acid substitutions; and ii) an LC-C comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 R1, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, LC-CDR2, comprising any one amino acid sequence of SEQ ID NOs: 141-161, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising any one of the amino acid sequences of SEQ ID NOS 162-182 and 247-250, or A complex comprising a HPV16-E7 peptide and an MHC class I protein comprising a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions A first scFv that specifically binds to b; and b) a second scFv that specifically binds to an antigen on the surface of the T cell Muzi -scFv bispecific anti E7MC antibodies are provided.

  In some embodiments, a) i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NO: A heavy chain variable domain sequence comprising HC-CDR3 comprising an amino acid sequence of any one of 99-119, 244 and 245; or up to about 5 (eg, about 1, 2, 3, 4 or Any of 5) variants thereof comprising amino acid substitutions; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; comprising any one amino acid sequence of SEQ ID NOs: 141-161 LC-CDR2; and LC-CD comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250 A light chain variable domain sequence comprising 3; or an HPV16-E7 comprising variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the LC-CDR sequence A tandem di-scFv bispecific comprising a first scFv that specifically binds to a complex comprising a peptide and an MHC class I protein, and b) a second scFv that specifically binds an antigen on the surface of a T cell. An anti-E7MC antibody is provided.

  In some embodiments, a) i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NO: A heavy chain variable domain sequence comprising HC-CDR3 comprising any one amino acid sequence of 99-119, 244 and 245; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; A light chain variable domain sequence comprising LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, Specific binding to a complex comprising HPV16-E7 peptide and MHC class I protein The first scFv that; and b) Tandemuji -scFv bispecific anti E7MC antibody comprising a second scFv that specifically bind to an antigen on the surface of T cells.

  In some embodiments, a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98% Or a variant thereof having sequence identity of 99%) and a light chain variable domain comprising any one amino acid sequence of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about A first scFv comprising a variant thereof having a sequence identity of either 96%, 97%, 98% or 99%) and b) a second scFv that specifically binds to an antigen on the surface of a T cell. A tandem-scFv bispecific anti-E7MC antibody comprising is provided.

  In some embodiments, a) a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and an amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243 Provided is a tandem di-scFv bispecific anti-E7MC antibody comprising a first scFv comprising a light chain variable domain and b) a second scFv that specifically binds an antigen on the surface of a T cell.

In some embodiments, a) a tandem comprising a first scFv that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) a second scFv that specifically binds CD3ε. -ScFv bispecific anti-E7MC antibodies are provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

In some embodiments, a) a first scFv that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) specific for CD3ε A tandem di-scFv bispecific anti-E7MC antibody comprising a second scFv that binds selectively is provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or variants thereof comprising substitutions of up to about 3 (eg, any of about 1, 2, or 3) amino acids HC-CDR2 comprising the amino acid sequence of number 184 or 185, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and any one of SEQ ID NOs: 186-188 An HC-CDR3 comprising an amino acid sequence, or a heavy chain variable domain sequence comprising a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and ii) of SEQ ID NO: 189 or 190 LC-CDR1 comprising an amino acid sequence, or that comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids HPV16 comprising a light chain variable domain comprising Liant and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or variants thereof comprising up to about 3 (eg, any of about 1, 2, or 3) amino acid substitutions A tandem di-scFv bispecific anti-E7MC antibody comprising a first scFv that specifically binds to a complex comprising an E7 peptide and an MHC class I protein, and b) a second scFv that specifically binds to CD3ε. Provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an amino acid sequence of any one of SEQ ID NOs: 186-188 A heavy chain variable domain sequence comprising HC-CDR3 comprising: and ii) comprising a light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 A tandem di-scFv bispecific anti-E7MC comprising: a first scFv that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein; and b) a second scFv that specifically binds to CD3ε. An antibody is provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) i) HC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 57-77, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof comprising amino acid substitutions, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR3 comprising a variant thereof, and an amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245, or up to about 5 (e.g., any of about 1, 2, 3, 4 or 5) Or) a heavy chain variable domain comprising a variant thereof comprising amino acid substitutions; and ii) an LC- comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246 DR1, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, Or a variant thereof comprising up to about 3 (eg, any of about 1, 2 or 3) amino acid substitutions, and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or A complex comprising a HPV16-E7 peptide and an MHC class I protein comprising a light chain variable domain comprising a variant thereof comprising up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acid substitutions A tandem di-s comprising a first scFv that specifically binds to b, and b) a second scFv that specifically binds to CD3ε A cFv bispecific anti-E7MC antibody is provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; A heavy chain variable domain sequence comprising HC-CDR3 comprising an amino acid sequence of any one of 99-119, 244 and 245; or up to about 5 (eg, about 1, 2, 3, 4 or Any of 5) variants thereof comprising amino acid substitutions; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; comprising any one amino acid sequence of SEQ ID NOs: 141-161 LC-CDR2; and LC- comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250 A light chain variable domain sequence comprising DR3; or an HPV16-E7 comprising variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids in the LC-CDR sequence Provided is a tandem di-scFv bispecific anti-E7MC antibody comprising a first scFv that specifically binds to a complex comprising a peptide and an MHC class I protein, and b) a second scFv that specifically binds to CD3ε. The In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; A heavy chain variable domain sequence comprising HC-CDR3 comprising any one amino acid sequence of 99-119, 244 and 245; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; A light chain variable domain sequence comprising LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, Specifically for complexes comprising HPV16-E7 peptides and MHC class I proteins First scFv that case, and b) Tandemuji -scFv bispecific anti E7MC antibody comprising a second scFv that specifically bind to CD3ε is provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98% Or a variant thereof having a sequence identity of any one) and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least about Tandem di-scFv containing a first scFv comprising a variant thereof having a sequence identity of either 96%, 97%, 98% or 99%) and b) a second scFv that specifically binds to CD3ε. Bispecific anti-E7MC antibodies are provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

  In some embodiments, a) a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, and an amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, A tandem di-scFv bispecific anti-E7MC antibody comprising a first scFv comprising a light chain variable domain comprising, and b) a second scFv that specifically binds CD3ε is provided. In some embodiments, the first scFv is fused to the second scFv via linkage by a peptide linker. In some embodiments, the peptide linker is between about 5 and about 20 amino acids in length (eg, any range between these values, including any of about 5, 10, 15 or 20). is there. In some embodiments, the peptide linker comprises (consists of in some embodiments) the amino acid sequence GGGGS. In some embodiments, the first scFv is human, humanized or semi-synthetic. In some embodiments, the second scFv is human, humanized or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized or semi-synthetic.

In some embodiments, the tandem-scFv bispecific anti-E7MC antibody is between about 0.1 pM and about 500 nM (eg, including any range between these values, about 0.1 pM, 1 Binds to complexes comprising HPV16-E7 peptides and MHC class I proteins with a K _d of 0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM or 500 nM). In some embodiments, the tandem di-scFv bispecific anti-E7MC antibody is between about 1 nM and about 500 nM (eg, including any range between these values, including about 1, 10, 25, 50 , with a _{K d} of any) of 75,100,150,200,250,300,350,400,450 or 500 nM, it binds to a complex containing HPV16-E7 peptides and MHC class I proteins.

Chimeric antigen receptor (CAR) and CAR effector cells In some embodiments, an anti-E7MC construct is a chimeric antigen receptor (CAR) comprising an anti-E7MC antibody portion (also referred to herein as “anti-E7MC CAR”). is there. Also provided are CAR effector cells (eg, T cells) comprising a CAR comprising an anti-E7MC antibody portion (also referred to herein as “anti-E7MC CAR effector cells”, eg, “anti-E7MC CAR T cells”).

  The anti-E7MC CAR comprises a) an extracellular domain comprising an anti-E7MC antibody moiety that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, and b) an intracellular signaling domain. The transmembrane domain can be between the extracellular domain and the intracellular domain.

  There may be a spacer domain between the extracellular domain of the anti-E7MC CAR and the transmembrane domain, or between the intracellular domain and the transmembrane domain of the anti-E7MC CAR. The spacer domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or intracellular domain in the polypeptide chain. The spacer domain can comprise up to about 300 amino acids, including for example from about 10 to about 100 or from about 25 to about 50 amino acids.

  The transmembrane domain can be from either a natural source or a synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane regions particularly used in the present invention are α, β, δ or γ chains of T cell receptors, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, It can be derived from CD64, CD80, CD86, CD134, CD137 or CD154 (ie, can include at least their transmembrane region (s)). In some embodiments, the transmembrane domain can be synthetic, in which case the transmembrane domain can comprise predominantly hydrophobic residues, such as leucine and valine. In some embodiments, a phenylalanine, tryptophan and valine triad can be found at each end of the synthetic transmembrane domain. In some embodiments, for example, having an amino acid length of between about 2 and about 10 (eg, any of about 2, 3, 4, 5, 6, 7, 8, 9 or 10) A short oligopeptide linker or polypeptide linker can form a link between the transmembrane domain of the anti-E7MC CAR and the intracellular signaling domain. In some embodiments, the linker is a glycine-serine bipartite.

  In some embodiments, a transmembrane domain that naturally binds to one of the sequences in the intracellular domain of the anti-E7MC CAR is used (eg, if the anti-E7MC CAR intracellular domain comprises a CD28 costimulatory sequence, The transmembrane domain of anti-E7MC CAR is derived from the CD28 transmembrane domain). In some embodiments, the transmembrane domain avoids binding of such a domain to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. Can be selected or modified by amino acid substitution.

  The intracellular signaling domain of anti-E7MC CAR is responsible for the activation of at least one of the normal effector functions of immune cells in which anti-E7MC CAR is placed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and directs the cell to perform specialized functions. Usually the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of an intact chain as long as it transmits effector function signals. Thus, the term “intracellular signaling sequence” is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce effector function signals.

  Examples of intracellular signaling domains for use in the anti-E7MC CAR of the present invention include T cell receptors (TCRs) and co-receptors that act in concert to initiate signal transduction following antigen receptor association. Cytoplasmic sequences, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capacity are included.

  It is known that signals generated via TCR alone are insufficient for full activation of T cells, and secondary or costimulatory signals are also required. Thus, T cell activation provides two distinct classes of intracellular signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary signaling sequences), and secondary or costimulatory signals As such, it can be said to be mediated by sequences that act in an antigen-independent manner (costimulatory signaling sequences).

  The primary signaling sequence regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling sequences that act in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine activation motif or ITAM. The anti-E7MC CAR construct comprises one or more ITAMs in some embodiments.

  Examples of ITAM-containing primary signaling sequences particularly used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d.

  In some embodiments, the anti-E7MC CAR comprises a primary signaling sequence derived from CD3ζ. For example, the intracellular signaling domain of CAR may comprise the CD3ζ intracellular signaling sequence itself, or any other desired intracellular signaling sequence (s) useful in the context of the anti-E7MC CAR of the invention. Can be combined. For example, the intracellular domain of an anti-E7MC CAR can include a CD3ζ intracellular signaling sequence and a costimulatory signaling sequence. Costimulatory signaling sequences include, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT , NKG2C, B7-H3, a part of the intracellular domain of costimulatory molecules including ligands that specifically bind to CD83, and the like.

  In some embodiments, the intracellular signaling domain of anti-E7MC CAR comprises an intracellular signaling sequence of CD3ζ and an intracellular signaling sequence of CD28. In some embodiments, the intracellular signaling domain of anti-E7MC CAR comprises a CD3ζ intracellular signaling sequence and a 4-1BB intracellular signaling sequence. In some embodiments, the intracellular signaling domain of anti-E7MC CAR comprises an intracellular signaling sequence of CD3ζ and an intracellular signaling sequence of CD28 and 4-1BB.

Thus, for example, in some embodiments, a) an extracellular domain comprising an anti-E7MC antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, b) a transmembrane domain, and c ) An anti-E7MC CAR comprising an intracellular signaling domain is provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence. In some embodiments, the anti-E7MC antibody portion comprises at least one (eg, at least 2, 3 or more) variants of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with the complex of either 4, 5, or 6. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

In some embodiments, a) an extracellular domain comprising an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, b An anti-E7MC CAR comprising a) a transmembrane domain and c) an intracellular signaling domain is provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1 HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOS: 186-188, or up to about 3 (eg, about 1, 2 or 3) Any) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) SEQ ID NO: 189 or 190 LC-CDR1 comprising the amino acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, b) a transmembrane domain, and c) An anti-E7MC CAR comprising an intracellular signaling domain is provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, A heavy chain variable domain sequence comprising HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185 and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 186 to 188; and ii) amino acids of SEQ ID NO: 189 or 190 An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising LC-CDR1 comprising the sequence and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191; b) an anti-E7MC CAR comprising an intracellular signaling domain; Provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 An HC-CDR3 comprising a sequence, or a variant thereof comprising substitutions of up to about 5 (eg any of about 1, 2, 3, 4 or 5) amino acids A heavy chain variable domain; and ii) LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof containing amino acid substitutions, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids LC-CDR3 comprising the variant and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising a substitution; b) a transmembrane domain; Anti E7MC CAR is provided comprising c) intracellular signaling domain. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 LC-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or up to about 5 in the LC-CDR sequence (eg, about Either 1, 2, 3, 4 or 5) an extracellular domain comprising an anti-E7MC antibody portion comprising a variant thereof containing amino acid substitutions; b) a transmembrane domain, and c) an anti-E7MC comprising an intracellular signaling domain CAR is provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 Anti E7MC CAR is provided comprising b) an intracellular signaling domain; extracellular domain containing anti E7MC antibody portion containing no light chain variable domain sequences. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 A heavy chain variable domain comprising an amino acid sequence, or variant thereof having at least about 95% (eg, at least about 96%, 97%, 98% or 99%) sequence identity, and SEQ ID NOs: 36-56 and A light chain variable domain comprising the amino acid sequence of any one of 238 to 243, or at least about 95% (eg, comprising at least about 96%, 97%, 98% or 99%) sequence identity An extracellular domain comprising an anti-E7MC antibody portion comprising the variant; b) a transmembrane domain, and c) a cell Anti E7MC CAR including signal transduction domain is provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 An extracellular domain comprising an anti-E7MC antibody portion comprising a heavy chain variable domain comprising an amino acid sequence and a light chain variable domain comprising any one amino acid sequence of SEQ ID NOs: 36-56 and 238-243; b) an intracellular signaling domain An anti-E7MC CAR is provided. In some embodiments, the intracellular signaling domain can activate immune cells. In some embodiments, the intracellular signaling domain comprises a primary signaling sequence and a costimulatory signaling sequence. In some embodiments, the primary signaling sequence comprises a CD3ζ intracellular signaling sequence. In some embodiments, the costimulatory signaling sequence comprises a CD28 intracellular signaling sequence. In some embodiments, the intracellular domain comprises a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence.

In some embodiments, a) an extracellular domain comprising an anti-E7MC antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, b) a transmembrane domain, and c) a CD3ζ intracellular An anti-E7MC CAR comprising an intracellular signaling domain comprising a signaling sequence and a CD28 intracellular signaling sequence is provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01.

In some embodiments, a) an extracellular domain comprising an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, b An anti-E7MC CAR comprising a) a transmembrane domain and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, Or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, about 1 HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOS: 186-188, or up to about 3 (eg, about 1, 2 or 3) Any) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) SEQ ID NO: 189 or 190 LC-CDR1 comprising the amino acid sequence, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or up to An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising substitution of about 3 (eg, any of about 1, 2 or 3) amino acids, b) a transmembrane domain, and c) An anti-E7MC CAR comprising an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) HC-CDR1, comprising the amino acid sequence of SEQ ID NO: 183, A heavy chain variable domain sequence comprising HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185 and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 186 to 188; and ii) amino acids of SEQ ID NO: 189 or 190 An extracellular domain comprising an anti-E7MC antibody portion, comprising LC-CDR1 comprising a sequence and a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, b) a transmembrane domain, and c) in a CD3ζ cell Intracellular containing a signaling sequence and a CD28 intracellular signaling sequence Anti E7MC CAR is provided comprising a signal transduction domain.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 An HC-CDR3 comprising a sequence, or a variant thereof comprising substitutions of up to about 5 (eg any of about 1, 2, 3, 4 or 5) amino acids A heavy chain variable domain; and ii) LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof containing amino acid substitutions, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids LC-CDR3 comprising the variant and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising a substitution; b) a transmembrane domain; Anti E7MC CAR is provided comprising the intracellular signaling domain comprising c) CD3 zeta intracellular signaling sequences and CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 LC-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or up to about 5 (eg, about Either 1, 2, 3, 4 or 5) an extracellular domain comprising an anti-E7MC antibody portion comprising a variant thereof containing amino acid substitutions; b) a transmembrane domain, and c) a CD3ζ intracellular signaling sequence and CD28 cells An anti-E7MC CAR comprising an intracellular signaling domain comprising an internal signaling sequence is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence; b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence. E7MC CAR is provided.

  In some embodiments, a) an anti-E7MC antibody moiety that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein comprising: i) any of SEQ ID NOs: 15-35 and 233-237 A heavy chain variable domain comprising one amino acid sequence, or variant thereof having at least about 95% (eg, at least about 96%, 97%, 98%, or 99%) sequence identity, and SEQ ID NOs: 36- A light chain variable domain comprising the amino acid sequence of any one of 56 and 238-243, or at least about 95% (eg including at least about 96%, 97%, 98% or 99%) sequence identity An extracellular domain comprising an anti-E7MC antibody portion comprising the variant thereof; b) a transmembrane domain, and c) Anti E7MC CAR is provided comprising the intracellular signaling domain containing D3ζ intracellular signaling sequences and CD28 intracellular signaling sequence.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 An extracellular domain comprising an anti-E7MC antibody portion comprising a heavy chain variable domain comprising an amino acid sequence and a light chain variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243; b) a transmembrane domain; c) An anti-E7MC CAR comprising an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence is provided.

  Effector cells (eg, lymphocytes, eg, T cells) that express anti-E7MC CAR are also provided herein.

  Also provided is a method of producing an effector cell that expresses an anti-E7MC CAR, the method comprising introducing into the effector cell a vector comprising a nucleic acid encoding the anti-E7MC CAR. In some embodiments, introducing the vector into the effector cell comprises transducing the effector cell with the vector. In some embodiments, introducing the vector into the effector cell comprises transfecting the effector cell with the vector. Transduction or transfection of the vector into effector cells can be carried out using any method known in the art.

Immunoconjugates Anti-E7MC constructs, in some embodiments, include immunoconjugates (also referred to herein as “anti-E7MC immunoconjugates”) that comprise an anti-E7MC antibody portion attached to an effector molecule. In some embodiments, the effector molecule is a therapeutic agent, eg, a cancer therapeutic agent that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In some embodiments, the effector molecule is a label that can produce a detectable signal either directly or indirectly.

  In some embodiments, an anti-E7MC immunoconjugate (also referred to herein as an “antibody-drug conjugate” or “ADC”) comprising an anti-E7MC antibody moiety and a therapeutic agent is provided. In some embodiments, the therapeutic agent is a toxin that is cytotoxic, cytostatic, or otherwise prevents or reduces the ability of the target cell to divide. Use of ADCs for local delivery of cytotoxic or cytostatic agents, ie drugs that kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Research 19: 605-614 ( 1999); Niculescu-Dubaz and Springer, Adv. Dr. Del. Rev. 26: 151-172 (1997); U.S. Pat. Delivery, and intracellular accumulation in target cells, where systemic administration of these unconjugated therapeutic agents is at an unacceptable level for normal cells as well as target cells that need to be eliminated (Baldwin et al., Lancet (March 15, 1986): 603-605 (1986); Thorpe (1985), "Antibody Carriers of Cytotoxic Agents in Cancer Therapies: A Review", Monoclonal Antibio A'84. (Ed.), Pages 475-506). As a result, maximum efficacy with minimal toxicity is sought. Importantly, since most normal cells do not present E7MC on their surface, they cannot bind anti-E7MC immunoconjugates and are protected from the killing effects of toxins or other therapeutic agents.

  Therapeutic agents used in anti-E7MC immunoconjugates include, for example, daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., Cancer Immunol. Immunother. 21: 183-187 (1986)). Toxins used in anti-E7MC immunoconjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as 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) ), Maytansinoids (EP1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996)), and calicheamicin (Lode et al., Can). . Er Res 58 Volume:. 2928 (1998); Hinman et al., Cancer Res 53 Volume: 3336-3342 (1993)) are included. Toxins can exert their cytotoxic and mitogenic effects by mechanisms including tubulin binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

  Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modedecin ) A chain, α-sarcin (α-sarcin), Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPIII and PAP-S), mordicica charantia inhibitor, crucin (curcin), crocin , Sapaonaria officinalis inhibitor, gelonin, mitgellin, restrictocin (restrictin) cin), phenol mycin (phenomycin), include Enomaishin (enomycin) and trichothecenes (tricothecenes). See, for example, WO 93/21232 published October 28, 1993.

  Anti-E7MC antibody moieties and one or more small molecule toxins such as calicheamicin, maytansinoids, dolastatin, auristatin, trichothecene and CC1065, and derivatives of these toxins having toxic activity Immunoconjugates are also contemplated herein.

  In some embodiments, an anti-E7MC immunoconjugate comprising a therapeutic agent having intracellular activity is provided. In some embodiments, the anti-E7MC immunoconjugate is internalized and the therapeutic agent is a cytotoxin that blocks cellular protein synthesis where it results in cell death. In some embodiments, the therapeutic agent comprises, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof. A cytotoxin comprising a polypeptide having ribosome inactivating activity. In some embodiments, when the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome inactivating activity, the anti-E7MC immunoconjugate is directed to the target cell such that the protein is cytotoxic to the cell. Must be internalized when joining.

  In some embodiments, an anti-E7MC immunoconjugate comprising a therapeutic agent that acts to disrupt DNA is provided. In some embodiments, therapeutic agents that act to disrupt DNA include, for example, enediyne (eg, calicheamicin and esperamicin) and non-enediyne small molecule drugs (eg, bleomycin, methidiumpropyl-EDTA). -Fe (II)). Other cancer therapeutics useful in accordance with this application include, without limitation, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, echinasaidin, duocarmycin / CC-1065 and bleomycin / pepleomycin. It is.

  The present invention further contemplates anti-E7MC immunoconjugates formed between an anti-E7MC antibody moiety and a compound having nucleolytic activity (eg, a ribonuclease or DNA endonuclease such as deoxyribonuclease; DNase).

  In some embodiments, the anti-E7MC immunoconjugate comprises an agent that acts to disrupt tubulin. Such agents may include, for example, rhizoxin / maytansine, paclitaxel, vincristine and vinblastine, colchicine, auristatin dolastatin 10 MMAE and perorside A.

  In some embodiments, the anti-E7MC immunoconjugate is, for example, Asaley NSC 167780, AZQ NSC 182986, BCNU NSC 409926, busulfan NSC 750, carboxyphthalatoplatinum NSC 271740, SC24N CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cis-platinum NSC 11875, clomezone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodison (cyc) 948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC 329680, hicanton NSC 142982, melphalan NSC 8806, methyl CCNU NSC 9441 mitomyc C , Nitrogen mustard NSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758, piperoman NSC 25154, porphyromycin NSC 56410, spirohydantoin mustard 72,112, including teroxirone (teroxirone) NSC 296934, tetraplatin (tetraplatin) NSC 363812, thiotepa NSC 6396, triethylenemelamine NSC 9706, alkylating agents include uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627.

  In some embodiments, a portion of the anti-E7MC immunoconjugate cancer therapeutic agent of the present application includes allocolchicine NSC 406042, halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC. 376128 (derived from NG-auristatin), maytansine NSC 153858, lysoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, tritylcysteine NSC 83298 tin sulfate 475 SC Is included without limitation It may include antimitotic agents.

  In some embodiments, the anti-E7MC immunoconjugate comprises camptothecin NSC 94600, camptothecin Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 642833, camptothecin derivative SC Derivatives NSC 295500, camptothecin derivatives NSC 249910, camptothecin derivatives NSC 606985, camptothecin derivatives NSC 374028, camptothecin derivatives NSC 176323, camptothecin derivatives NSC 295501, camptothecin derivatives NSC 606172, camptothecin derivatives NS C 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610457, camptothecin derivative NSC 606499, camptothecin derivative NSC 61046, camptothecin derivative NSC 3604 830 A topoisomerase I inhibitor.

  In some embodiments, the anti-E7MC immunoconjugate comprises doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 2499992, anthrapyrazole derivative NSC 355644, pyrazoloacridine 6r SC (Bisantrene) HCL NSC 337766, Daunorubicin NSC 82151, Dexoxorubicin NSC 267469, Mitoxantrone NSC 301739, Menogalyl NSC 269148, N, N-dibenzyldaunomycin NSC 268242, Oxanthrazol 91 (oxanthrazol 91) 4, including topoisomerase II inhibitors Rubidazon (rubidazone) NSC 164011, VM-26 NSC 122819, and VP-16 NSC 141540 are included without limitation.

  In some embodiments, the anti-E7MC immunoconjugate comprises L-alanosin NSC 153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, aminopterin Derivatives NSC 134033, antifol NSC 633713, antifall NSC 623017, Baker's soluble antifol NSC 139105, dichloroallyl Lawson NSC 134771 SC, 9083 NSC 126771 SC ur) (prodrug) NSC 148958, 5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N- (phosphonoacetyl) -L-aspartate (PALA) NSC 224131, pyrazofurin ( pyrazofurin) NSC 143095, trimetrexate NSC 352122, 3-HP NSC 95678, 2'-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, α-TGDR NSC 71651, aphidicolin glycinate NSC 303812, ara-C NSC 63878, 5-aza-2'-deoxycytidine NSC 127716, β-TGDR NSC 71261, Cyclocytidine NSC 145668, Guanazole NSC 1895, Hydroxyurea NSC 32065, Inosine glycodialdehyde NSC 118994, Macbecin III pIol NSC Including RNA or DNA antimetabolite, including without limitation thioguanine NSC 752 and thiopurine NSC 755.

In some embodiments, the anti-E7MC immunoconjugate comprises a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include radioactive isotopes of ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb, and Lu.

  In some embodiments, the anti-E7MC antibody moiety can be conjugated to a “receptor” (eg, streptavidin) for use in tumor pretargeting, wherein the antibody-receptor conjugate is Followed by removal of unbound conjugate from the circulation using a scavenger and then administration of a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, radionuclide). The

  In some embodiments, the anti-E7MC immunoconjugate can comprise an anti-E7MC antibody moiety conjugated to a prodrug activating enzyme. In some such embodiments, the prodrug activating enzyme converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO81 / 01145) into an active drug such as an anticancer drug. Such anti-E7MC immunoconjugates are useful in some embodiments in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymes that can be conjugated to antibodies include alkaline phosphatases useful for converting phosphate-containing prodrugs to free drugs; aryl sulfatases useful for converting sulfate-containing prodrugs to free drugs; non-toxic 5-fluoro Cytosine deaminase useful for converting cytosine to the anticancer drug 5-fluorouracil; proteases useful for converting peptide-containing prodrugs to free drugs, such as serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (e.g. , Cathepsins B and L); D-alanyl carboxypeptidases useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving fermentations useful for converting glycosylated prodrugs to free drugs Β-galactosidase and neuraminidase; β-lactamases useful for converting drugs derivatized with β-lactams to free drugs; and penicillin amidases such as phenoxyacetyl groups or phenylacetyl at their amine nitrogens, respectively. Penicillin V amidase and penicillin G amidase useful for converting a group derivatized drug to a free drug include, but are not limited to. In some embodiments, the enzyme can be covalently linked to the antibody moiety by recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature 312: 604-608 (1984).

  In some embodiments, the therapeutic portion of the anti-E7MC immunoconjugate can be a nucleic acid. Nucleic acids that can be used include, but are not limited to, antisense RNA, genes, or nucleic acid analogs, such as other polynucleotides including thioguanine and thiopurine.

The application further provides an anti-E7MC immunoconjugate comprising an anti-E7MC antibody moiety conjugated to an effector molecule, wherein the effector molecule is a label that can produce a signal that can be detected indirectly or directly. These anti-E7MC immunoconjugates can be used for research or diagnostic applications, eg, for in vivo detection of cancer. The label is preferably capable of producing a detectable signal either directly or indirectly. For example, the label can be radiopaque or can be a radioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I, ¹²⁵ I, ¹³¹ I; fluorescence (fluorophore) or chemiluminescence ( Chromophor) compounds such as fluorescein isothiocyanate, rhodamine or luciferin; enzymes such as alkaline phosphatase, β-galactosidase or horseradish peroxidase; imaging agents; or metal ions. In some embodiments, the label is for a radioactive atom for scintigraphic studies, such as ⁹⁹ Tc or ¹²³ I, or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI). For example, zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 can be complexed to various metal chelators, for example, conjugated to antibodies for PET imaging (WO2011 / 056983).

  In some embodiments, the anti-E7MC immunoconjugate is indirectly detectable. For example, a secondary antibody that is specific for an anti-E7MC immunoconjugate and contains a detectable label can be used to detect the anti-E7MC immunoconjugate.

Thus, for example, in some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) an anti-E7MC immunoconjugate comprising an effector molecule. Provided. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the effector molecule is covalently linked to the anti-E7MC antibody moiety. In some embodiments, the effector molecule is a therapeutic agent selected from the group consisting of, for example, drugs, toxins, radioisotopes, proteins, peptides and nucleic acids. In some embodiments, the effector molecule is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the cancer therapeutic agent is selected from the group consisting of, for example, ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P and ²¹² Pb. It is a highly radioactive atom. In some embodiments, the effector molecule is a label that can produce a detectable signal either directly or indirectly. In some embodiments, the label is a radioisotope selected from the group consisting of, for example, ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I, ¹²⁵ I, and ¹³¹ I. In some embodiments, the anti-E7MC antibody portion is an scFv. In some embodiments, the anti-E7MC antibody portion is human, humanized or semi-synthetic. In some embodiments, the anti-E7MC antibody portion comprises at least one (eg, at least 2, 3 or more) variants of HPV16-E7 peptide having an MHC class I protein and one amino acid substitution (eg, a conservative amino acid substitution). Cross-react with the complex of either 4, 5, or 6. In some embodiments, the anti-E7MC antibody portion comprises at least one complex (eg, any of at least 2, 3, 4 or 5) comprising an HPV16-E7 peptide and a different subtype of MHC class I protein. Cross react.

In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01, and b) an effector molecule An anti-E7MC immunoconjugate comprising is provided. In some embodiments, the effector molecule is covalently linked to the anti-E7MC antibody moiety. In some embodiments, the effector molecule is a therapeutic agent selected from the group consisting of, for example, drugs, toxins, radioisotopes, proteins, peptides and nucleic acids. In some embodiments, the effector molecule is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the cancer therapeutic agent is selected from the group consisting of, for example, ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P and ²¹² Pb. It is a highly radioactive atom. In some embodiments, the effector molecule is a label that can produce a detectable signal either directly or indirectly. In some embodiments, the label is a radioisotope selected from the group consisting of, for example, ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I, ¹²⁵ I, and ¹³¹ I. In some embodiments, the anti-E7MC antibody portion is an scFv. In some embodiments, the anti-E7MC antibody portion is human, humanized or semi-synthetic.

  In some embodiments, a) i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or variants, sequences comprising up to about 3 (eg, any of about 1, 2, or 3) amino acid substitutions HC-CDR2 comprising the amino acid sequence of number 184 or 185, or variants thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and any one of SEQ ID NOs: 186-188 An HC-CDR3 comprising an amino acid sequence, or a heavy chain variable domain sequence comprising a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and ii) of SEQ ID NO: 189 or 190 LC-CDR1 containing the amino acid sequence, or substitution of up to about 3 (eg, about 1, 2 or 3) amino acids And a light chain variable domain comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof comprising up to about 3 (eg, any of about 1, 2 or 3) amino acid substitutions. An anti-E7MC immunoconjugate comprising an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) an effector molecule is provided.

  In some embodiments, a) i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an amino acid sequence of any one of SEQ ID NOs: 186-188 A heavy chain variable domain sequence comprising HC-CDR3 comprising: and ii) comprising a light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 An anti-E7MC immunoconjugate comprising an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) an effector molecule is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1, comprising any of the amino acid sequences of SEQ ID NOs: 78-98, or variants thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids HC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids, and any one amino acid of SEQ ID NOs: 99-119, 244 and 245 An HC-CDR3 comprising a sequence, or a variant thereof comprising substitutions of up to about 5 (eg any of about 1, 2, 3, 4 or 5) amino acids A heavy chain variable domain; and ii) LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) ) Variants thereof containing amino acid substitutions, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, or substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids LC-CDR3 comprising the variant and any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250, or up to about 5 (eg, any of about 1, 2, 3, 4 or 5) amino acids An anti-E7MC antibody portion comprising a light chain variable domain comprising a variant thereof comprising a substitution, and b) an anti-E7MC comprising an effector molecule Takeno conjugates are provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; or variant thereof comprising substitutions of up to about 5 (eg, about 1, 2, 3, 4 or 5) amino acids in the HC-CDR sequence; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence of any one of SEQ ID NOs: 141 to 161 LC-CDR2; and a light chain variable domain sequence comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or up to about 5 (eg, about An anti-E7MC immunoconjugate comprising either 1, 2, 3, 4 or 5) an anti-E7MC antibody portion comprising a variant thereof containing an amino acid substitution, and b) an effector molecule is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising HPV16-E7 peptide and MHC class I protein, i) the amino acid sequence of any one of SEQ ID NOs: 57-77 HC-CDR1 comprising: an HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and a heavy chain comprising HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245 A variable domain sequence; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and SEQ ID NO: 162- LC-CDR3 comprising the amino acid sequence of any one of 182 and 247-250 Anti E7MC antibody portion containing no light chain variable domain sequence, and b) anti E7MC immunoconjugate comprising an effector molecule is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 A heavy chain variable domain comprising an amino acid sequence, or variant thereof having at least about 95% (eg, at least about 96%, 97%, 98%, or 99%) sequence identity, and SEQ ID NOs: 36-56 and A light chain variable domain comprising the amino acid sequence of any one of 238 to 243, or variant thereof having at least about 95% (eg, at least about 96%, 97%, 98% or 99%) sequence identity And an anti-E7MC immunoconjugate comprising an effector molecule It is provided.

  In some embodiments, a) an anti-E7MC antibody portion that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, comprising any one of SEQ ID NOs: 15-35 and 233-237 An anti-E7MC immunoconjugate comprising an heavy chain variable domain comprising an amino acid sequence and a light chain variable domain comprising any one amino acid sequence of SEQ ID NOs: 36-56 and 238-243, b) an effector molecule Provided.

Nucleic acid molecules encoding nucleic acid anti-E7MC constructs or anti-E7MC antibody portions are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full length anti-E7MC antibody is provided. In some embodiments, a multispecific anti-E7MC molecule (eg, multispecific anti-E7MC antibody, bispecific anti-E7MC antibody or bispecific T cell engager anti-E7MC antibody) or a portion of a polypeptide thereof ( A nucleic acid (or set of nucleic acids) encoding a polypeptide portion is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding an anti-E7MC CAR is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a portion of an anti-E7MC immunoconjugate or polypeptide thereof is provided.

  The application also includes variants to these nucleic acid sequences. For example, a variant includes a nucleotide sequence that hybridizes to a nucleic acid sequence encoding an anti-E7MC construct or anti-E7MC antibody portion of the present application under at least moderately stringent hybridization conditions.

  The present invention also provides a vector into which the nucleic acid of the present invention has been inserted.

  Briefly summarized, expression of an anti-E7MC construct (eg, anti-E7MC CAR) or a portion of a polypeptide thereof with a natural or synthetic nucleic acid encoding a portion of the anti-E7MC construct or a polypeptide thereof is expressed by, for example, a promoter Achieved by inserting the nucleic acid into an appropriate expression vector so as to be operably linked to 5 ′ and 3 ′ regulatory elements (eg, a lymphocyte-specific promoter) and a 3 ′ untranslated region (UTR). obtain. The vector may be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for the regulation of expression of the desired nucleic acid sequence.

  The nucleic acids of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present invention provides gene therapy vectors.

  Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly targeted vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

  Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Have been described. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (eg, WO01 / 96584; WO01 / 29058). And U.S. Patent No. 6,326,193).

  Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of interest either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In some embodiments, a lentiviral vector is used. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from onco-retrovirus such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

  Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically these are located in a region 30-110 bp upstream of the start site, but some promoters have recently been shown to contain functional elements also downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline.

  An example of a suitable promoter is the early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α). However, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus early promoter, Rous sarcoma virus promoters and other constitutive promoter sequences can also be used, including but not limited to human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter . Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters can turn on expression of operably linked polynucleotide sequences if such expression is desired, or turn off expression if expression is not desired. A simple molecular switch. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

  In order to assess the expression of a polypeptide or a portion thereof, the expression vector introduced into the cell is used to identify and select the expressing cell from a population of cells that are required to be transfected or infected via a viral vector. To facilitate, either a selectable marker gene or reporter gene or both may also be included. In other embodiments, the selectable marker can be carried on separate pieces of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

  Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene encodes a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression is revealed by certain easily detectable properties, such as enzymatic activity. It is a gene. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (eg, Ui-Tel et al., 2000 FEBS Letters 479 Volume: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, the construct with the smallest 5 'flanking region showing the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

  Methods for introducing and expressing genes in cells are known in the art. With respect to expression vectors, the vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method in the art. For example, an expression vector can be transferred into a host cell by physical, chemical or biological means.

  Physical methods for introducing the polynucleotide into the host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are 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, introduction of the polynucleotide into the host cell is performed by calcium phosphate transfection.

  Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, eg, human cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus 1, adenovirus, adeno-associated virus, and the like. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

  Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipids including oil-in-water emulsions, micelles, mixed micelles and liposomes. A base system is included. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).

  When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, can be interspersed within the lipid bilayer of the liposome, can be bound to the liposome via a linking molecule that binds both the liposome and the oligonucleotide, Can be trapped in, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included as a suspension in lipids, included with micelles Alternatively, it can be complexed with micelles or otherwise associated with lipids. The lipid, lipid / DNA or lipid / expression vector association composition is not limited to any particular structure in solution. For example, these compositions may exist in a bilayer structure, as micelles, or in a “collapsed” structure. They can also simply be scattered throughout the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that include long chain aliphatic hydrocarbons and derivatives thereof such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

  Regardless of the method used to introduce exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitors of the present invention, to confirm the presence of the recombinant DNA sequence in the host cell. In addition, various assays can be performed. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; for example by immunological means (ELISA and Western blot) or of the invention Included are “biochemical” assays such as detecting the presence or absence of a particular peptide, according to the assays described herein for identifying agents that fall within the scope.

MHC class I proteins MHC class I proteins are one of the two major classes of major histocompatibility complex (MHC) molecules (the other is MHC class II) and are almost all nucleated in the body. Found in cells. Their function is to display intracellularly derived protein fragments on T cells; healthy cells are ignored, but cells containing foreign proteins are attacked by the immune system. Since MHC class I proteins present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often referred to as the cytosolic pathway or the intrinsic pathway. Class I MHC molecules bind peptides generated primarily from the degradation of cytosolic proteins by the proteasome. The MHC I: peptide complex is then inserted into the plasma membrane of the cell. The peptide is bound to the extracellular portion of class I MHC molecules. Thus, the function of class I MHC is to display intracellular proteins on cytotoxic T cells (CTL). However, Class I MHC can also present peptides generated from exogenous proteins in a process known as cross-presentation.

  MHC class I proteins consist of two polypeptide chains, α and β2-microglobulin (β2M). These two chains are non-covalently linked through the interaction of b2m and the α3 domain. Only the α chain is polymorphic and is encoded by the HLA gene, whereas the b2m subunit is not polymorphic and is encoded by the β-2 microglobulin gene. The α3 domain extends to the plasma membrane and interacts with the CD8 co-receptor of T cells. The α3-CD8 interaction holds the MHC I molecule in place and the T cell receptor (TCR) on the surface of the cytotoxic T cell binds its α1-α2 heterodimeric ligand and is coupled to the peptide Are checked for antigenicity. The α1 and α2 domains fold to form a groove for the peptide to bind. MHC class I proteins bind peptides that are 8-10 amino acids long.

  The human leukocyte antigen (HLA) gene is a human version of the MHC gene. The three major MHC class I proteins in humans are HLA-A, HLA-B and HLA-C, and the three major MHC class I proteins are HLA-E, HLA-F and HLA-G . HLA-A is ranked the fastest evolving coding sequence among genes in humans. As of December 2013, there were 2432 known HLA-A alleles encoding 1740 active proteins and 117 null proteins. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger alpha chain constituent of HLA-A. Variations in the HLA-A α chain are important for HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for a peptide of a particular structure, more diverse HLA means more diverse antigens that are “presented” on the cell surface, and a subset of the population can be located anywhere. Increase the likelihood of being resistant to a given alien intruder. This reduces the likelihood that a single pathogen has the ability to wipe out the entire human population. Each individual can express up to two types of HLA-A from each of their parents. Some individuals inherit the same HLA-A from their parents and reduce their individual HLA diversity; however, the majority of individuals receive two different copies of HLA-A. This same pattern applies to all HLA groups. In other words, humans can express only one or two of 2432 known HLA-A alleles.

All alleles receive a classification of at least 4 digits, for example HLA-A ^* 02 ^: 12. A shows the HLA gene to which the allele belongs. There are many HLA-A alleles, so classification by serotype simplifies categorization. The next pair of digits indicates this assignment. For ^{example, HLA-A * 02: 02} , HLA-A * 02:04 and ^HLA-A * 02: 324 are all members of the ^(specified by * 02 prefix) A2 serotype. This group is the main factor responsible for HLA compatibility. All numbers after this cannot be determined by serotyping and are specified via gene sequencing. The second set of digits indicates which HLA protein is produced. These are assigned in the order of discovery, and as of December 2013, 456 different HLA-A02 proteins are known (HLA-A ^* 02: 01 to HLA-A ^* 02: 456 names are assigned). . The shortest possible HLA name includes both of these details. Each extension beyond that indicates a nucleotide change that may or may not change the protein.

In some embodiments, the anti-E7MC antibody portion specifically binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the MHC class I protein is HLA-A, HLA-B, HLA-C , HLA-E, HLA-F or HLA-G. In some embodiments, the MHC class I protein is HLA-A, HLA-B or HLA-C. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein is HLA-B. In some embodiments, the MHC class I protein is HLA-C. In some embodiments, the MHC class I protein is 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, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is any one of HLA-A ^* 02: 01-555, eg, 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 H LA-A ^* 02 ^: 24. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. HLA-A ^* 02 ^: 01 is expressed in 39-46% of all Caucasians, thus indicating an appropriate selection of MHC class I proteins for use in the present invention.

HPV16-E7 peptides suitable for use in generating anti-E7MC antibody moieties are, for example, using HLA-A ^* 02 ^: 01 binding motifs and proteasomes and immune-proteasomes (using computer predictive models known to those skilled in the art). can be determined based on the presence of a cleavage site for (immune-proteasome). To predict MHC binding sites, such models include IEDB (Vita et al., The immunoepitope database (IEDB) 3.0. Nucleic Acids Res. October 9, 2014. pi: gku938), ProPred1 (Sing and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMICS 17 (12): 1236-1237, described in more detail in 2001) and SYFPEITHIse (Schuler et. -Cell Epitope Prediction., Immunoinformatics ethods in Molecular Biology, 409 Volume (No. 1): 75-93 pages, but are not limited to, including but see 2007).

  Once a suitable peptide is identified, peptide synthesis can be performed according to protocols well known to those skilled in the art. Due to their relatively small size, the peptides of the invention can be synthesized directly in solution or on a solid support according to conventional peptide synthesis techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. Synthesis of peptides in solution phase has become a well-established procedure for large-scale production of synthetic peptides and is therefore a suitable alternative for preparing the peptides of the present invention (eg, John Morrow). See Stewart and Martin et al. Application of Almez-mediated Aidation Reactions to Solution Phase Peptide Synthesis, Tetrahedron Letters 39, 1517-1520.

  The binding activity of candidate HPV 16-E7 peptides can be tested using an antigen processing-deficient T2 cell line that increases HLA-A expression when stabilized by a peptide in the antigen presenting groove. T2 cells are pulsed with the candidate peptide for a time sufficient to stabilize HLA-A expression on the cell surface and used, for example, specific for HLA-A, using any method known in the art. It can be measured by immunostaining with a fluorescently labeled monoclonal antibody (eg, BB7.2) followed by fluorescence activated cell sorting (FACS) analysis.

Preparation of anti-E7MC antibody and anti-E7MC antibody portion In some embodiments, the anti-E7MC antibody or anti-E7MC antibody portion is a monoclonal antibody. Monoclonal antibodies use hybridoma methods such as those described, for example, by Kohler and Milstein, Nature, 256: 495 (1975) and Sergeeva et al., Blood, 117 (16): 4262-4272. Prepared using the phage display methods described herein and in the examples below, or using recombinant DNA methods (see, eg, US Pat. No. 4,816,567). Can be done.

  In hybridoma methods, a hamster, mouse or other suitable host animal typically produces an immunizing agent to raise lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Immunized with. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent can include a polypeptide or fusion protein of the protein of interest, or a complex comprising at least two molecules, such as a complex comprising HPV16-E7 peptide and MHC class I protein. In general, peripheral blood lymphocytes ("PBL") are used when cells of human origin are desired, or spleen cells or lymph node cells are used when a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press, 1986), pages 59-103. Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin and thymidine, which prevents the growth of HGPRT-deficient cells. Contains (“HAT medium”).

  In some embodiments, the immortalized cell line is efficiently fused, supports stable high level expression of the antibody by the selected antibody-producing cells, and is sensitive to a medium such as HAT medium. In some embodiments, the immortal cell line can be obtained from, for example, a mouse bone marrow, which is a mouse bone marrow, which can be obtained from the Salk Institute Cell Distribution Center, San Diego, California, and American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. Kozbor, J. et al. Immunol. 133: 3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc .: New York, 1987) 51-63.

  The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the polypeptide. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies is described, for example, in Munson and Pollard, Anal. Biochem. 107: 220 (1980).

  After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Godding, above. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

  Monoclonal antibodies secreted by subclones are isolated or purified from culture medium or ascites by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Can be done.

  Anti-E7MC antibodies or antibody portions can also be identified by screening combinatorial libraries for antibodies having the desired activity (s). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001), for example, McCafferty et al., Nature. 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, Human Press, Towota, NJ, 2003); Sidhu et al., J. Biol. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Biol. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Biol. Immunol. Methods 284 (1-2): 119-132 (2004).

  In one particular phage display method, Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994), repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined into a phage library, This can then be screened for antigen-binding phage. Phages typically display antibody fragments as either single chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), a single source of a broad range of non-self antigens and also antibodies to self antigens, without any immunity The naïve repertoire can be cloned (eg, from a human). Finally, the naive library is described in Hoogenboom and Winter, J.A. Mol. Biol. 227: 381-388 (1992), which encodes a highly variable CDR3 region and uses an unrearranged V gene segment to achieve rearrangement in vitro. It can also be made synthetically by cloning from stem cells and using PCR primers containing random sequences. Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005/0079574, 2005/0119455, and 2005/0266000. No. 2007/0117126, No. 2007/0160598, No. 2007/0237764, No. 2007/0292936 and No. 2009/0002360.

Antibodies or antigen-binding fragments thereof can be prepared by screening the library for antibodies specific for complexes comprising HPV16-E7 peptides and MHC class I proteins using phage display. The library is at least 1 × 10 ⁹ (eg, at least about 1 × 10 ⁹ , 2.5 × 10 ⁹ , 5 × 10 ⁹ , 7.5 × 10 ⁹ , 1 × 10 ¹⁰ , 2.5 × 10 ¹⁰ , It can be a human scFv phage display library with a diversity of unique human antibody fragments (either 5 × 10 ¹⁰ , 7.5 × 10 ¹⁰ or 1 × 10 ¹¹ ). In some embodiments, the library is a naive human library constructed from DNA extracted from human PMBC and spleen from healthy donors, including all human heavy and light chain subfamilies. In some embodiments, the library is constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients and patients with infectious diseases. A naive human library. In some embodiments, the library is a semi-synthetic human library, wherein the heavy chain CDR3 is fully randomized and all amino acids (except cysteine) are in any given position. (See, for example, Hoet, RM, et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library is about 5 to about 24 (eg, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23 or 24) having an amino acid length. In some embodiments, the library is a non-human phage display library.

Phage clones that bind E7MC with high affinity can be obtained by repetitive binding of phage to E7MC that binds to a solid support (eg, beads for solution panning or mammalian cells for cell panning) followed by non-binding. Selection can be by removal of bound phage and elution of specifically bound phage. In one example of solution panning, E7MC can be biotinylated for immobilization to a solid support. Biotinylated E7MC is mixed with a phage library and a solid support, such as streptavidin-conjugated Dynabeads M-280, and then the E7MC-phage-bead complex is isolated. The bound phage clones are then eluted and are expressed in a suitable host cell, eg, E. coli, for expression and purification. used to infect E. coli XL1-Blue. In one example of cell panning, T2 cells (TAP-deficient, HLA-A ^* 02 ^: 01+ lymphoblast cell line) loaded with E7MC HPV16-E7 peptide are mixed with the phage library, after which the cells are collected The bound clones are then eluted and used to infect appropriate host cells for expression and purification. Panning is performed using either solution panning, cell panning, or a combination of both to enrich for phage clones that specifically bind to E7MC (eg, about 2, 3, 4, 5, 6 Or any round). Enriched phage clones can be tested for specific binding to E7MC by any method known in the art including, for example, ELISA and FACS.

  Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention may be prepared using conventional procedures (eg, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody). Can be easily isolated and sequenced. The hybridoma cells or the E7MC-specific phage clones of the present invention can function as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which then produces a monkey COS that does not produce a host cell, eg, another immunoglobulin protein, in order to obtain synthesis of monoclonal antibodies in the recombinant host cell. Transfected into cells, Chinese hamster ovary (CHO) cells or myeloma cells. DNA may be obtained, for example, by using human heavy and light chain constant domains and / or framework region coding sequences in place of homologous non-human sequences (US Pat. No. 4,816,567; Morrison et al., Supra). Or by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domains of the antibodies of the invention, or can be used in place of the variable domains of one antigen binding site of the antibodies of the invention to create chimeric bivalent antibodies.

  The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is truncated at any point in the Fc region, generally to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or deleted to prevent crosslinking.

  In vitro methods are also suitable for preparing monovalent antibodies. Digestion of the antibody to produce its fragments, particularly Fab fragments, can be accomplished using any method known in the art.

  Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

Human antibodies and humanized antibodies The anti-E7MC antibody or antibody portion can be a humanized antibody or a human antibody. Humanized forms of non-human (eg, murine) antibodies typically comprise chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab) that contain minimal sequence derived from non-human immunoglobulin. ', F (ab') ₂ , scFv or other antigen-binding subsequence of the antibody). Humanized antibodies are those in which residues from the recipient CDR are replaced by residues from the CDRs of a non-human species (donor antibody), eg, mouse, rat or rabbit, having the desired specificity, affinity and ability. Human immunoglobulin (recipient antibody). In some examples, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody can comprise at least one, typically substantially all of the two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. However, all or substantially all of the FR region is of human immunoglobulin consensus sequence. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. 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).

  Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, typically taken from an “import” variable domain. According to some embodiments, humanization is performed by Winter and co-workers (Jones et al., Nature, 321: by using rodent CDRs or CDR sequences instead of the corresponding sequences of human antibodies 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Can be done. Accordingly, such “humanized” antibodies are those in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (US Pat. No. 4,816,567). . In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies. is there.

  As an alternative to humanization, human antibodies can be generated. For example, upon immunization, it is now possible to produce transgenic animals (eg, mice) that are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. 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. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97/17852. See Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice, in which the endogenous immunoglobulin genes are partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425. No .; and 5,661,016 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, In tern. Rev. Immunol. 13: 65-93 (1995).

  Human antibodies can also be obtained by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275) or in the art including phage display libraries. It can be generated by using various known techniques. Hoogenboom and Winter, J.A. Mol. Biol. 227: 381 (1991); Marks et al., J. Biol. Mol. Biol. 222: 581 (1991). The techniques of Cole et al. And Boerner et al. Are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R., et al. Liss, 77 (1985) and Boerner et al. Immunol. 147 (1): 86-95 (1991).

Multispecific antibodies In some embodiments, the anti-E7MC construct is a multispecific antibody. Suitable methods for making multispecific (eg, bispecific) antibodies are well known in the art. For example, the production of bispecific antibodies can be based on the co-expression of two immunoglobulin heavy / light chain pairs, where each of the two pairs has a different specificity and due to the association is a heterodimeric antibody (See, eg, Milstein and Cuello, Nature, 305: 537-539 (1983); WO 93/088829, and Traunecker et al., EMBO J. 10: 3655 (1991)). Due to the random sorting of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which is the correct bispecific It has a structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in WO 93/08829 and Traunecker et al., EMBO, 10: 3655-3659 (1991). Alternatively, heavy and light chain combinations can be directed by utilizing species-restricted pairing (eg, Lindhofer et al., J. Immunol. 155: 219-225 (1995). ), Heavy chain pairing can be directed through the use of “knob-in-hole” manipulation of the CH3 domain (eg, US Pat. No. 5,731,168; Ridgway et al., Protein Eng). 9 (7): 617-621 (1996)). Multispecific antibodies can also be made by manipulating the electrostatic steering effect to create antibody Fc heterodimeric molecules (see, eg, WO2009 / 089004A1). In yet another method, a stable bispecific antibody can be generated by controlled Fab arm exchange, in which two parent antibodies with distinct antigen specificities and matched point mutations in the CH3 domain are generated. , Allowing for separation, reassembly and reoxidation to form highly pure bispecific antibodies, mixed in reducing conditions. Labrigin et al., Proc. Natl. Acad. Sci. 110 (13): 5145-5150 (2013). Such antibodies comprising a mixture of heavy / light chain pairs are also referred to herein as “heteromultimeric antibodies”.

  Antibodies with different specificities or antigen-binding fragments thereof can also be chemically cross-linked to produce multispecific heteroconjugate antibodies. For example, two F (ab ') 2 molecules, each with specificity for a different antigen, can be chemically linked. Pulllarkat et al., Trends Biotechnol. 48: 9-21 (1999). Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection. WO91 / 00360; WO92 / 003733; EP03089. It is contemplated that these antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in US Pat. No. 4,676,980. .

  In some embodiments, multispecific antibodies can be prepared using recombinant DNA technology. For example, bispecific antibodies can be engineered by fusing two scFvs, for example by fusing two scFvs via a peptide linker to give a tandem scFv. An example of a tandem scFv is a bispecific T cell engager. A bispecific T cell engager links anti-CD3 scFv to a target cell surface antigen, eg, a scFv specific for a tumor associated antigen (TAA), resulting in T cell redirection to the target cell Is produced. Mack et al., Proc. Natl. Acad. Sci. 92: 7021-7025 (1995); Brischwein et al., Mol. Immunol. 43 (8): 1129-1143 (2006). By shortening the length of the peptide linker between the two variable domains, they can be prevented from self-assembling, forced to pair with a domain on the second polypeptide, diabody (Db) and This results in a compact bispecific antibody called. Holliger et al., Proc. Natl. Acad. Sci. 90: 6444-6448 (1993). Each of the two polypeptides of Db contains VH linked to VL by a linker that is too short to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one polypeptide are forced to pair with the complementary VL and VH domains of another polypeptide, thereby providing two antigen-binding sites. Form. In this form of modification, two polypeptides are joined by another peptide linker to yield a single chain diabody (scDb). In yet another modification of the Db format, the dual affinity retargeting (DART) bispecific antibody optionally precedes the C-terminal cysteine residue with an optional domain that drives assembly of the desired heterodimeric structure. And can be generated by introducing a disulfide bond between the cysteine residues at the C-terminus of each polypeptide. Veri et al., Arthritis Rheum. 62 (7): 1933-1943 (2010). A dual variable domain immunoglobulin (DVD-Ig ™) in which the target binding variable domains of two monoclonal antibodies are combined via a naturally occurring linker to obtain a tetravalent bispecific antibody is also Are known in the art. Gu and Ghayur, Methods Enzymol. 502: 25-41 (2012). In yet another format, the dock-and-lock (DNL) bispecific antibody is human cAMP-dependent protein kinase (PKA) with a peptide (AD2) derived from the anchoring domain of human A kinase anchor protein (AKAP). It is prepared by utilizing dimerization of a peptide derived from the regulatory subunit of (DDD2). Rossi et al., Proc. Natl. Acad. Sci. 103: 6841-6846 (2006).

  Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Koselny et al. Immunol. 148 (5): 1547-1553 (1992). This method can also be utilized for the production of antibody homodimers.

Anti-E7MC Variants In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion from the amino acid sequence of the antibody and / or insertion into such amino acid sequence and / or substitution of residues within such amino acid sequence. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final construct has the desired characteristics, eg, antigen binding.

  In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Amino acid substitutions can be introduced into the antibody of interest and the product can be screened for the desired activity, eg, retained / improved antigen binding, decreased immunogenicity or improved ADCC or CDC.

Conservative substitutions are shown in Table 5 below.

Amino acids can be grouped into different classes according to common side chain properties:
a. Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
c. Acidity: Asp, Glu;
d. Basic: His, Lys, Arg;
e. Residues that affect chain orientation: Gly, Pro;
f. Aromatic: Trp, Tyr, Phe.

  Non-conservative substitutions involve exchanging one member of these classes for another class.

  Exemplary substitution variants are affinity matured antibody moieties that can be conveniently generated using, for example, phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and variant antibody portions are displayed on phage and screened for specific biological activities (eg, binding affinity). Changes (eg, substitutions) can be made in HVRs, for example, to improve the affinity of the antibody portion. Such changes include HVR “hot spots”, ie, residues encoded by codons that are frequently mutated during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196). 2008)) and / or in specificity-determining residues (SDR) and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, by Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (Edited by O'Brien et al., Human Press, Totowa, NJ (2001)). )It is described in.

  In some embodiments of affinity maturation, diversity is selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Introduced into the generated variable gene. A secondary library is then created. The library is then screened to identify variants of any antibody portion that have the desired affinity. Another method for introducing diversity involves an HVR-oriented approach in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

  In some embodiments, substitutions, insertions or deletions may be present in one or more HVRs as long as such changes do not substantially reduce the ability of the antibody moiety to bind to the antigen. For example, conservative changes (eg, conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such changes may be outside the HVR “hot spot” or SDR. In some embodiments of the variant VH and VL sequences provided above, any of each HVR is unaltered or includes 1, 2, or 3 amino acid substitutions.

  Useful methods for the identification of residues or regions of antibody moieties that can be targeted for mutagenesis are described by Cunningham and Wells (1989) Science 244: 1081-1085, as “ Called “Alanine Scanning Mutagenesis”. In this method, one residue or group of target residues (eg, charged residues, eg, arg, asp, his, lys and glu) is identified and whether the interaction between the antibody moiety and the antigen is affected. To determine whether it is replaced by a neutral or negatively charged amino acid (eg alanine or polyalanine). Additional substitutions can be introduced at amino acid positions that clearly show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody subcomplex can be determined to identify contact points between the antibody moiety and the antigen. Such contact residues and neighboring residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine if they contain the desired property.

  Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from 1 residue to a polypeptide containing 100 or more residues, and intra-sequence insertion of single or multiple amino acid residues Is included. Examples of terminal insertions include antibody moieties having an N-terminal methionyl residue. Other insertional variants of the antibody portion include fusions to the N- or C-terminus of the antibody portion to an enzyme (eg, for ADEPT) or to a polypeptide that increases the serum half-life of the antibody portion.

Fc region variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of a full-length anti-E7MC antibody provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced antibody-dependent cellular cytotoxicity (ADCC) effector function that is often associated with binding to an Fc receptor (FcR). In some embodiments, the Fc region variant has reduced ADCC effector function. There are many examples of changes or mutations to the Fc sequence that can alter effector function. See, for example, WO 00/42072 and Shields et al. J Biol. Chem. 9 (2): 6591-6604 (2001) describes antibody variants with improved or weakened binding to FcR. The contents of these publications are specifically incorporated herein by reference.

  Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (eg, cancer cells) whose membrane surface antigens are bound by specific antibodies (eg, anti-E7MC antibodies). It is. Typical ADCC involves the activation of NK cells by antibodies. NK cells express CD16, an Fc receptor. This receptor recognizes and binds to a portion of the antibody Fc bound to the surface of the target cell. The most common Fc receptor on the surface of NK cells is called CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody results in NK cell activation, release of cytolytic granules and subsequent apoptosis of the target cells. The ADCC contribution to tumor cell killing can be measured using a specific test using NK-92 cells transfected with high affinity FcR. Results are compared to wild type NK-92 cells that do not express FcR.

  In some embodiments, the present invention contemplates anti-E7MC construct variants comprising an FC region that possesses some but not all effector functions, whereby such variants are in vivo halved of the anti-E7MC construct. Although phase is important, certain effector functions (eg, CDC and ADCC) have become desirable candidates for applications where unnecessary or harmful effects are present. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay is performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. obtain. NK cells, the primary cell for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991), summarized in Table 3 on page 464. A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is US Pat. No. 5,500,362 (eg, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83). Volume: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); see US Pat. No. 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987). )It is described in. Alternatively, non-radioactive assay methods can be used (eg, ACTI ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); And CytoTox 96 ™ non- Radioactive cytotoxicity assay (see Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be determined in vivo, eg, Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998) and can be evaluated in animal models such as those disclosed. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO2006 / 0298779 and WO2005 / 100402. CDC assays can be performed to assess complement activation (eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101). : 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (eg, Petkova, SB, et al., Int'l. Immunol. 18 (12)). : 1759-1769 (2006)).

  Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056). issue). Such Fc variants include amino acid positions 265, 269, 270, 297, and so-called “DANA” Fc variants (US Pat. No. 7,332,581) with substitutions of residues 265 and 297 to alanine. Fc variants having substitutions at 2 or more of 327 are included.

  Certain antibody variants have been described that have improved or weakened binding to FcR (eg, US Pat. No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9). Volume (2): 6591-6604 (2001)).

  In some embodiments, an anti-E7MC construct (eg, full length anti-E7MC antibody) variant comprising a variant Fc region comprising one or more amino acid substitutions that improve ADCC is provided. In some embodiments, the variant Fc region comprises one or more amino acid substitutions that improve ADCC, and these substitutions are at positions 298, 333 and / or 334 of the variant Fc region (residues of residues). EU numbering). In some embodiments, an anti-E7MC construct (eg, full length anti-E7MC antibody) variant comprises the following amino acid substitutions in its variant Fc region: S298A, E333A and K334A.

  In some embodiments, see, for example, US Pat. No. 6,194,551, WO 99/51642 and Idusogie et al. Immunol. 164: 4178-4184 (2000), modified (ie, either improved or attenuated) C1q binding and / or complement dependent cytotoxicity (CDC) The changes that give rise to are made in the Fc region.

  In some embodiments, an anti-E7MC construct comprising a variant Fc region comprising one or more amino acid substitutions that increase half-life and / or improve binding to a neonatal Fc receptor (FcRn) (eg, full-length anti-E7MC Antibody) variants are provided. Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). These antibodies include an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413 Including substitutions at one or more of 424 or 434, eg, substitution of residue 434 of the Fc region (US Pat. No. 7,371,826).

  For other examples of Fc region variants, see Duncan and Winter, Nature 322: 738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; See also 29351.

  Anti-E7MC constructs (eg, full length anti-E7MC antibodies) comprising any of the Fc variants described herein or combinations thereof are contemplated.

Glycosylation variants In some embodiments, the anti-E7MC constructs provided herein are altered to increase or decrease the extent to which the anti-E7MC construct is glycosylated. Addition or deletion of glycosylation sites to the anti-E7MC construct is facilitated by altering the amino acid sequence of a portion of the anti-E7MC construct or a polypeptide thereof such that one or more glycosylation sites are created or removed. Can be achieved.

  If the anti-E7MC construct contains an Fc region, the carbohydrate attached to it can be altered. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are commonly linked by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the anti-E7MC constructs of the invention can be made to create anti-E7MC construct variants with certain improved properties.

  In some embodiments, an anti-E7MC construct (eg, full-length anti-E7MC antibody) variant comprising an Fc region is provided, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which is ADCC. Can improve function. Specifically, anti-E7MC constructs having reduced fucose compared to the amount of fucose on the same anti-E7MC construct produced in wild type CHO cells are contemplated herein. That is, they are produced by native CHO cells (eg, CHO cells that produce a native glycosylation pattern, eg, CHO cells that contain a native FUT8 gene) that have a lower amount of fucose than they have. It is characterized by having. In some embodiments, the anti-E7MC construct is a construct in which less than about 50%, 40%, 30%, 20%, 10% or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an anti-E7MC construct can be 1% -80%, 1% -65%, 5% -65% or 20% -40%. In some embodiments, the anti-E7MC construct is free of fucose in any of the N-linked glycans thereon, i.e., the anti-E7MC construct is completely fucose-free or has no fucose or non-fucose. An afucosylated construct. The amount of fucose is measured by MALDI-TOF mass spectrometry as described in, for example, WO 2008/077546. All sugar structures attached to Asn297 (eg, complexes, hybrids and high mannose structures) As determined by calculating the average amount of fucose in the sugar chain at Asn297. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 is also in position 297 due to minor sequence variations in the antibody. About ± 3 amino acids upstream or downstream of, ie, between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, eg, US Patent Publication No. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140. US 2004/0110704; US 2004/0110282; US 2004/0108865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/031742; WO 2002/031140; Okazaki et al. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., In particular Example 11) and knockout cell lines such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (eg Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006) Year); and WO 2003/085107).

  An anti-E7MC construct (eg, full length anti-E7MC antibody) variant further comprises a bisected oligosaccharide, eg, a biantennary oligosaccharide attached to the Fc region of the anti-E7MC construct is bisected by GlcNAc. Such anti-E7MC construct (eg, full length anti-E7MC antibody) variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US2005 / 0123546 (Umana et al.) And Ferrara et al., Biotechnology and Bioengineering, Vol. 93. (No. 5): 851 to 861 (2006). Also provided are anti-E7MC construct (eg, full-length anti-E7MC antibody) variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such anti-E7MC construct variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

  In some embodiments, an anti-E7MC construct (eg, full length anti-E7MC antibody) variant comprising an Fc region is capable of binding to FcγRIII. In some embodiments, an anti-E7MC construct (eg, full-length anti-E7MC antibody) variant comprising an Fc region has ADCC activity in the presence of human effector cells, or otherwise comprises a human wild-type IgG1 Fc region. Has increased ADCC activity in the presence of human effector cells compared to anti-E7MC constructs (eg, full length anti-E7MC antibodies).

Cysteine engineered variants In some embodiments, it may be desirable to create a cysteine engineered anti-E7MC construct (eg, a full-length anti-E7MC antibody) in which one or more amino acid residues are replaced with cysteine residues. In some embodiments, the substituted residue is present at an accessible site of the anti-E7MC construct. By substituting these residues with cysteine, a reactive thiol group is placed at an accessible site of the anti-E7MC construct to create an anti-E7MC immunoconjugate as further described herein. It can be used to conjugate the construct to other moieties, such as drug moieties or linker-drug moieties. Cysteine engineered anti-E7MC constructs (eg, full-length anti-E7MC antibodies) can be generated, for example, as described in US Pat. No. 7,521,541.

Derivatives In some embodiments, the anti-E7MC constructs provided herein can be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of the anti-E7MC construct include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3. , 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide (Polypropylene oxide) / ethylene oxide copolymers, polyoxyethylated polyols (eg glycero ), Polyvinyl alcohol, as well as mixtures thereof without limitation. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the anti-E7MC construct can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymer used for derivatization will depend on the particular property or function of the anti-E7MC construct to be improved, whether the anti-E7MC construct derivative will be used therapeutically under defined conditions, etc. It can be determined based on considerations including but not limited to.

  In some embodiments, a conjugate of an anti-E7MC construct and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In some embodiments, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11160-11605 (2005)). The radiation can be of any wavelength and does not harm normal cells, but the non-proteinaceous part up to a temperature at which cells proximal to the anti-E7MC construct-non-proteinaceous part are killed. This includes, but is not limited to, the wavelength of heating.

CAR Effector Cell Preparation In one aspect, the present invention provides effector cells (eg, lymphocytes, eg, T cells) that express anti-E7MC CAR. Exemplary methods for preparing effector cells (eg, T cells) that express anti-E7MC CAR (eg, anti-E7MC CAR effector cells, eg, anti-E7MC CAR T cells) are provided herein.

  In some embodiments, the anti-E7MC CAR effector cells (eg, T cells) are anti-E7MC CAR (eg, CARs comprising anti-E7MC antibody portions and CD28 and CD3ζ intracellular signaling sequences) (eg, lentis). Viral vectors) can be generated by introducing into effector cells (eg, T cells). In some embodiments, the anti-E7MC CAR effector cells (eg, T cells) of the invention can replicate in vivo, and HPV16-E7 positive diseases (eg, cancer, eg, squamous cell carcinoma, Cause long-term persistence that can lead to sustained control of cervical or anal cancer).

  In some embodiments, the invention uses genes that express anti-E7MC CAR for the treatment of patients with HPV16-E7 positive disease or at risk of having HPV16-E7 positive disease using lymphocyte infusion It relates to administering modified T cells. In some embodiments, autologous lymphocyte infusion is used in the treatment. Autologous PBMC are collected from patients in need of treatment, and T cells are activated and expanded using methods described in the art and known in the art, and then the patient It is injected back into the back.

In some embodiments, the anti-E7MC CAR T cell comprises an anti-E7MC antibody portion.
Expresses CAR (also referred to herein as “anti-E7MC CAR T cells”). In some embodiments, the anti-E7MC CAR T cells express an anti-E7MC CAR comprising an extracellular domain comprising an anti-E7MC antibody portion and an intracellular domain comprising CD3ζ and CD28 intracellular signaling sequences. The anti-E7MC CAR T cells of the present invention can undergo robust in vivo T cell expansion and can establish E7MC specific memory cells that persist at high levels for extended periods in blood and bone marrow. In some embodiments, the anti-E7MC CAR T cells of the invention injected into a patient can eliminate E7MC-presenting cells, such as E7MC-presenting cancer cells, in vivo in patients with HPV16-E7 positive disease. In some embodiments, the anti-E7MC CAR T cells of the invention injected into a patient are presented in vivo in patients with HPV16-E7 positive disease that are refractory to at least one conventional treatment. E7MC-presenting cells such as cancer cells can be excluded.

Prior to T cell expansion and genetic modification, a source of T cells is obtained from the subject. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor. In some embodiments of the invention, any number of T cell lines available in the art may be used. In some embodiments of the invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll ™ separation. In some embodiments, cells from the individual's circulating blood are obtained by apheresis. Apheresis products typically include lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a buffer or medium appropriate for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution may lack calcium, may lack magnesium, or may lack many, but not all, divalent cations. As one skilled in the art will readily appreciate, the washing step is performed by methods known to those skilled in the art, for example, semi-automated “flow-through” centrifuges (eg, Cobe 2991 cell processor, Baxter CytoMate or Haemonetics according to the manufacturer's instructions). It can be achieved by using Cell Saver 5). After washing, the cells can be resuspended in various biocompatible buffers such as Ca ²⁺ free, Mg ²⁺ PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesired components of the apheresis sample can be removed and the cells can be resuspended directly in the culture medium.

In some embodiments, T cells lyse erythrocytes and deplete monocytes, for example, by centrifugation through a PERCOLL ™ gradient or by counterflow centrifugal elution. Isolated from peripheral blood lymphocytes. Certain subpopulations of T cells, such as CD3 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are anti-CD3 / anti-CD28 (ie, 3 × 28) conjugated beads, eg, DYNABEADS®, for a period sufficient for positive selection of the desired T cells. Isolated by incubation with M-450 CD3 / CD28 T. In some embodiments, the period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or more, and all integer values therebetween. In some embodiments, the period is at least 1, 2, 3, 4, 5 or 6 hours. In some embodiments, the time period is 10-24 hours. In some embodiments, the incubation period is 24 hours. For isolation of T cells from patients with leukemia, the use of longer incubation times, eg 24 hours, can increase cell yield. Longer incubation times can be used in any situation where there are fewer T cells compared to other cell types, eg, in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Can be used to isolate T cells. In addition, the use of longer incubation times can increase the efficiency of CD8 ⁺ T cell capture. Thus, by simply shortening or extending this time, T cells become bound to CD3 / CD28 beads and / or by increasing or decreasing the bead to T cell ratio, The population can be advantageously or preferentially selected at the beginning of the culture or at other points during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subpopulations can be advantageously obtained at the start of culture or at other desired time points. Or it may be preferentially selected. One skilled in the art recognizes that multiple rounds of selection can also be used in the context of the present invention. In some embodiments, it may be desirable to perform a selection procedure and use “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to negatively selected cells. One method uses a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells, cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry. It is. For example, to enrich CD4 + cells by negative selection, monoclonal antibody cocktails typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4 ⁺ , CD25 ⁺ , CD62Lhi, GITR ⁺ and FoxP3 ⁺ . Alternatively, in some embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

For isolation of the desired population of cells by positive or negative selection, the cell concentration and surface (eg, particles such as beads) can be varied. In some embodiments, it is desirable to significantly reduce the volume at which the beads and cells are mixed together (ie, increase the concentration of cells) to ensure maximum contact between the cells and the beads. It can be rare. For example, in some embodiments, a concentration of about 2 billion cells / ml is used. In some embodiments, a concentration of about 1 billion cells / ml is used. In some embodiments, greater than about 100 million cells / ml are used. In some embodiments, a concentration of cells of about 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In some embodiments, a cell concentration of about 75, 80, 85, 90, 95 or 100 million cells / ml is used. In some embodiments, a concentration of about 125 or about 150 million cells / ml is used. Using high concentrations can result in increased cell yield, cell activation and cell expansion. Furthermore, the use of high cell concentrations is more likely from cells that can weakly express the target antigen of interest, eg, CD28 negative T cells, or from samples in which many tumor cells are present (ie, leukemia blood, tumor tissue, etc.). Enable efficient capture. Such a population of cells can have therapeutic value and is desired to be obtained. For example, using a high concentration of cells allows for more efficient selection of CD8 ⁺ T cells that usually have weaker CD28 expression.

  In some embodiments of the invention, T cells are obtained directly from the patient after treatment. In this regard, following certain cancer treatments, particularly treatment with drugs that damage the immune system, immediately after treatment during a period in which the patient is usually recovering from treatment, the quality of T cells obtained is: It has been observed that their ability to expand in vivo can be optimal or improved. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a favorable state for enhanced engraftment and in vivo expansion. Accordingly, in the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage during this recovery phase. Further, in some embodiments, mobilization (eg, mobilization with GM-CSF) and conditioning regimen may be used in a subject to repopulate, recirculate, regenerate and / or expand proliferation of a particular cell type, particularly treatment. It can be used to create a preferred state during a later defined time window. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

  T cells, for example, before or after genetic modification of T cells to express the desired anti-E7MC CAR, are described, for example, in US Pat. Nos. 6,352,694; 6,534,055; No. 6,692,964; No. 5,858,358; No. 6,887,466; No. 6,905,681; No. 7,144,575; No. 7,067,318 No. 7,172,869; No. 7,232,566; No. 7,175,843; No. 5,883,223; No. 6,905,874; No. 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005 can generally be used to activate and expand.

In general, the T cells of the present invention are expanded by contact with a surface having an agent that stimulates a CD3 / TCR complex associated signal bound to it and a ligand that stimulates a costimulatory molecule on the surface of the T cell. In particular, T cell populations can be produced, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof immobilized on the surface, or an anti-CD2 antibody, or in combination with a calcium ionophore (eg, bryostatin). ) Can be stimulated by contact. For costimulation of accessory molecules on the surface of T cells, ligands that bind accessory molecules are used. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies to stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besanson, France) and can be used in a manner similar to other methods commonly known in the art. as can other methods commonly known in the art) (Berg et al., Transplant Proc. 30 (8): 3975-3777, 1998; Haanen et al., J. Exp. Med. 190 (9): 1319). 1328, 1999; Garland et al., J. Immunol. Meth. 227 (1-2): 53-63, 1999).

Immunoconjugate preparation Anti-E7MC immunoconjugates can be prepared using any method known in the art. See, for example, WO 2009/067800, WO 2011/133886 and US Patent Publication No. 20143222129, which are incorporated herein by reference in their entirety.

  The anti-E7MC antibody portion of the anti-E7MC immunoconjugate can be “coupled” to the effector molecule by any means by which the anti-E7MC antibody portion can be associated with or linked to the effector molecule. For example, the anti-E7MC antibody portion of the anti-E7MC immunoconjugate can be coupled to the effector molecule by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare anti-E7MC immunoconjugates. The method used to conjugate the anti-E7MC antibody moiety and the effector molecule allows the binding protein to bind to the effector molecule without interfering with the ability of the binding protein to bind the antigen on the target cell. ) Must be possible.

  The anti-E7MC antibody portion of the anti-E7MC immunoconjugate can be indirectly linked to the effector molecule. For example, the anti-E7MC antibody portion of an anti-E7MC immunoconjugate can be directly linked to a liposome containing one of several types of effector molecules. The effector molecule (s) and / or anti-E7MC antibody moiety can also be bound to a solid surface.

  In some embodiments, the anti-E7MC antibody portion of the anti-E7MC immunoconjugate and the effector molecule are both proteins and can be conjugated using techniques well known in the art. Hundreds of crosslinkers are available that can conjugate two proteins (see, for example, “Chemistry of Protein Conjugation and Crosslinking”, 1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generally selected based on reactive functional groups available on or inserted on the anti-E7MC antibody moiety and / or effector molecule. In addition, in the absence of reactive groups, photoactivatable crosslinkers can be used. In certain instances, it may be desirable to include a spacer between the anti-E7MC antibody portion and the effector molecule. Cross-linking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipimidate and bis (diazobenzidine) and heterobifunctional agents: m maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m maleimidobenzoyl- N-hydroxysuccinimide is included.

  In some embodiments, the anti-E7MC antibody portion of the anti-E7MC immunoconjugate can be engineered with specific residues for chemical attachment of effector molecules. Specific residues used in the chemical coupling of molecules known in the art include lysine and cysteine. The crosslinker is selected based on the reactive functional group inserted on the anti-E7MC antibody moiety and available on the effector molecule.

  Anti-E7MC immunoconjugates can also be prepared using recombinant DNA technology. In such a case, the DNA sequence encoding the anti-E7MC antibody portion is fused to the DNA sequence encoding the effector molecule, resulting in a chimeric DNA molecule. The chimeric DNA sequence is transfected into a host cell that expresses the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.

  Examples of binding effector molecules that are labels to binding proteins include Hunter et al., Nature 144: 945 (1962); David et al., Biochemistry 13: 1014 (1974); Pain et al., J. Biol. Immunol. Meth. 40: 219 (1981); Nygren, J. et al. 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 Xenografics In Athymic. Enzymol. 121: 802-16 (1986).

A radioactive label or other label can be incorporated into the immunoconjugate in a known manner. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis, for example using a suitable amino acid precursor containing fluorine-19 instead of hydrogen. Labels such as ⁹⁹ Tc or ¹²³ I, ¹⁸⁶ Re, ¹⁸⁸ Re and ¹¹¹ In can be attached via cysteine residues in the peptide. Yttrium-90 can be linked via a lysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80: 49-57 (1978)) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscinography” (Chatal, CRC Press 1989) describes other methods in detail.

  Immunoconjugates of antibody moieties and cytotoxic agents include various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimide) Methyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (eg dimethyl adipimidate HCI), active esters (eg disuccinimidyl suberate), aldehydes (eg Glutaraldehyde), bis-azide compounds (for example, bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (for example, bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (for example, 2,6-dii). Cyanate toluene), and bis - active fluorine compounds (e.g., may be made using the 1,5-difluoro-). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary for the conjugation of radionuclides to antibodies. Chelating agent. For example, see WO94 / 11026. The linker may be a “cleavable linker” that facilitates release of the cytotoxic drug in the cell. For example, acid labile linkers, peptidase sensitive linkers, photodegradable linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US Pat. No. 5,208,020) Can be used.

  The anti-E7MC immunoconjugates of the invention are cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL, USA) MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl- ( ADCs prepared using 4-vinylsulfone) benzoate) are explicitly contemplated but not limited thereto. See pages 467-498 of the Applications Handbook and Catalog, 2003-2004.

Pharmaceutical Compositions Compositions comprising anti-E7MC constructs (eg, pharmaceutical compositions, also referred to herein as formulations) are also provided herein. In some embodiments, the composition further comprises cells (eg, effector cells, eg, T cells) associated with the anti-E7MC construct. In some embodiments, a pharmaceutical composition comprising an anti-E7MC construct and a pharmaceutically acceptable carrier is provided. In some embodiments, the pharmaceutical composition further comprises cells (eg, effector cells, eg, T cells) associated with the anti-E7MC construct.

  A suitable formulation of an anti-E7MC construct is an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's) in the form of a lyophilized formulation or an aqueous solution having the desired degree of purity. s Pharmaceutical Sciences 16th edition, Osol, A. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, including buffers such as phosphates, citrates and other organic acids; ascorbic acid and Antioxidants including methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; Resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; parent Sex polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™ ), PLURONICS ™ or polyethylene glycol (PEG). Exemplary formulations are described in WO 98/56418 which is expressly incorporated herein by reference. A lyophilized formulation adapted for subcutaneous administration is described in WO 97/04801. Such lyophilized formulations can be reconstituted to a high protein concentration with a suitable diluent, and the reconstituted formulation can be administered subcutaneously to the individual being treated herein. Lipofectin or liposomes can be used to deliver the anti-E7MC constructs of the invention into cells.

  The formulations herein may have, as necessary for the particular indication being treated, one or more active compounds in addition to the anti-E7MC construct, preferably activities with complementary activities that do not adversely affect each other Compounds can also be included. For example, it may be desirable to further provide an antineoplastic agent, growth inhibitory agent, cytotoxic agent or chemotherapeutic agent in addition to the anti-E7MC construct. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of anti-E7MC construct present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1-99% of the dosages used so far.

  The anti-E7MC construct is also a colloid drug delivery system in microcapsules prepared by, for example, coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl methacrylate) microcapsules, respectively. It can be entrapped in (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. et al. (1980). Sustained release preparations can be prepared.

  Sustained release preparations of anti-E7MC constructs can be prepared. Suitable examples of sustained release preparations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies (or fragments thereof), which matrix is in the form of a molded article, eg, a film or microcapsule. . Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and ethyl. Copolymers of L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and Poly-D-(-)-3-hydroxybutyric acid is included. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow release of molecules for over 100 days, certain hydrogels release proteins over shorter time periods. If encapsulated antibodies remain in the body for a long time, they denature or aggregate as a result of exposure to moisture at 37 ° C., resulting in loss of biological activity and possible changes in immunogenicity. Can result. Rational strategies can be devised for the stabilization of anti-E7MC constructs, depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is intermolecular SS bond formation via thio-disulfide interconversion, stabilization modifies sulfhydryl residues, freeze-drys from acidic solutions, By developing a specific polymer matrix composition using a suitable additive and using appropriate additives.

  In some embodiments, the anti-E7MC construct is formulated in a buffer comprising citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination of the above. The In some embodiments, the anti-E7MC construct is formulated in a buffer comprising about 100 mM to about 150 mM glycine. In some embodiments, the anti-E7MC construct is formulated in a buffer comprising about 50 mM to about 100 mM NaCl. In some embodiments, the anti-E7MC construct is formulated in a buffer comprising about 10 mM to about 50 mM acetate. In some embodiments, the anti-E7MC construct is formulated in a buffer comprising about 10 mM to about 50 mM succinate. In some embodiments, the anti-E7MC construct is formulated in a buffer comprising about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-E7MC construct is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-E7MC construct is formulated in a buffer comprising 10 mM citrate, 100 mM NaCl, 100 mM glycine and 0.01% polysorbate 80, which formulation has a pH of 5.5. It is.

  Formulations used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through a sterile filtration membrane.

Methods of treatment using anti-E7MC constructs Anti-E7MC constructs and / or compositions of the invention include, for example, HPV16-E7 positive cancers (eg, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, Diseases and / or disorders associated with HPV16-E7 expression, including vulvar cancer, penile cancer, head and neck cancer or oropharyngeal cancer (also referred to herein as “HPV16-E7 positive” disease or disorder) ) Can be administered to an individual (eg, a mammal such as a human). Accordingly, the application provides, in some embodiments, a method of treating an HPV16-E7 positive disease (eg, cancer, eg, squamous cell carcinoma) in an individual, wherein the individual has an effective amount of the present specification. Administering a composition (eg, pharmaceutical composition) comprising an anti-E7MC construct comprising an anti-E7MC antibody moiety, such as any one of the anti-E7MC constructs described in the literature. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the cancer is, for example, from squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. Selected from the group consisting of

For example, in some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. There is provided a method comprising administering a composition comprising an anti-E7MC construct comprising an anti-E7MC antibody moiety. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual is administered an effective amount of HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02 ^: 01. There is provided a method comprising administering a composition comprising an anti-E7MC construct comprising an anti-E7MC antibody moiety that specifically binds to the comprising complex. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. Administering a composition comprising an anti-E7MC construct comprising an antibody moiety, wherein the anti-E7MC antibody moiety comprises i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or up to about 3 (eg, about Variants thereof containing amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, or up to about 3 (eg, any of about 1, 2 or 3) amino acids HC-CDR3 comprising the variant thereof, and the amino acid sequence of any one of SEQ ID NOs: 186-188, Or a heavy chain variable domain sequence comprising a variant thereof comprising substitutions of up to about 3 (eg, any of about 1, 2 or 3) amino acids; and ii) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 Or a variant thereof comprising a substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or up to about 3 (eg, about 1 A method is provided comprising a light chain variable domain comprising a variant thereof comprising amino acid substitutions (either 2 or 3). In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. An antibody portion, i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and HC comprising any one amino acid sequence of SEQ ID NOs: 186 to 188 An anti-E7MC comprising a heavy chain variable domain sequence comprising CDR3; and ii) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, and a light chain variable domain comprising LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 Administering a composition comprising an anti-E7MC construct comprising an antibody moiety. The law is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising an HPV16-E7 peptide and an MHC class I protein. An antibody portion, i) HC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 57-77, or up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids Variants thereof comprising HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 78-98, or up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids Variants thereof comprising substitutions; and HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245; or up to about 5 For example, any of about 1, 2, 3, 4, or 5) a heavy chain variable domain sequence comprising a variant thereof containing amino acid substitutions; and ii) an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids; comprising any one amino acid sequence of SEQ ID NOs: 141-161 LC-CDR2, or a variant thereof comprising substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids; and any one of SEQ ID NOs: 162-182 and 247-250 LC-CDR3 comprising an amino acid sequence; or substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids Comprising administering a composition comprising an anti E7MC constructs comprising anti E7MC antibody portion comprising a light chain variable domain sequence comprising Musono variants are provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. An antibody portion comprising: i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NOs: 99-119 HC-CDR3 comprising any one amino acid sequence of 244 and 245; or substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids within the HC-CDR sequence A heavy chain variable domain sequence comprising the variant thereof; and ii) the amino acid of any one of SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising the sequence; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or An anti-E7MC construct comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence comprising a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2 or 3) amino acids within the LC-CDR sequence A method is provided comprising administering a composition. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. An antibody portion comprising: i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NOs: 99-119 A heavy chain variable domain sequence comprising HC-CDR3 comprising any one amino acid sequence of 244 and 245; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; SEQ ID NO: 141 LC-CDR2 comprising the amino acid sequence of any one of ˜161; and SEQ ID NOs: 162-1 And a method comprising administering an anti-E7MC construct comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence comprising LC-CDR3 comprising an amino acid sequence of any one of 2 and 247-250. . In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. Administering a composition comprising an anti-E7MC construct comprising an antibody moiety, wherein the anti-E7MC antibody moiety comprises a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237; Or a variant thereof having at least about 95% sequence identity, such as at least about 96%, 97%, 98% or 99%, and any one amino acid of SEQ ID NOs: 36-56 and 238-243 A light chain variable domain comprising the sequence, or at least about 95% (eg, at least about 96%, 97% Including its variants with 98% or 99% sequence identity either), a method is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. Administering a composition comprising an anti-E7MC construct comprising an antibody moiety, wherein the anti-E7MC antibody moiety comprises a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237; And a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243 is provided. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the composition further comprises cells (eg, effector cells) associated with the anti-E7MC construct. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments of any of the methods for treating HPV16-E7 positive disease described above, the anti-E7MC construct is conjugated to a cell (eg, immune cell, eg, T cell) prior to administration to the individual. . Thus, for example, a method of treating an HPV16-E7 positive disease in an individual comprising: a) conjugating any one of the anti-E7MC constructs described herein with a cell (eg, an immune cell, eg, a T cell) Thus forming an anti-E7MC construct / cell conjugate, and b) administering to the individual an effective amount of a composition comprising the anti-E7MC construct / cell conjugate. In some embodiments, the cell is from an individual. In some embodiments, the cell is not derived from an individual. In some embodiments, the anti-E7MC construct and the cell are conjugated by covalent linkage with a molecule on the surface of the cell. In some embodiments, the anti-E7MC construct and the cell are conjugated by non-covalent binding to a molecule on the surface of the cell. In some embodiments, the anti-E7MC construct is conjugated to the cell by inserting a portion of the anti-E7MC construct into the outer membrane of the cell. In some embodiments, the anti-E7MC construct is not naturally occurring. In some embodiments, the anti-E7MC construct is a full length antibody. In some embodiments, the anti-E7MC construct is a multispecific (eg, bispecific) molecule. In some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, the individual is a mammal (eg, a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. In some embodiments, the individual is less than about 60 years old (e.g., less than about 50 years old, less than about 40 years old, less than about 30 years old, less than about 25 years old, less than about 20 years old, less than about 15 years old, or about 10 years old). Any one under age). In some embodiments, the individual is over about 60 years old (eg, including any of over about 70 years old, over about 80 years old, over about 90 years old, or over about 100 years old). In some embodiments, the individual has a disease or disorder described herein (eg, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck Or one or more of the cancers of the oropharynx) or is genetically susceptible to it. In some embodiments, the individual has one or more risk factors associated with one or more of the diseases or disorders described herein.

  The application provides, in some embodiments, an anti-E7MC construct (eg, an anti-E7MC described herein) in a cell that presents on its surface a complex comprising an HPV16-E7 peptide and an MHC class I protein in an individual. A method of delivering any one of the constructs) comprising administering to an individual a composition comprising an anti-E7MC construct. In some embodiments, the anti-E7MC construct to be delivered is associated with a cell (eg, an effector cell, eg, a T cell).

  HPV16-E7 positive cancer (eg squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer or oropharyngeal cancer) or HPV16-E7 Many diagnostic methods are known in the art for any other disease that exhibits expression and the clinical picture of those diseases. Such methods include, but are not limited to, immunohistochemical examination, PCR, and fluorescence in situ hybridization (FISH), for example.

  In some embodiments, an anti-E7MC construct and / or composition of the invention is treated with a second agent, third agent, or fourth agent (to treat a disease or disorder associated with HPV16-E7 expression ( For example, an antineoplastic agent, growth inhibitory agent, cytotoxic agent, or chemotherapeutic agent). In some embodiments, the anti-E7MC construct is administered in combination with an agent that increases the expression of MHC class I protein and / or enhances the surface presentation of HPV16-E7 peptide by MHC class I protein. In some embodiments, agents include, for example, IFN receptor agonists, Hsp90 inhibitors, enhancers of p53 expression, and chemotherapeutic agents. In some embodiments, the agent is an IFN receptor agonist, including, for example, IFNγ, IFNβ, and IFNα. In some embodiments, the agent is, for example, tanespimycin (17-AAG), alvespimycin (17-DMAG), letaspimycin (IPI-504), IPI-493, CNF2024 / BIIB021, MPC-3100, Hsp90 inhibition including Debio 0932 (CUDC-305), PU-H71, Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL888 It is an agent. In some embodiments, the agent is an enhancer of p53 expression including, for example, 5-fluorouracil and nutrine-3. In some embodiments, the agent is a chemotherapeutic agent including, for example, topotecan, etoposide, cisplatin, paclitaxel, and vinblastine.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein cells expressing HPV16-E7 typically comprise a complex comprising HPV16-E7 protein and MHC class I protein. Individuals that do not present on their surface or that present at a relatively low level are given a composition comprising an anti-E7MC construct to increase expression of MHC class I protein and / or HPV16 by MHC class I protein. -A method is provided comprising administering in combination with an agent that enhances the surface presentation of the E7 peptide. In some embodiments, agents include, for example, IFN receptor agonists, Hsp90 inhibitors, enhancers of p53 expression, and chemotherapeutic agents. In some embodiments, the agent is an IFN receptor agonist, including, for example, IFNγ, IFNβ, and IFNα. In some embodiments, the agent is, for example, tanespimycin (17-AAG), alvespimycin (17-DMAG), letaspimycin (IPI-504), IPI-493, CNF2024 / BIIB021, MPC-3100, Hsp90 inhibition including Debio 0932 (CUDC-305), PU-H71, Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL888 It is an agent. In some embodiments, the agent is an enhancer of p53 expression including, for example, 5-fluorouracil and nutrine-3. In some embodiments, the agent is a chemotherapeutic agent including, for example, topotecan, etoposide, cisplatin, paclitaxel, and vinblastine.

  Cancer treatment includes, for example, tumor regression, tumor weight or size reduction, time to progression, duration of survival, progression-free survival, response rate, duration of response, quality of life, protein expression And / or by activity. Techniques for determining the effectiveness of the therapy can be used including, for example, measurement of response by radiographic imaging.

  In some embodiments, the effectiveness of treatment is measured as a percentage of tumor growth inhibition (% TGI), which is equal to Equation 100− (T / C × 100), where T is the tumor being treated. Relative tumor volume, and C is the average relative tumor volume of untreated tumors). In some embodiments, the% TGI is 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 greater than 95%.

Methods of Dosing and Administering the Anti-E7MC Construct Composition The dose of the anti-E7MC construct composition administered to an individual (eg, a human) can vary depending on the particular composition, the mode of administration, and the type of disease being treated. In some embodiments, the amount of the composition is an amount effective to produce the desired response (eg, partial response or complete response). In some embodiments, the amount of anti-E7MC construct composition is sufficient to provide a complete response in the individual. In some embodiments, the amount of anti-E7MC construct composition is sufficient to provide a partial response in the individual. In some embodiments, the amount of anti-E7MC construct composition administered (eg, when administered alone) is about 20%, about 20% of the population of individuals treated with the anti-E7MC construct composition. 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 64%, about 65%, about 70%, about 75%, about 80% Sufficient to provide an overall response rate greater than either about 85% or about 90%. An individual's response to treatment with the methods described herein can be determined, for example, based on RECIST levels.

  In some embodiments, the amount of the composition is sufficient to prolong the progression free survival of the individual. In some embodiments, the amount of the composition is sufficient to prolong the overall survival of the individual. In some embodiments, the amount of the composition (eg, when administered alone) is about 50%, about 60% in the population of individuals treated with the anti-E7MC construct composition. Sufficient to provide a clinical benefit of greater than either about 70%, or about 77%.

  In some embodiments, the amount of the composition alone or in combination with the second agent, third agent, and / or fourth agent, the corresponding tumor size in the same subject prior to treatment, At least about 10%, about 20%, about 30%, about 40% compared to the number of cancer cells, or tumor growth rate, or compared to the corresponding activity in other subjects not receiving treatment Reduce tumor size, reduce the number of cancer cells, or about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 100%, or An amount sufficient to reduce the growth rate of the tumor. The magnitude of this effect can be measured using standard methods such as in vitro assays with purified enzymes, cell-based assays, animal models, or human tests.

  In some embodiments, the amount of anti-E7MC construct (eg, full-length anti-E7MC antibody, multispecific anti-E7MC molecule, anti-E7MC CAR, or anti-E7MC immunoconjugate) in the composition is a toxicological effect (ie, A level that induces (beyond a clinically acceptable level of toxicity), or a level at which potential side effects can be controlled or tolerated when the composition is administered to an individual.

  In some embodiments, the amount of the composition is near the maximum tolerated dose (MTD) of the composition according to the same dosing regimen. In some embodiments, the amount of the composition is greater than either about 80%, about 90%, about 95%, or about 98% of the MTD.

  In some embodiments, the amount of anti-E7MC construct (eg, full length anti-E7MC antibody, multispecific anti-E7MC molecule, anti-E7MC CAR, or anti-E7MC immunoconjugate) in the composition is from about 0.001 μg to about It is included in the range of 1000 μg.

  In some embodiments of any of the above aspects, an effective amount of an anti-E7MC construct (eg, full-length anti-E7MC antibody, multispecific anti-E7MC molecule, anti-E7MC CAR, or anti-E7MC immunoconjugate) in the composition. Is in the range of about 0.1 μg to about 100 mg per kg of total body weight.

  The anti-E7MC construct composition can be administered to an individual (eg, a human), for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal. Can be administered by a variety of routes including transmucosal and transdermal. In some embodiments, sustained continuous release formulations of the compositions can be used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraportally. In some embodiments, the composition is administered intraarterially. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic artery infusion.

Anti-E7MC CAR Effector Cell Therapy This application relates to the use of anti-E7MC CAR to redirect the specificity of effector cells (eg, primary T cells) to complexes comprising HPV16-E7 peptides and MHC class I proteins. Also provide. Accordingly, the present invention provides a method of stimulating an effector cell-mediated response (eg, a T cell-mediated immune response) to a target cell population or tissue comprising cells presenting E7MC in a mammal comprising the steps of: Also provided is a method comprising administering an effector cell (eg, T cell) that expresses an anti-E7MC CAR.

  Anti-E7MC CAR effector cells (eg, T cells) that express anti-E7MC CAR can be injected into a recipient in need thereof. The injected cells can kill cells presenting E7MC in the recipient. In some embodiments, unlike antibody therapy, anti-E7MC CAR effector cells (eg, T cells) can replicate in vivo, resulting in long-term tumor control that can result in sustained tumor control. Sustainability is provided.

  In some embodiments, the anti-E7MC CAR effector cell is an anti-E7MC CAR T cell that is capable of undergoing robust in vivo T cell expansion and is sustainable over time. In some embodiments, the anti-E7MC CAR T cells of the invention develop into specific memory T cells that can be reactivated to inhibit any further tumor formation or growth.

  The anti-E7MC CAR T cells of the present invention may also function as a type of vaccine for ex vivo immunization and / or in vivo therapy in mammals. In some embodiments, the mammal is a human.

  For ex vivo immunization, at least one of the following is performed in vitro prior to administering the cell to the mammal: i) expansion of the cell, ii) the nucleic acid encoding the anti-E7MC CAR. Introduction into cells and / or iii) cryopreservation of cells.

  Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (ie, transduced or transfected in vitro) using the vectors expressing the anti-E7MC CAR disclosed herein. To do). Anti-E7MC CAR cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human and the anti-E7MC CAR cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem cells and hematopoietic progenitor cells described in US Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method of expansion of cells ex vivo. Briefly, ex vivo culture and expansion of T cells comprises (1) collecting mammalian-derived CD34 ⁺ hematopoietic stem cells and hematopoietic progenitor cells from a peripheral blood collection or bone marrow explant; and (2 ) Expanding such cells ex vivo. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand are used for cell culture and expansion. Can be used.

  In addition to using cell-based vaccines for ex vivo immunity, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

  The anti-E7MC CAR effector cells (eg, T cells) of the present invention can be either alone or as a pharmaceutical composition in combination with diluents and / or other components or cell populations such as IL-2 or other cytokines. Can be administered. Briefly, the pharmaceutical composition of the present invention comprises anti-E7MC CAR effector cells (eg, T cells) with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. May be included in combination. Such compositions include, for example, buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants A chelating agent such as EDTA or glutathione; an adjuvant (eg, aluminum hydroxide); and a preservative. In some embodiments, the anti-E7MC CAR effector cell (eg, T cell) composition is formulated for intravenous administration.

The exact amount of the anti-E7MC CAR effector cell (eg, T cell) composition of the invention to be administered will depend on the physician's age, weight, tumor size, degree of infection or metastasis, and individuality of the condition. Can be determined by taking into account the differences. In some embodiments, the anti E7MC CAR effector cells (eg, T cells) a pharmaceutical composition comprising a ⁴ to about 10 ⁹ body weight 1kg per cell to about 10, such as weight 1kg per cell to about ¹⁰⁴ to about ¹⁰⁵ including pieces, about 10 ⁵ to about 10 ^6, about ¹⁰⁶ to about ^107, from about 10 ⁷ to about 10 ^8, or about 10 ⁸ to about 10 ⁹ (all integer values within these ranges )) At any dose. The anti-E7MC CAR effector cell (eg, T cell) composition can also be administered multiple times at these dosages. Cells can be administered by using generally known infusion techniques in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med., 319: 1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting treatment accordingly.

  In some embodiments, activated anti-E7MC CAR T cells are administered to a subject, after which blood is again drawn (or apheresis is performed), from which T cells are activated according to the present invention, to the patient. It may be desirable to reinject the activated and expanded T cells. This process can be performed multiple times every few weeks. In some embodiments, T cells can be activated from 10 cc to 400 cc of blood draw. In some embodiments, T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc blood draws.

  Administration of anti-E7MC CAR effector cells (eg, T cells) can be done in any convenient manner, including by aerosol inhalation, injection, oral ingestion, blood transfusion, implantation or implantation. The compositions described herein may be administered to a patient, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. Can be administered. In some embodiments, an anti-E7MC CAR effector cell (eg, T cell) composition of the invention is administered to a patient by intradermal or subcutaneous injection. In some embodiments, an anti-E7MC CAR effector cell (eg, T cell) composition of the invention is i. v. Administer by injection. The composition of anti-E7MC CAR effector cells (eg, T cells) can be injected directly into the tumor, lymph node, or site of infection.

Thus, for example, in some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual is specific for a complex comprising an effective amount of a) an HPV16-E7 peptide and an MHC class I protein. Expressing an anti-E7MC CAR comprising an extracellular domain comprising an anti-E7MC antibody moiety that binds electrically, b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence There is provided a method comprising administering a composition comprising effector cells (eg, T cells). In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-A ^* 02 ^: 01. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual includes an effective amount of a) HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A ^* 02: An extracellular domain comprising an anti-E7MC antibody moiety that specifically binds to a complex comprising 01, b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence Administering a composition comprising an effector cell (eg, T cell) that expresses an anti-E7MC CAR comprising: In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion, i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, or a variant thereof comprising substitution of up to about 3 (eg, about 1, 2 or 3) amino acids HC-CDR2 comprising the amino acid sequence of number 184 or 185, or variants thereof comprising substitutions of up to about 3 (eg, any of about 1, 2, or 3) amino acids, and any one of SEQ ID NOs: 186-188 HC-CDR3 containing one amino acid sequence, or up to about 3 (eg, any of about 1, 2, or 3) amino acid positions A heavy chain variable domain sequence comprising a variant thereof comprising: and ii) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or up to about 3 (eg, any of about 1, 2, or 3) amino acids Light chain variable comprising a variant thereof including substitutions, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or substitutions of up to about 3 (eg, any of about 1, 2, or 3) amino acids Expressing an anti-E7MC CAR comprising an extracellular domain comprising an anti-E7MC antibody portion comprising a domain, b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence Administering a composition comprising effector cells (e.g., T cells) , A method is provided. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion comprising: i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 183, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and any one amino acid sequence of SEQ ID NOs: 186 to 188 A heavy chain variable domain sequence comprising HC-CDR3 comprising; and ii) comprising a light chain variable domain comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191 , An extracellular domain comprising an anti-E7MC antibody portion, b) a transmembrane domain, and c) A method comprising administering a composition comprising an effector cell (eg, T cell) expressing an anti-E7MC CAR, comprising an intracellular signaling domain comprising a D3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence is provided. Is done. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion, i) HC-CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 57-77, or at most about 5 (eg, any of about 1, 2, 3, 4, or 5) ) Variants thereof containing amino acid substitutions, HC-CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 78-98, or up to about 5 (eg, any of about 1, 2, 3, 4, or 5) Variants thereof comprising amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 99-119, 244 and 245, or at most A heavy chain variable domain comprising a variant thereof comprising 5 (eg, any of about 1, 2, 3, 4, or 5) amino acid substitutions; and ii) an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246 Or an amino acid sequence of any one of SEQ ID NOs: 141-161 comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids LC-CDR2 comprising, or a variant thereof comprising substitution of up to about 3 (eg, any of about 1, 2, or 3) amino acids, and any one amino acid sequence of SEQ ID NOs 162-182 and 247-250 LC-CDR3 containing, or its vacancies containing up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acid substitutions An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain comprising ant; b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence A method is provided comprising administering a composition comprising effector cells (eg, T cells) that express anti-E7MC CAR. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion, i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NO: 99 HC-CDR3 comprising any one amino acid sequence of ˜119, 244 and 245; or up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids within the HC-CDR sequence A heavy chain variable domain sequence comprising that variant containing substitutions; and ii) the amino acid of any one of SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising the sequence; LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161; and LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or LC A cell comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence comprising a variant thereof comprising a substitution of up to about 5 (eg, any of about 1, 2, 3, 4, or 5) amino acids within the CDR sequence An effector cell (eg, T cell) expressing an anti-E7MC CAR comprising an outer domain; b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence A method is provided comprising administering a composition. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion, i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57-77; HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98; and SEQ ID NO: 99 A heavy chain variable domain sequence comprising HC-CDR3 comprising any one amino acid sequence of ˜119, 244 and 245; and ii) LC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 120-140 and 246; LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161; and SEQ ID NO: 162 An extracellular domain comprising an anti-E7MC antibody portion comprising a light chain variable domain sequence comprising LC-CDR3 comprising an amino acid sequence of any one of 182 and 247-250; b) a transmembrane domain, and c) CD3ζ intracellular signaling A method is provided comprising administering a composition comprising an effector cell (eg, T cell) that expresses an anti-E7MC CAR comprising a sequence and an intracellular signaling domain comprising a CD28 intracellular signaling sequence. In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating an HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a) a complex comprising HPV16-E7 peptide and MHC class I protein. An anti-E7MC antibody portion, i) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about 95% (eg, at least about 96%, 97%, 98 A variant thereof having a sequence identity of either 1% or 99%) and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or at least about 95% (eg, at least Variants thereof having a sequence identity of about 96%, 97%, 98% or 99%) An extracellular domain comprising an anti-E7MC antibody portion; b) a transmembrane domain, and c) an effector expressing an anti-E7MC CAR comprising an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence A method is provided comprising administering a composition comprising cells (eg, T cells). In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

  In some embodiments, a method of treating HPV16-E7 positive disease in an individual, wherein the individual specifically binds to an effective amount of a complex comprising HPV16-E7 peptide and MHC class I protein. An antibody portion comprising a) a heavy chain variable domain comprising any one amino acid sequence of SEQ ID NOs: 15-35 and 233-237 and a light chain comprising any one amino acid sequence of SEQ ID NOs: 36-56 and 238-243 An anti-E7MC CAR comprising an extracellular domain comprising an anti-E7MC antibody portion comprising a chain variable domain; b) a transmembrane domain, and c) an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence. A step of administering a composition comprising an effector cell to be expressed (eg, a T cell). A method is provided that includes: In some embodiments, the HPV 16-E7 positive disease is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer . In some embodiments, the cancer is HPV16-E7 positive squamous cell carcinoma. In some embodiments, the individual is a human.

Cancer In some embodiments, anti-E7MC constructs and anti-E7MC CAR cells may be useful for treating HPV16-E7 positive cancer. Cancers that can be treated using any of the methods described herein include tumors that are not vascularized or not yet substantially vascularized, as well as vascularized tumors. The cancer can include non-solid tumors (eg, hematological tumors, such as leukemias and lymphomas), or can include solid tumors. Types of cancer treated with the anti-E7MC constructs and anti-E7MC CAR cells of the present invention include, but are not limited to, cancer (carcinoma), blastoma, and sarcoma, and certain leukemias or lymphatic systems. Examples include malignant tumors, benign tumors and malignant tumors, and malignancies such as sarcomas, cancers, and melanomas. Adult tumor / cancer and childhood tumor / cancer are also included.

  A hematological cancer is a cancer of the blood or bone marrow. Examples of blood (or hematogenous cancers) include acute leukemias (eg, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, as well as myeloblastic leukemia, preosteoblastic leukemia) Leukemias, including red blood cells, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (eg, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia) Augmentation, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and spinal cord Examples include dysplasia.

  A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The names of the different types of solid tumors are derived from the cell types that form them (eg, sarcomas, cancers, and lymphomas). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial, mesothelioma, Ewing sarcoma, leiomyosarcoma, striated muscle Tumor, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid cancer, Papillary thyroid cancer, pheochromocytoma, sebaceous gland cancer, papillary cancer, papillary carcinoma, medullary cancer, bronchogenic cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer (eg Cervical cancer and preinvasive cervical dysplasia), anal, anal canal or anorectal cancer, vaginal cancer, vulvar cancer (eg squamous cell carcinoma, carcinoma in situ, adenocarcinoma and Fibrosarcoma), penile cancer, oropharyngeal cancer, head cancer (eg squamous cell carcinoma), cervical cancer Eg squamous cell carcinoma), testicular cancer (eg seminoma, teratomas, fetal cancer, teratocarcinoma, choriocarcinoma, sarcoma, Leydig celloma, fibroma, fibroadenoma, adenomatous tumor and lipoma), bladder Cancer, melanoma, uterine cancer (eg, endometrial cancer), urothelial cancer (eg, squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral and bladder cancer), and CNS Tumors (eg, glioma (eg, brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, Medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retina Blastoma and brain metastases).

  Cancer treatment includes, for example, tumor regression, tumor weight or size reduction, time to progression, duration of survival, progression-free survival, response rate, duration of response, quality of life, protein expression And / or by activity. Techniques for determining the effectiveness of the therapy can be used including, for example, measurement of response by radiographic imaging.

Methods for Diagnosis and Imaging Using Anti-E7MC Constructs Labeled anti-E7MC antibody moieties that specifically bind to E7MC on the surface of cells and derivatives and analogs thereof can be used for the above diseases and disorders such as cancer. Diseases and / or disorders associated with the expression of HPV16-E7, including any of the above, can be used to detect, diagnose, or monitor for diagnostic purposes. For example, the anti-E7MC antibody portion of the invention can be used in diagnostic or imaging assays in situ, in vivo, ex vivo, and in vitro.

  Further embodiments of the invention include methods of diagnosing a disease or disorder associated with the expression of HPV16-E7 in an individual (eg, a mammal such as a human). The method includes detecting cells presenting E7MC in the individual. In some embodiments, a method of diagnosing a disease or disorder associated with expression of HPV16-E7 in an individual (eg, a mammal such as a human), wherein (a) the individual is in an effective amount of the above embodiment. Administering a labeled anti-E7MC antibody moiety according to any of the above; and (b) determining the level of labeling in the individual, wherein the level of labeling above the threshold level indicates that the individual has a disease or disorder A method is provided that includes the steps of: The threshold level is detected, for example, according to the diagnostic method described above in a first set of individuals with a disease or disorder and a second set of individuals without a disease or disorder, and the first set and second It can be determined by various methods, including by setting a threshold at a level that allows discrimination between sets. In some embodiments, the threshold level is zero and the method includes determining the presence or absence of the label in the individual. In some embodiments, the method preferentially concentrates the labeled anti-E7MC antibody portion at a site where E7MC is expressed in an individual at a time interval after administration of step (a) ( And removing the unbound labeled anti-E7MC antibody portion). In some embodiments, the method further comprises subtracting the background level of the label. The background level is, for example, by detecting the label in the individual prior to administration of the labeled anti-E7MC antibody portion, or by detecting the label according to the diagnostic method described above in an individual having no disease or disorder. Can be determined by various methods, including: In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is, for example, from squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. Selected from the group consisting of

The anti-E7MC antibody portion of the invention can be used to assay the level of cells presenting E7MC in a biological sample using methods known to those skilled in the art. Suitable antibody labels are known in the art and include enzyme labels as those, such as glucose oxidase; Iodine ^{(131 I, 125 I, 123} I, 121 I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), Tritium ( ³ H), indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ In), technetium ( ⁹⁹ Tc, ^{99 m} Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd) , Molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), samarium ( ¹⁵³ Sm), lutetium ( ¹⁷⁷ Lu), gadolinium ( ¹⁵⁹ Gd), promethium ( ¹⁴⁹ Pm), lanthanum ( ¹⁴⁰ La), ytterbium ⁽¹⁷⁵ Yb), holmium ⁽¹⁶⁶ Ho , Yttrium ⁽⁹⁰ Y), scandium ⁽⁴⁷ Sc), rhenium ^{(186 Re,} 188 ^Re), praseodymium ⁽¹⁴² Pr), rhodium ⁽¹⁰⁵ Rh), and radioactive isotopes, such as ruthenium ⁽⁹⁷ Ru); luminol; fluorescein and Fluorescent labels such as rhodamine; as well as biotin.

  Techniques known in the art can be applied to the labeled anti-E7MC antibody portion of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugate agents (eg, US Pat. Nos. 5,756,065; 5,714,631; 696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; No. 5,342,604; 5,274,119; 4,994,560; and 5,808,003). In addition to the above assays, various in vivo and ex vivo assays are available to those skilled in the art. For example, cells in a subject's body can be exposed to an anti-E7MC antibody portion that is optionally labeled with a detectable label, eg, a radioisotope, and binding of the anti-E7MC antibody portion to the cell can be achieved, for example, Can be assessed by external scanning for radioactivity, or by analyzing a sample (eg, a biopsy or other biological sample) derived from a subject that has been previously exposed to an anti-E7MC antibody portion.

Articles of Manufacture and KitsIn some embodiments of the invention, for delivering an anti-E7MC construct to cells presenting E7MC on its surface, or for isolating or detecting cells presenting E7MC in an individual. Treatment of HPV16-E7 positive diseases such as squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer or oropharyngeal cancer Articles of manufacture containing useful materials are provided. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container may be formed from a variety of materials such as glass or plastic. In general, a container holds a composition that is effective in the treatment of the diseases or disorders described herein and may have a sterile access port (eg, the container may be punctured with an intravenous solution bag or hypodermic needle. It may be a vial with a stopcock that can be opened). At least one active agent in the composition is an anti-E7MC construct of the invention. The label or package insert indicates that the composition is to be used to treat a particular condition. The label or package insert further comprises instructions regarding administration of the anti-E7MC construct composition to the patient. Articles of manufacture and kits comprising the combinatorial therapy described herein are also contemplated.

  The package insert contains information about indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products, which are customarily included in packages of commercial therapeutic products Point to instructions to do. In some embodiments, the package insert is such that the composition comprises HPV16-E7 positive cancer (eg, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, , Head and neck cancer or oropharyngeal cancer).

  In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

  Delivering an anti-E7MC construct to cells presenting E7MC on its surface for various purposes, optionally in combination with an article of manufacture, eg, for the treatment of HPV16-E7 positive diseases or disorders described herein Also provided are kits useful for isolating or detecting cells presenting E7MC in an individual. The kits of the invention comprise one or more containers comprising an anti-E7MC construct composition (or unit dosage form and / or article of manufacture), and in some embodiments, another agent (eg, as described herein). Instructions) and / or instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of the selection of individuals suitable for treatment. The instructions supplied in the kits of the present invention are generally instructions written on a label or package insert (eg, a paper sheet included in the kit), but are machine readable instructions (eg, magnetic or optical). Instructions held on a typical storage disk) are also allowed.

  For example, in some embodiments, the kit includes an anti-E7MC construct (eg, full-length anti-E7MC antibody, multispecific anti-E7MC molecule (eg, bispecific anti-E7MC antibody), or anti-E7MC immunoconjugate). Including a composition. In some embodiments, the kit includes: a) a composition comprising an anti-E7MC construct, and b) an effective amount of at least one other agent that increases MHC class I protein expression and / or Other agents that enhance the surface presentation of HPV16-E7 peptides by MHC class I proteins (eg, IFNγ, IFNβ, IFNα, or Hsp90 inhibitors) are included. In some embodiments, the kit comprises a) a composition comprising an anti-E7MC construct, and b) for the treatment of HPV16-E7 positive diseases including, for example, squamous cell carcinoma, cervical cancer or anal cancer. Instructions for administering the anti-E7MC construct composition to the individual. In some embodiments, the kit comprises a) a composition comprising an anti-E7MC construct, b) an effective amount of at least one other agent that increases expression of MHC class I protein and / or MHC. Other agents that enhance the surface presentation of HPV16-E7 peptides by class I proteins (eg, IFNγ, IFNβ, IFNα, or Hsp90 inhibitors), and c) eg, squamous cell carcinoma, cervical cancer or anal cancer Instructions for administering an anti-E7MC construct composition and other drug (s) to an individual for the treatment of HPV16-E7 positive diseases, including: The anti-E7MC construct and other drug (s) may be present in separate containers or may be present in a single container. For example, a kit may comprise one separate composition, two or more compositions, wherein one composition comprises an anti-E7MC construct and another composition comprises another agent. The composition containing this may be included.

  In some embodiments, the kit comprises a) an anti-E7MC construct (eg, full length anti-E7MC antibody, multispecific anti-E7MC molecule (eg, bispecific anti-E7MC antibody), or anti-E7MC immunoconjugate). A composition, and b) combining an anti-E7MC construct with a cell (eg, a cell from an individual, eg, an immune cell) to form a composition comprising the anti-E7MC construct / cell conjugate to form an anti-E7MC construct / cell conjugate composition Instructions for administering the product to an individual for the treatment of HPV16-E7 positive disease (including for example squamous cell carcinoma, cervical cancer or anal cancer). In some embodiments, the kit comprises a) a composition comprising an anti-E7MC construct, and b) a cell (eg, a cytotoxic cell). In some embodiments, the kit comprises a) a composition comprising an anti-E7MC construct, b) a cell (eg, a cytotoxic cell), and c) an anti-E7MC construct / cell conjugate combining the anti-E7MC construct with a cell. And an anti-E7MC construct / cell conjugate composition is administered to an individual for the treatment of HPV16-E7 positive diseases including, for example, squamous cell carcinoma, cervical cancer or anal cancer Including instructions on In some embodiments, the kit comprises a composition comprising an anti-E7MC construct associated with a cell (eg, a cytotoxic cell). In some embodiments, the kit comprises a) a composition comprising an anti-E7MC construct associated with cells (eg, cytotoxic cells), and b) eg, squamous cell carcinoma, cervical cancer or anal cancer Including instructions for administering the composition to an individual for the treatment of HPV16-E7 positive diseases. In some embodiments, the association is by conjugation of molecules on the surface of the cell with the anti-E7MC construct. In some embodiments, the association is by inserting a portion of the anti-E7MC construct into the outer membrane of the cell.

  In some embodiments, the kit is an anti-E7MC construct (eg, full-length anti-E7MC antibody, multispecific anti-E7MC molecule (eg, bispecific anti-E7MC antibody), anti-E7MC CAR, or anti-E7MC immunoconjugate). Or a nucleic acid (or set of nucleic acids) encoding the polypeptide portion. In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-E7MC construct or polypeptide portion thereof, and b) a host cell for expressing the nucleic acid (or set of nucleic acids) (eg, Effector cells). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-E7MC construct or a polypeptide portion thereof, and b) i) an anti-E7MC construct in a host cell (eg, an effector cell, eg, Ii) preparing a composition comprising an anti-E7MC construct or a host cell expressing an anti-E7MC construct; and iii) a composition comprising a host cell expressing an anti-E7MC construct or anti-E7MC construct. Instructions for administering the product to an individual for the treatment of HPV16-E7 positive diseases including, for example, squamous cell carcinoma, cervical cancer or anal cancer. In some embodiments, the host cell is from an individual. In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an anti-E7MC construct or polypeptide portion thereof; b) a host cell for expressing the nucleic acid (or set of nucleic acids) (eg, Effector cells), and c) i) expressing an anti-E7MC construct in a host cell, ii) preparing an anti-E7MC construct or a host cell expressing an anti-E7MC construct, and iii) an anti-E7MC construct. Or instructions on administering a composition comprising host cells expressing an anti-E7MC construct to an individual for the treatment of HPV16-E7 positive diseases including, for example, squamous cell carcinoma, cervical cancer or anal cancer Including.

  In some embodiments, the kit includes a nucleic acid encoding an anti-E7MC CAR. In some embodiments, the kit comprises a vector comprising a nucleic acid encoding an anti-E7MC CAR. In some embodiments, the kit comprises a) a vector comprising a nucleic acid encoding an anti-E7MC CAR, and b) i) introducing the vector into an effector cell such as a T cell derived from an individual, ii) an anti-E7MC Preparing a composition comprising CAR effector cells, and iii) anti-E7MC CAR effector cell compositions for the treatment of HPV16-E7 positive diseases including, for example, squamous cell carcinoma, cervical cancer or anal cancer Instructions for administering to an individual.

  The kit of the invention is placed in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. The kit may optionally also provide additional components such as buffers and explanatory information. Accordingly, the application also provides articles of manufacture including vials (eg, sealed vials), bottles, jars, flexible packaging, and the like.

  Instructions regarding the use of the anti-E7MC construct composition generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (eg, a multiple dose package), or a sub-unit dose. For example, the effective treatment of an individual can be, for example, 1 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 Anti-E7MC as disclosed herein in a dosage sufficient to provide an extended period of time, either 4 months, 5 months, 7 months, 8 months, 9 months or more Kits containing constructs (eg, full length anti-E7MC antibodies, multispecific anti-E7MC molecules (eg, bispecific anti-E7MC antibodies), anti-E7MC CAR, or anti-E7MC immunoconjugates) can be provided. The kit also includes multiple unit doses of the anti-E7MC construct and pharmaceutical composition and instructions for use, and may be packaged in an amount sufficient for storage and use in a pharmacy, eg, hospital pharmacy and dispensing pharmacy. Good.

Exemplary Embodiments Embodiment 1 In some embodiments, a complex comprising a human papillomavirus subtype 16 (HPV16) E7 peptide and a major histocompatibility (MHC) class I protein (HPV16-E7 / MHC class I complex or E7MC) An isolated anti-E7MC construct comprising an antibody moiety that specifically binds to is provided.

  Embodiment 2. FIG. In some further embodiments of embodiment 1, the HPV16-E7 / MHC class I complex is present on the cell surface.

  Embodiment 3. FIG. In some further embodiments of embodiment 1, the HPV16-E7 / MHC class I complex is present on the surface of a cancer cell.

  Embodiment 4 FIG. In some further embodiments of any one of embodiments 1-3, the MHC class I protein is human leukocyte antigen (HLA) -A.

  Embodiment 5. FIG. In some further embodiments of embodiment 4, the MHC class I protein is HLA-A02.

Embodiment 6. FIG. In some further embodiments of embodiment 5, the MHC class I protein is HLA-A ^* 02: 01, HLA-A ^* 02: 02, HLA-A ^* 02: 06, HLA-A ^* 02: 07 and Selected from the group consisting of HLA-A ^* 02 ^: 11.

Embodiment 7. FIG. In some further embodiments of embodiment 6, the MHC class I protein is HLA-A ^* 02 ^: 01.

  Embodiment 8. FIG. In some further embodiments of any one of embodiments 1 to 7, the antibody moiety comprises a second MHC class I protein having an HLA allele different from the HPV16-E7 peptide and the MHC class I protein. Cross-react with.

  Embodiment 9. FIG. In some further embodiments of any one of embodiments 1-8, the HPV16-E7 peptide is 8-12 amino acids in length.

  Embodiment 10 FIG. In some further embodiments of any one of embodiments 1 to 9, the HPV 16-E7 peptide is derived from an E7 protein of human papillomavirus subtype 16.

  Embodiment 11. FIG. In some further embodiments of any one of embodiments 1 to 10, the HPV 16-E7 peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-14.

  Embodiment 12 FIG. In some further embodiments of embodiment 10, the HPV16-E7 peptide has an amino acid sequence of YMLDLQPET (SEQ ID NO: 4).

  Embodiment 13. FIG. In some further embodiments of embodiment 12, the isolated anti-E7MC construct cross-reacts with a complex comprising a variant of HPV16-E7 peptide having the amino acid sequence of YMLVQPETET (SEQ ID NO: 11) and an MHC class I protein. To do.

  Embodiment 14 FIG. In some further embodiments of any one of embodiments 1 to 13, the antibody moiety is human, humanized or semi-synthetic.

  Embodiment 15. FIG. In some further embodiments of any one of embodiments 1 to 14, the antibody moiety is a full-length antibody, Fab, Fab ', (Fab') 2, Fv or single chain Fv (scFv).

  Embodiment 16. FIG. In some further embodiments of any one of embodiments 1-15, the antibody moiety binds to an HPV16-E7 / MHC class I complex with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM.

  Embodiment 17. FIG. In some further embodiments of any one of embodiments 1-16, the isolated anti-E7MC construct binds to an HPV16-E7 / MHC class I complex with a Kd of about 0.1 pM to about 500 nM.

Embodiment 18. FIG. In some further embodiments of any one of embodiments 1 to 17, the antibody moiety is
i) heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of GF / G / YSS / TFSS / TSYA / G (SEQ ID NO: 183), Or a variant thereof containing up to about 3 amino acid substitutions, I / N / I-X-X-G-G / T / IT / A / P or I-S-X-S / D-G HC-CDR2 comprising the amino acid sequence of / NG / SNT / I / K (SEQ ID NO: 184 or 185), or variants thereof containing up to about 3 amino acid substitutions, and ARS / RY / S / G-Y / V-Y / W-G-X-Y-D, A-R-G-X-X-X-Y-Y / G / S or A-R-G- HC-CDR3 comprising any one amino acid sequence of XXXYQ / WWSXDD (SEQ ID NO: 186 to 188), or about 3 at most A heavy chain variable domain comprising a variant thereof containing a substitution of noic acid; and ii) an amino acid of NIGSGN / K or LRS / NXY (SEQ ID NO: 189 or 190) A light chain complementarity determining region (LC-CDR) 1 comprising a sequence, or variant thereof comprising substitutions of up to about 3 amino acids, and A / Q / NS / A / VW / Y / RD- Light chain variable comprising LC-CDR3 comprising the amino acid sequence of S / DSL / S / GXXXXV (SEQ ID NO: 191), or variants thereof containing substitutions of up to about 3 amino acids Contains a domain, where X can be any amino acid.

Embodiment 19. FIG. In some further embodiments of any one of embodiments 1 to 17, the antibody moiety is
i) HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 57-77, or variants thereof containing substitutions of up to about 5 amino acids, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98 Or a variant thereof comprising a substitution of up to about 5 amino acids, and HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245; or a variant thereof comprising a substitution of up to about 5 amino acids A heavy chain variable domain comprising; and ii) any LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or variants thereof comprising substitutions of up to about 5 amino acids, SEQ ID NOs: 141-161 LC-CDR2 containing one amino acid sequence, or up to about 3 amino acid positions A light chain variable domain comprising a variant thereof including LC, and LC-CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5 amino acid substitutions.

Embodiment 20. FIG. In some further embodiments of any one of embodiments 1 to 17, the antibody moiety is
i) HC-CDR1 comprising any one amino acid sequence of SEQ ID NOs: 57 to 77, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78 to 98, and any of SEQ ID NOs: 99 to 119, 244 and 245 A heavy chain (HC) variable domain comprising HC-CDR3 comprising any one amino acid sequence; or variants thereof comprising substitutions of up to about 5 amino acids in the HC-CDR region; and ii) SEQ ID NOs: 120-140 and 246 LC-CDR1 comprising any one amino acid sequence, LC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 141-161, and LC comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250 A light chain (LC) variable domain comprising CDR3; or in the LC-CDR region Including variants thereof containing up to about 5 amino acid substitutions.

  Embodiment 21. FIG. In some further embodiments of embodiment 19 or 20, the antibody portion comprises: a) a heavy chain variable domain comprising an amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or SEQ ID NOs: 15-35 and A variant thereof having at least about 95% sequence identity to any one of 233-237; and b) a light chain comprising any one of the amino acid sequences of SEQ ID NOs: 36-56 and 238-243 Variable domains, or variants thereof having at least about 95% sequence identity to any one of SEQ ID NOs: 36-56 and 238-243.

  Embodiment 22. FIG. In some further embodiments of embodiment 21, the antibody portion comprises a heavy chain variable domain comprising any one amino acid sequence of SEQ ID NOs: 15-35 and 233-237 and any of SEQ ID NOs: 36-56 and 238-243. A light chain variable domain comprising one amino acid sequence.

  Embodiment 23. FIG. In some further embodiments of any one of embodiments 1-22, the isolated anti-E7MC construct is a full-length antibody.

  Embodiment 24. FIG. In some further embodiments of any one of embodiments 1 to 23, the isolated anti-E7MC construct is monospecific.

  Embodiment 25. FIG. In some further embodiments of any one of embodiments 1 to 23, the isolated anti-E7MC construct is multispecific.

  Embodiment 26. FIG. In some further embodiments of embodiment 25, the isolated anti-E7MC construct is bispecific.

  Embodiment 27. FIG. In some further embodiments of embodiment 25 or 26, the isolated anti-E7MC construct is a tandem scFv, diabody (Db), single chain diabody (scDb), dual affinity retargeting (DART). Antibodies, double variable domain (DVD) antibodies, knob-in-hole (KiH) antibodies, dock-and-lock (DNL) antibodies, chemically cross-linked antibodies, heteromultimeric antibodies or heteroconjugate antibodies.

  Embodiment 28. FIG. In some further embodiments of embodiment 27, the isolated anti-E7MC construct is a tandem scFv comprising two scFvs linked by a peptide linker.

  Embodiment 29. FIG. In some further embodiments of embodiment 28, the peptide linker comprises the amino acid sequence GGGGS.

  Embodiment 30. FIG. In some further embodiments of any one of embodiments 25-29, the isolated anti-E7MC construct further comprises a second antibody portion that specifically binds to a second antigen.

  Embodiment 31. FIG. In some further embodiments of embodiment 30, the second antigen is an antigen on the surface of a T cell.

  Embodiment 32. FIG. In some further embodiments of embodiment 31, the second antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.

  Embodiment 33. FIG. In some further embodiments of embodiment 31, the second antigen is CD3ε, wherein the isolated anti-E7MC construct comprises an N-terminal scFv specific for the HPV16-E7 / MHC class I complex and A tandem scFv containing a C-terminal scFv specific for CD3ε.

  Embodiment 34. FIG. In some further embodiments of embodiment 31, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, and natural killer T cells.

  Embodiment 35. FIG. In some further embodiments of embodiment 30, the second antigen is an antigen on the surface of natural killer cells, neutrophils, monocytes, macrophages or dendritic cells.

  Embodiment 36. FIG. In some further embodiments of any one of embodiments 1-22, the isolated anti-E7MC construct is a chimeric antigen receptor.

  Embodiment 37. FIG. In some further embodiments of embodiment 36, the chimeric antigen receptor comprises an extracellular domain comprising an antibody portion, a transmembrane domain, and an intracellular signaling comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence. Domain.

  Embodiment 38. FIG. In some further embodiments of any one of embodiments 1-22, the isolated anti-E7MC construct is an immunoconjugate comprising an antibody moiety and an effector molecule.

  Embodiment 39. FIG. In some further embodiments of embodiment 37, the effector molecule is a therapeutic agent selected from the group consisting of drugs, toxins, radioisotopes, proteins, peptides and nucleic acids.

  Embodiment 40. FIG. In some further embodiments of embodiment 39, the therapeutic agent is a drug or toxin.

  Embodiment 41. FIG. In some further embodiments of embodiment 38, the effector molecule is a label.

  Embodiment 42. FIG. In some embodiments, a pharmaceutical composition comprising the isolated anti-E7MC construct according to any one of embodiments 1 to 40 is provided.

  Embodiment 43. FIG. In some embodiments, host cells expressing the isolated anti-E7MC construct according to any one of embodiments 1-41 are provided.

  Embodiment 44. FIG. In some embodiments, a nucleic acid encoding a polypeptide component of the isolated anti-E7MC construct according to any one of embodiments 1-41 is provided.

  Embodiment 45. FIG. In some embodiments, a vector comprising the nucleic acid of embodiment 44 is provided.

  Embodiment 46. FIG. In some embodiments, effector cells expressing the isolated anti-E7MC construct of embodiment 36 or 37 are provided.

  Embodiment 47. In some further embodiments of embodiment 46, the effector cells are T cells.

  Embodiment 48. FIG. In some embodiments, a method for detecting a cell that presents a complex comprising an HPV16-E7 peptide and an MHC class I protein on its surface, wherein the cell is isolated as described in embodiment 41. There is provided a method comprising the steps of contacting with an anti-E7MC construct and detecting the presence of a label on the cell.

  Embodiment 49. In some embodiments, there is provided a method for treating an individual having an HPV16-E7 positive disease, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 42. The

  Embodiment 50. FIG. In some embodiments, there is provided a method for treating an individual having an HPV16-E7 positive disease, comprising administering to the individual an effective amount of the effector cells of embodiment 46 or 47. Is done.

Embodiment 51. FIG. In some embodiments, a method of diagnosing an individual having an HPV16-E7 positive disease comprising:
a) administering an effective amount of an isolated anti-E7MC construct according to embodiment 41 to the individual; and b) determining the level of label in the individual, wherein the level of label above the threshold level A method is provided comprising a step wherein the level indicates that the individual has an HPV16-E7 positive disease.

Embodiment 52. FIG. In some embodiments, a method of diagnosing an individual having an HPV16-E7 positive disease comprising:
a) contacting a sample from an individual with the isolated anti-E7MC construct of embodiment 41; and b) determining the number of cells bound to the isolated anti-E7MC construct in the sample. Wherein a value of the number of cells associated with the isolated anti-E7MC construct above a threshold level indicates that the individual has an HPV16-E7 positive disease is provided. The

  Embodiment 53. FIG. In some further embodiments of embodiments 49-52, the HPV16-E7 positive disease is an HPV16-E7 positive cancer.

  Embodiment 54. FIG. In some further embodiments of embodiment 53, the HPV 16-E7 positive cancer is squamous cell carcinoma.

  Embodiment 55. FIG. In some further embodiments of embodiment 53, the HPV 16-E7 positive cancer is cervical cancer, anogenital cancer, head and neck cancer or oropharyngeal cancer.

  Those skilled in the art will appreciate that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the invention, but of course should not be construed as limiting its scope in any way.

Materials Cell samples, cell lines and antibodies Cell lines include CaSki (ATCC CRL-1550; HLA-A2 ⁺ , HPV16 ⁺ ), head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2 ⁺ , HPV16 ⁺ ), liver adenocarcinoma cell line SK-HEP-1 (ATCC HTB-52; HLA-A2 ⁺ , HPV16 ⁻ ), cervical squamous cell carcinoma cell line SiHa (ATCC HTB-35; HLA-A2 ⁻ , HPV16 ⁺ ), cervical cancer cell line C33A (ATCC HTB-31; HLA-A2 ⁺ , HPV16 ⁻ ), cervical cancer cell line MS-751 (ATCC HTB-34; HLA-A2 ⁺ , HPV16 ⁻ ), uterus cervical adenocarcinoma cell line ^{HeLa (ATCC CCL-2; HLA} -A2 -, HPV16 -), hepatocyte GanHoso Strain ^{Hep3B (ATCC HB-8064; HLA} -A2 -, HPV16 -), hepatocellular carcinoma cell line ^{HepG2 (ATCC HB-8065; HLA} -A2 -, HPV16 -), lymphoma cell lines Raji (ATCC CCL-86; HLA- A2 ^-, HPV16 ^-), T cell leukemia cell line ^{Jurkat (ATCC TIB-152; HLA} -A2 -, HPV16 -), lymphoma cell lines ^{Daudi (ATCC CCL-213; HPA} -A2 -, HPV16 -), leukemia cell line ^{K562 (ATCC CCL-243; HLA} -A2 -, HPV16 -), and lymphoblastoid cell line ^{T2 (ATCC CRL-1992; HLA} -A2 +, HPV16 -) are included. T2 is a TAP deficient cell line. Cell lines were cultured at 37 ° C./5% CO ₂ in RPMI 1640 supplemented with 5% FCS, penicillin, streptomycin, 2 mmol / L glutamine, and 2-mercaptoethanol.

All peptides were purchased and purchased from Genemed Synthesis, Inc. (San Antonio, Tex.). The purity of the peptide was greater than 90%. The peptide was dissolved in DMSO, diluted with saline to 5 mg / mL, and frozen at -180 ° C. Biotinylated single chain HPV16-E7 peptide / HLA-A ^* 02 ^: 01 complex and control peptide / HLA-A ^* 02 ^: 01 complex were transformed into recombinant HLA-A02 and beta-2 microglobulin (β2M) (approximately 2M ) And the peptide was synthesized by refolding. Eleven control peptides (SEQ ID NO: 193-203) that bind HLA-A ^* 02 ^: 01 were generated from the following 10 genes: BCR, BTG2, CALR, CD247, CTSG, DDX5, HLA-E, IFI30, PPP2R1B, SSR1.

Example 1
Biotinylated ^{HPV16-E7 / HLA-A *} 02:01 complex monomer prepared biotinylated ^{HPV16-E7 / HLA-A *} 02:01 complex monomers were prepared according to standard protocols (John D. Altman And Mark M. Davis, Current Protocols in Immunology 17.3.1 to 13.333, 2003). Briefly, the gene encoding full-length human beta-2 microglobulin (β2m) was synthesized by Genewiz and cloned into the vector pET-27b. BirA substrate peptide (BSP) was added to the C-terminus of HLA-A ^* 02 ^: 01 extracellular domain (ECD). Similarly, DNA encoding HLA-A ^* 02 ^: 01 ECD-BSP was synthesized by Genewiz and cloned into the vector pET-27b. A vector expressing human β2m and a vector expressing HLA-A ^* 02 ^: 01 ECD-BSP were separately prepared in E. coli. E. coli BL21 cells were transformed and the expressed protein was isolated from bacterial cultures as inclusion bodies. Peptide ligand HPV16-E7 peptide 11-19 was refolded using human β2m and HLA-A ^* 02 ^: 01 ECD-BSP to generate HPV16-E7 peptide / HLA-A ^* 02 ^: 01 complex monomer. Formed. Folded peptide / HLA-A ^* 02 ^: 01 monomer was concentrated by ultrafiltration and further purified by size exclusion chromatography. HiPrep 26/60 Sephacryl S-300 HR was equilibrated using 1.5 column volumes of Hyclone Dulbecco's Phosphate Buffered Saline solution (Thermo Scientific, catalog number SH30000282). The crude sample was loaded and eluted using 1 column volume. The first peak corresponding to the misfolded aggregate elutes at approximately 111 mL, the peak corresponding to the properly folded MHC complex is observed at 212 mL, and the peak corresponding to free β2M is observed at 267 mL (FIG. 1). Peptide / HLA-A ^* 02 ^: 01 monomer was biotinylated by BirA mediated enzymatic reaction and then purified by high resolution anion exchange chromatography. Biotinylated peptide / HLA-A ^* 02 ^: 01 monomer was stored in PBS at −80 ° C.

  SDS-PAGE of the purified HPV16-E7 peptide / MHC complex can be performed to determine protein purity. For example, 1 μg of protein complex is mixed with 2.5 μL of NuPAGE LDS Sample Buffer (Life Technologies, NP0008) and made up to 10 μL using deionized water. Samples are heated at 70 ° C. for 10 minutes and then loaded onto the gel. Gel electrophoresis is performed at 180V for 1 hour.

(Example 2)
Selection and characterization of scFv specific for HPV16-E7 / HLA-A ^* 02 ^: 01 complex.
A collection of human scFv antibody phage display libraries (diversity = 10 × 10 ¹⁰ ) constructed by Eureka Therapeutics were used to select human mAbs specific for HPV16-E7 / HLA-A ^* 02 ^: 01. Fifteen fully human phage scFv libraries were used to pan against the HPV16-E7 / HLA-A ^* 02 ^: 01 complex. Solution panning and cell panning were used instead of conventional plate panning to reduce the conformational change of the MHC1 complex introduced by immobilizing the protein complex on the plastic surface. In solution panning, the biotinylated antigen is first mixed with a human scFv phage library after extensive washing with PBS buffer, and then the antigen-scFv antibody phage complex is combined with streptavidin-conjugated Dynabeads M-280. Pulled down through the magnetic rack. The bound clones were then eluted and used to infect E. coli XL1-Blue cells. For cell panning, T2 cells loaded with HPV16-E7 peptide were first mixed with a human scFv phage library. T2 cells are a TAP-deficient HLA-A ^* 02 ^: 01+ lymphoblast cell line. To load the peptide, T2 cells were pulsed overnight with peptide (50 μg / ml) in serum-free RPMI 1640 medium in the presence of 20 μg / ml β2M. After extensive washing with PBS, peptide loaded T2 cells with bound scFv antibody phage were spun down. The bound clones were then eluted and used to infect E. coli XL1-Blue cells. The phage clone expressed in bacteria was then purified. ScFv phage clones that specifically bound HPV16-E7 / HLA-A ^* 02 ^: 01 after performing 3-4 rounds of panning using either solution panning, cell panning or a combination of solution panning and cell panning Enriched.

Streptavidin ELISA plates were coated with biotinylated HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex monomer or biotinylated C3 control peptide / HLA-A ^* 02 ^: 01 monomer, respectively did. The C3 peptide is a human nucleoprotein p68-derived peptide YLLPAIVHI (SEQ ID NO: 193). Individual phage clones from the enriched phage display panning pool for the HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex were incubated in the coated plates. Binding of phage clones was detected by anti-M13 antibody conjugated with HRP and developed using HRP substrate. Absorbance at 450 nm was read. ELISA screening of 1272 phage clones enriched from phage panning identified 145 positive clones. FIG. 2 provides an example of phage clone binding to biotinylated HPV16-E7 / HLA-A ^* 02 ^: 01 monomer in an ELISA assay. DNA sequencing of 145 ELISA positive phage clones identified 41 unique clones. Specific and unique clones were analyzed for flow cytometry (FACS analysis) using live T2 cells loaded with HPV16-E7 for their binding to HLA-A ^* 02: 01 / peptide complexes on the surface of live cells. Further testing by FIG. 3 demonstrates the binding of representative positive clones to T2 cells loaded with HPV16-E7 11-19 peptide or to T2 cells loaded with a control peptide (C3 peptide). Controls included T2 cells without peptide loading (cells only) and horse anti-mouse IgG control conjugated with R-PE (secondary antibody only). Briefly, T2 cells loaded with HPV16-E7 11-19 peptide or C3 peptide are first stained with purified scFv phage clones, followed by a second staining with mouse anti-M13 mAb. And third staining with equine anti-mouse IgG conjugated with R-PE from Vector Labs. Each staining step was performed on ice for 30-60 minutes and the cells were washed twice between staining. Of the 41 clones tested, 24 specifically recognized T2 cells loaded with HPV16-E7. These 24 phage clones specifically bind to T2 cells loaded with HPV16-E7, both T2 cells loaded with C3 peptide in the HLA-A ^* 02 ^: 01 situation, and T2 cells without peptide loading. I did not recognize.

(Example 3)
Characterization of FACS-positive HPV16-E7 specific phage clones Cross-reactivity to HPV16-E7 peptide 11-19 variants The HPV16-E7 11-19 peptides selected in the present invention are highly conserved among various HPV16 strains . In one study, this peptide was found to be fully conserved in 16 of the 17 known HPV16-E7 protein sequences. A single amino acid mutation within E7 11-19 was identified in the remaining sequence. This variant was then found in one of the 35 HPV16 infected cervical cancer or cervicitis patients analyzed (Zhang GL et al., Database (Oxford). 2014: bau031, 2014). Clones selected from FACS binding analysis on T2 cells loaded with HPV16-E7 peptide 11-19 were cross-reacted to this HPV16-E7 peptide 11-19 variant / HLA-A ^* 02 ^: 01 complex on the surface of living cells. Sex is further characterized by FACS analysis using live T2 cells loaded with variant HPV16-E7 peptide. This variant peptide differs from the conserved HPV16-E7 11-19 peptide by a single amino acid at position 5. The variant peptide sequence is YMLDQPQPET (SEQ ID NO: 11), while the conserved HPV16-E7 11-19 sequence is YMLDLQPET (SEQ ID NO: 4).

Briefly, T2 cells are loaded with the conserved HPV16-E7 11-19 peptide, variant HPV16-E7 11-19 peptide, or β2M. T2 cells loaded with peptides were stained with purified scFv phage clones, followed by a second staining with mouse anti-M13 mAb and equine anti-mouse IgG conjugated with Vector Labs R-PE. The third staining used is performed. Each staining step is performed on ice for 30-60 minutes, and the cells are washed twice between each staining.
Epitope mapping by alanine walking

To accurately investigate the epitope recognized by the mAb, HPV16-E7 peptide with an alanine substitution at positions 1, 5 and 8 was pulsed onto T2 cells. Antibody phage clones were then tested by FACS analysis for binding to T2 cells loaded with these peptides. The FACS mean fluorescence intensity (MFI) values for each FACS assay are shown in Table 6. K07 helper phage is a negative control indicating that phage alone without scFv display on the surface of phage particles does not bind to any of the T2 cell populations loaded with the peptide. Antibody BB7.2 recognizes the HLA-A02 alpha chain. Peptide binding to the MHC complex stabilizes the cell surface MHC complex. Thus, T2 cells loaded with MHC binding peptides enhanced BB7.2 binding signal (as shown in the first column of Table 6) compared to T2 cells without peptide loading. The BB7.2 binding data shows that alanine substituted peptides can still bind HLA-A ^* 02 ^: 01 molecules on the T2 cell surface. All the antibodies tested recognized a small conformational epitope formed by the HPV16-E7 peptide and its surrounding MHC alpha chain residues, but the key peptide residues that interact with the various antibodies were quite different. . For example, clone number 4 is expected to bind to the N-terminal half of the HPV16-E7 peptide because alanine substitution at position 1 or 5 dramatically reduced binding to peptide loaded T2 cells. . In contrast, alanine substitution at position 8 did not change the binding of the same clone to the HLA-A ^* 02 ^: 01 complex. On the other hand, clone number 11 was insensitive to alanine substitution at positions 1 and 8 but its binding was completely suppressed by alanine substitution at position 5. FIG. 4 provides an example of FACS analysis showing the binding of phage clone number 11 to T2 cells loaded with various HPV16-E7 peptides. Controls included T2 cells without peptide loading (cells only) and horse anti-mouse IgG control conjugated with R-PE (secondary antibody only). Antibody binding to T2 cells loaded with these different peptides was tested by FACS assay.

Assessment of antibody binding specificity for endogenous peptides On average, nucleated cells in the human body each express about 500,000 different peptide / MHC class I complexes. In order to develop anti-peptide / MHCI-complex antibodies into anti-cancer drugs with high specificity and therapeutic index, the antibody specifically recognizes the target peptide / MHCI complex, but the MHCI molecule itself or the cell surface It is essential not to recognize MHCI molecules bound to the other peptides presented above. For this study, the relevant MHCI molecule is HLA-A ^* 02 ^: 01. Early in our phage panning and screening, we eliminated antibodies bound to the HLA-A ^* 02 ^: 01 molecule alone (see, eg, FIGS. 2 and 3). The top phage clones are 11 endogenous HLA-A ^* 02 derived from proteins normally expressed in multiple types of nucleated human cells, eg, globin alpha chain, beta chain, nucleoprotein p68, etc .: Also screened against the 01 peptide. Recombinant peptides / HLA-A ^* 02 ^: 01 complexes folded with 11 endogenous peptides (Table 7, SEQ ID NO: 193-203) were separately coated on streptavidin plates and antibody binding was analyzed by ELISA. Determined by. Briefly, individual phage clones were incubated on peptide / HLA-A ^* 02 ^: 01 complex coated plates. Binding of phage clones was detected by anti-M13 antibody conjugated with HRP and developed using HRP substrate. Absorbance at 450 nm was read. As shown in FIG. 5, the HPV16-E7 peptide / HLA-A ^* 02 ^: 01 specific antibody phage clone bound the HPV16-E7 peptide / HLA-A ^* 02 ^: 01 complex, but the endogenous peptide Did not bind the HLA-A ^* 02 ^: 01 complex folded using The inventors have identified that the antibodies identified are specific for the HPV16-E7 peptide / HLA-A ^* 02 ^: 01 complex and HLA-A bound to other HLA-A ^* 02 ^: 01 constrained peptides. ^* Concludes that 02:01 molecule is not recognized.

Example 4
Engineering Bispecific Antibodies Bispecific antibodies (BsAbs) were generated using the scFv sequence of HPV16-E7 / HLA-A ^* 02 ^: 01 specific phage clones. BsAb is a single chain bispecific antibody containing the scFv sequence of HPV16-E7 / HLA-A ^* 02 ^: 01 specific phage clone at the N-terminus and anti-human CD3ε mouse monoclonal scFv at the C-terminus (Brischwein, K. et al., Molecular Immunology 43: 1129-1143, 2006). The DNA fragment encoding HPV16-E7 scFv and the DNA fragment encoding anti-human CD3ε scFv were synthesized by Genewiz and subcloned into the Eureka mammalian expression vector pGSN-Hyg using standard DNA techniques. A 6 histamine tag was inserted at the C-terminus for antibody purification and detection. Chinese hamster ovary (CHO) cells were transfected with a BsAb expression vector and then cultured for 7 days for BsAb antibody production. CHO cell supernatants containing secreted HPV16-E7 BsAb molecules were collected. BsAb was purified by FPLC AKTA system using HisTrap HP column (GE Healthcare). Briefly, CHO cell cultures are clarified and loaded onto the column using a low imidazole concentration (20 mM) and then bound BsAb protein using isocratic high imidazole concentration elution buffer (500 mM). Was eluted. The purity and molecular weight of the purified HPV16-E7 BsAb was determined by gel electrophoresis under reducing conditions. 4 μg of protein was mixed with 2.5 μL of NuPAGE LDS Sample Buffer (Life Technologies, NP0008) and made up to 10 μL with deionized water. The sample was heated at 70 ° C. for 10 minutes and then loaded onto the gel. Gel electrophoresis was performed at 180V for 1 hour. A band of about 50 KD is observed as the main band on the gel (Figure 6).

  Antibody aggregation can be assessed by size exclusion chromatography (SEC). For example, 50 μL of sample is injected into a SEC column (eg, Agilent, BioSEC-3, 300A, 4.6 × 300 mm) and at the same time, Dulbecco's phosphate buffered saline (Fisher Scientific, SH30028.FS) adjusted to pH 7.0. And a buffer consisting of 0.2 M arginine. BsAbs with high molecular weight aggregation less than 10% are selected for further characterization.

(Example 5)
Characterization of HPV16-E7 BsAb Antibody Binding Affinity of HPV16-E7 BsAb Antibody The binding affinity of HPV16-E7 BsAb antibody to the recombinant HPV16-E7 / HLA-A ^* 02 ^: 01 complex is measured, for example, by Surface Plasma Resonance ( BiaCore). Binding parameters between HPV16-E7 BsAb and HPV16-E7 / HLA-A ^* 02 ^: 01 complex are used, for example, using His Capture Kit (GE Healthcare, catalog number 28995056) in Biacore X100 (GE Healthcare), Measure according to manufacturer's protocol for multicycle kinetic measurements. All proteins used in the assay are diluted using HBS-E buffer. For example, 1 μg / mL of HPV16-E7 BsAb is immobilized by flowing the solution through the flow cell 2 at 2 μL per minute for 2 minutes on a sensor chip previously functionalized with an anti-histidine antibody. Binding to the HPV16-E7 / A ^* 02 ^: 01 complex was analyzed, for example, at 0.19 μg / mL, 0.38 μg / mL, 7.5 μg / mL, 15 μg / mL, and 30 μg / mL, each run being Consists of 3 minutes of association and 3 minutes of dissociation at 30 μL per minute. At the end of the cycle, the surface is regenerated using the regeneration buffer from His Capture Kit. After the kinetic measurement, the surface is regenerated using the regeneration solution from the kit. Data is analyzed using BiaCore X-100 evaluation software using 1: 1 binding site mode. Next, the binding parameters (association rate constant k _a , dissociation constant k _d , and equilibrium dissociation constant K _d ) are calculated.

T cell killing assay using peptide pulsed T2 cells Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega). Human T cells purchased from AllCells were activated and expanded using CD3 / CD28 Dynabeads (Invitrogen) according to the manufacturer's protocol. Activated T cells (ATC) were cultured and maintained in RPMI 1640 medium with 10% FBS plus 30 U / ml IL-2 and used on days 7-14. T cells were> 99% CD3 ⁺ by FACS analysis. Activated T cells (effector cells) and T2 cells loaded with the target peptide were co-cultured with 1 μg / ml or 0.2 μg / ml BsAb for 16 hours in a 5: 1 ratio. P2 loaded T2 cells are incubated overnight with either 50 μg / ml of target HPV16-E7 11-19 peptide (YMLDLQPET, SEQ ID NO: 4) or negative control AFP158 peptide (FMNKFIYEI, SEQ ID NO: 192) Prepared. A negative control AFP158 / HLA-A ^* 02 ^: 01 specific BsAb was also included. Cytotoxicity was determined by measuring LDH activity in the culture supernatant. As shown in FIG. 7, clones No. 2, No. 5, No. 17, and No. 40 killed T2 cells loaded with HPV16-E7 peptide in a selective and dose-dependent manner. The negative control anti-AFP158 / HLA-A ^* 02 ^: 01 BsAb specifically directed killing of T2 cells loaded with AFP158 peptide.

T cell killing assay using cancer cell lines Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega). Human T cells were purchased from AllCells and activated and expanded using CD3 / CD28 Dynabeads (Invitrogen) according to the manufacturer's protocol. Activated T cells (ATC) were cultured and maintained in RPMI 1640 medium with 10% FBS plus 30 U / ml IL-2 and used on days 7-14. Activated T cells (effector cells) and target cancer cells in a 5: 1 ratio at various concentrations of BsAb (eg, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.0016 μg / Ml BsAb) for 16 hours. Cytotoxicity was then determined by measuring LDH activity in the culture supernatant.

The target cancer cells tested were cervical cancer cell line CaSki (ATCC CRL-1550; HLA-A2 ⁺ , HPV16 ⁺ ) and cervical cancer cell line MS-751 (ATCC HTB-34; HLA-A2 ⁺ , HPV16 ^- ) Included. As shown in FIG. 8A, multiple clones (eg, 2, 41 and US-7) were tested in HPV-16 ⁺ cell line CaSki, HPV-16 ⁻ cell line MS at 0.2 μg / ml BsAb. Mediated selective killing for -751. BsAb-mediated killing of target positive CaSki is dose dependent as shown in FIG. 8B.

Additional target cancer cells that can be tested include head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2 ⁺ , HPV16 ⁺ ), liver adenocarcinoma cell line SK-HEP-1 (ATCC HTB- 52; HLA-A2 ⁺ , HPV16 ⁻ ), cervical squamous cell carcinoma cell line SiHa (ATCC HTB-35; HLA-A2 ⁻ , HPV16 ⁺ ), cervical cancer cell line C33A (ATCC HTB-31; HLA -A2 ^+, HPV16 ^-), cervical adenocarcinoma cell line ^{HeLa (ATCC CCL-2; HLA} -A2 -, HPV16 -), hepatocellular carcinoma cell line ^{Hep3B (ATCC HB-8064; HLA} -A2 -, HPV16 - ), hepatocellular carcinoma cell line ^{HepG2 (ATCC HB-8065; HLA} -A2 -, HPV16 -), phosphorus Pas lymphoma cell lines ^{Raji (ATCC CCL-86; HLA} -A2 -, HPV16 -), T cell leukemia cell line ^{Jurkat (ATCC TIB-152; HLA} -A2 -, HPV16 -), lymphoma cell lines Daudi (ATCC CCL-213 ^{; HPA-A2 -, HPV16 -} ), and leukemia cell line ^{K562 (ATCC CCL-243; HLA} -A2 -, HPV16 -) are included.

Cross-reactivity of HPV16-E7 BsAb antibodies against multiple HLA-A02 alleles Human MHCI molecules are composed of six class isoforms, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and It consists of HLA-G. The heavy chain genes of HLA-A, HLA-B, and HLA-C are highly polymorphic. For each isoform, the HLA genes are further grouped according to heavy chain sequence similarity. For example, HLA-A is divided into different alleles such as HLA-A01, HLA-A02, and HLA-A03. For the HLA-A02 allele, there are multiple subtypes such as HLA-A ^* 02: 01, HLA-A ^* 02: 02, and the like. Sequence differences between the different subtypes of the HLA-A02 group are limited to only a few amino acids. Thus, in many cases, a peptide that binds to the HLA-A ^* 02 ^: 01 molecule can form a complex with multiple subtypes of the HLA-A02 allele. As shown in Table 8 (http://www.allelefrequencies.net/), HLA-A ^* 02 ^: 01 is the dominant HLA-A02 subtype among Caucasian populations, but in Asia, ALA ^{* 02: 05, a * 02} : 06, a * 02:07 and ^a * 02:11 also is a common HLA-A02 subtype. The ability of the HPV16-E7 antibody to recognize not only the HPV16-E7 peptide but also other subtypes of HLA-A02 in the context of HLA-A ^* 02 ^: 01 may benefit from treatment with the HPV16-E7 antibody drug The patient population is greatly expanded. To determine cross-reactivity, HPV16-E7 / MHC class I complexes with other subtypes of the HLA-A02 allele are generated and HPV16-E7 / HLA-A ^* 02 for these other complexes: The binding affinity of the 01 specific antibody is tested. The binding affinity is determined using, for example, ForteBio Octet QK. Briefly, 5 μg / mL of biotinylated HPV16-E7 peptide / HLA-A02 MHC complex with various subtypes is loaded onto a streptavidin biosensor. After washing away excess antigen, the BsAb is tested for association and dissociation kinetics at, for example, 10 μg / mL. Binding parameters are calculated using a 1: 1 binding site, partial fitting model.

(Example 6)
Generation of T cells (CAR-T) presenting HPV16-E7 / HLA-A ^* 02 ^: 01 specific chimeric antigen receptor Chimeric antigen receptor therapy (CAR-T therapy) is a new form of targeted immunotherapy is there. This combines the elaborate targeting specificity of monoclonal antibodies with the powerful cytotoxicity and long-lasting afforded by cytotoxic T cells. This technique allows T cells to acquire long-term novel antigen specificity independent of endogenous TCR. According to clinical trials, neuroblastoma (Louis CU et al., Blood 118 (23): 6050-6056), B-ALL (Maude SL et al., N. Engl. J. Med. 371 (16): 1507-1517, 2014), CLL (Brentjens RJ et al., Blood 118 (18): 4817-4828, 2011), and B cell lymphoma (Kochenderfer JN et al., Blood. 116 (20)): 4099-4102 (2010) shows the clinically significant antitumor activity of CAR-T therapy. One study reported a 90% complete response rate in 30 B-ALL patients treated with CD19-CART therapy (Maude SL et al., Supra).

To further explore the potency of HPV16-E7 / HLA-A ^* 02 ^: 01 specific antibodies, CARs expressing HPV16-E7 scFv are constructed and transduced into T cells. For example, a HPV16-E7 / HLA-A ^* 02 ^: 01 specific CAR is constructed using a lentiviral CAR expression vector. Anti-HPV16-E7 / HLA-A ^* 02 ^: 01 scFv has a CD28 signaling domain and TCRζ engineered in cis to provide intracellular T cell stimulation signals and to activate T cells Transplanted to generation CAR (Mackall CL et al., Nat. Rev. Clin. Oncol. 11 (12): 693-703, 2014). FIG. 9 provides a schematic diagram of the HPV16-E7 CAR construct.

(Example 7)
Characterization of HPV16-E7 CAR-T cells In vitro cytotoxicity studies of HPV16-E7 CAR-T cells Lentiviruses containing HPV16-E7 / HLA-A ^* 02 ^: 01 specific chimeric antigen receptors, for example, It is generated by transfecting 293T cells with a CAR vector. Human T cells are used for transduction after stimulation with CD3 / CD28 beads (Dynabeads®, Invitrogen) for 2 days in the presence of 30 U / ml interleukin-2. Concentrated lentivirus is applied to T cells in a 6-well plate coated with Retrolectin (Takara) for 72 hours. Transduction efficiency is assessed by FACS using biotinylated HPV16-E7 tetramer and streptavidin conjugated with PE. Repeated FACS analysis is performed at 72 hours and every 3-4 days thereafter.

Functional evaluation of transduced T cells (HPV16-E7 CAR-T cells) is performed using LDH Cytotoxicity Assay. Effector to target ratios used include, for example, 5: 1 and 10: 1. Target cell lines include, for example, cervical cancer cell line CaSki (ATCC CRL-1550; HLA-A2 ⁺ , HPV16 ⁺ ), head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2 ⁺ , HPV16 ⁺ ), liver adenocarcinoma cell line SK-HEP-1 (ATCC HTB-52; HLA-A2 ⁺ , HPV16 ⁻ ), cervical squamous cell carcinoma cell line SiHa (ATCC HTB-35; HLA-A2 ⁻ , HPV16 ⁺ ), cervical cancer cell line C33A (ATCC HTB-31; HLA-A2 ⁺ , HPV16 ⁻ ), cervical cancer cell line MS-751 (ATCC HTB-34; HLA-A2 ⁺ , HPV16 ⁻ ), cervical adenocarcinoma cell line ^{HeLa (ATCC CCL-2; HLA} -A2 -, HPV16 -), hepatocellular carcinoma cell lines ^{Hep3B (ATCC HB-8064; HLA} -A2 -, HPV16 -), hepatocellular carcinoma cell line ^{HepG2 (ATCC HB-8065; HLA} -A2 -, HPV16 -), lymphoma cell lines Raji (ATCC CCL-86; HLA -A2 ^-, HPV16 ^-), T cell leukemia cell line ^{Jurkat (ATCC TIB-152; HLA} -A2 -, HPV16 -), lymphoma cell lines ^{Daudi (ATCC CCL-213; HPA} -A2 -, HPV16 -), and leukemia cell lines ^{K562 (ATCC CCL-243; HLA} -A2 -, HPV16 -) may include. As a control, SK-HEP-1-MiniG was transformed into SK-HEP-1 with the HPV16-E7 peptide expression minigene cassette resulting in high level cell surface expression of the HPV16-E7 / HLA-A ^* 02 ^: 01 complex. Generate by introducing. The specificity and efficiency with which HPV16-E7 CAR expressing T cells kill target positive cancer cells is determined.

(Example 8)
Generation and Characterization of Full-Length IgG1 HPV16-E7 Antibody Full-length human IgG1 of selected phage clones is produced as described, for example, in HEK293 and Chinese hamster ovary (CHO) cell lines (Tomimatsu K. et al., Biosci. Biotechnol Biochem. 73 (7): 1465-1469, 2009). Briefly, antibody variable regions are subcloned into a mammalian expression vector along with corresponding human lambda or kappa light chain constant region sequences and human IgG1 constant region sequences. The same cloning strategy is applied to generate a chimeric HPV16-E7 full-length antibody with a murine IgG1 heavy chain constant region and a light chain constant region. The molecular weight of the purified full-length IgG antibody is measured by electrophoresis under both reducing and non-reducing conditions. SDS-PAGE of purified HPV16-E7 mouse chimeric IgG1 antibody is performed to determine protein purity. Briefly, 2 μg of protein is mixed with 2.5 μL of NuPAGE LDS Sample Buffer (Life Technologies, NP0008) and made up to 10 μL with deionized water. Samples are heated at 70 ° C. for 10 minutes and then loaded onto the gel. Gel electrophoresis is performed at 180V for 1 hour.

HPV16-E7 chimeric IgG1 antibody is tested by flow cytometry for binding to SK-HEP-1 cells presenting HPV16-E7. SK-HEP-1 is an HLA-A ^* 02 ^: 01 positive and HPV16-E7 negative cell line. HPV16-E7 minigene cassette is transfected into SK-HEP-1 cells to generate SK-HEP-1-miniG cells displaying HPV16-E7. 10 μg / mL of antibody is added to the cells and placed on ice for 1 hour. After washing, anti-mouse IgG (H + L) conjugated with R-PE (Vector Labs # EI-2007) is added to detect antibody binding. The binding affinity of the mouse chimeric IgG1 HPV16-E7 antibody is determined by ForteBio Octet QK. 5 μg / mL of biotinylated HPV16-E7 peptide / HLA-A ^* 02 ^: 01 complex is loaded onto a streptavidin biosensor. After washing away excess antigen, mouse chimeric full length antibodies are tested for association and dissociation kinetics at 10 μg / mL. Binding parameters are calculated using a 1: 1 binding site, partial fitting model.

HPV16-E7 specific and negative control (eg ET901) mouse chimeric IgG1 tested for binding to HPV16-E7 / HPA-A ^* 02: 01, HPV16-E7 recombinant protein and free HPV16-E7 peptide in ELISA assay To do. The antibodies are tested, for example, starting at 100 ng / mL, 3 × serial dilution, 8 total concentrations. Biotinylated HPV16-E7 / A ^* 02 ^: 01 MHC is coated onto streptavidin plates at 2 μg / mL, HPV16-E7 protein is coated at 2 μg / mL, and HPV16-E7 peptide is coated at 40 ng / mL. The ability of the full-length anti-HPV16-E7 / HLA-A ^* 02 ^: 01 antibody to recognize only HPV16-E7 peptide in the context of HLA-A02 and not bind to recombinant HPV16-E7 protein or free HPV16-E7 peptide decide.

Example 9
In vivo efficacy studies HPV16-E7 CAR-T cell treatment in mice HLA-A02 ⁺ / HPV16-E7 ⁺ cancer cell lines (eg, CaSki or UM-SCC-104) subcutaneous (sc) xenograft model. , SCID-beige (no functional T cells, B cells, NK cells) are generated in mice. Average s. c. When the tumor volume reaches 200 mm ³ , the animals are randomized. Animals are treated with 60 mg / kg cyclophosphamide (by intraperitoneal route) 24 hours prior to CART administration. (Ii) no treatment, (ii) 10 ⁷ mock-transduced CAR T cells, once weekly for 4 weeks, (iii) 10 ⁷ anti-E7MC CAR T cells, once weekly for 4 weeks, Or (iv) split the mice into 4 groups (n = 8-10 mice / group) that receive one of 2 × 10 ⁶ anti-E7MC CAR T cells, once a week for 4 weeks . Each group of animals was analyzed for tumor volume, adverse response, human cytokine profile, CAR T cell invasion, tumor histopathology for human CD3 ⁺ cells in tumors and organs, serum HPV16-E7, body weight and general health status. Monitor (eating, walking, daily activities).

(Example 10)
Affinity maturation of anti-HPV16-E7 antibody agents This example demonstrates affinity maturation of anti-HPV16-E7 antibody agents. In particular, this example illustrates the generation of a series of antibody variants by incorporating random mutations into a representative anti-HPV16-E7 antibody agent (clone US-7) followed by screening and characterization of antibody variants. To demonstrate.

Generation of variant phage library DNA encoding the anti-HPV16-E7 clone US-7 scFv was subjected to random mutagenesis using the GeneMorph II Random Mutagenesis kit (Agilent Technologies) according to the manufacturer's specifications. After mutagenesis, the DNA sequence was cloned into a phagemid vector expressing scFv to construct a variant human antibody phage library containing approximately 5 × 10 ⁸ unique phage clones. On average, variant clones have a 2 nucleotide variation compared to the parental anti-HPV16-E7 clone with variations ranging from 1 to 4 nucleotides per scFv sequence.

Cell Panning HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex as described in Example 2 using a human phage scFv library with variants generated from clone US-7 Panned against. In particular, cell panning was used. The human scFv phage library was first mixed with T2 cells loaded with 50 ug / ml of a pool of 20 different endogenous peptides (P20, SEQ ID NOs: 193-212) as a negative control panning. The negative control depleted human scFv phage library was then loaded with HPV16-E7 11-19 peptide (first round 1.5 ug / ml, second round 0.8 ug / ml, third round 0.4 ug / ml). Mixed with loaded T2 cells. To load the peptide, T2 cells were pulsed overnight in the presence of 20 μg / ml β2M with peptide in serum-free RPMI 1640 medium. After extensive washing with PBS, peptide loaded T2 cells with bound scFv antibody phage were spun down. The bound clones were then eluted and used to infect E. coli XL1-Blue cells. The phage clone expressed in bacteria was then purified. Three rounds of panning were performed to enrich for scFv phage clones that specifically bound to HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01.

Streptavidin ELISA plates were coated with biotinylated HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex monomer or biotinylated P20 control peptide / HLA-A ^* 02 ^: 01 monomer . Individual phage clones from the enriched phage display panning pool for the HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 complex were incubated in the coated plates. Binding of phage clones was detected by anti-M13 antibody conjugated with HRP and developed using HRP substrate. Absorbance at 450 nm was read. Fifteen positive clones were identified by ELISA screening of 135 phage clones enriched from phage panning. DNA sequencing of 15 ELISA positive phage clones identified 11 unique clones. Specific and unique clones were analyzed for flow cytometry using live T2 cells loaded with HPV16-E7 11-19 peptide for their binding to HLA-A ^* 02: 01 / peptide complexes on the surface of live cells. Further testing was done by (FACS analysis). Controls included T2 cells without peptide loading (cells only) and horse anti-mouse IgG control conjugated with R-PE (secondary antibody only). Briefly, T2 cells loaded with HPV16-E7 11-19 peptide or P20 peptide pool were first stained with a purified scFv phage clone, followed by a second staining with mouse anti-M13 mAb. And third staining with equine anti-mouse IgG conjugated with Vector Labs R-PE. Each staining step was performed on ice for 30-60 minutes, and the cells were washed twice between staining steps. Of the 10 unique clones tested, 8 specifically recognized T2 cells loaded with HPV16-E7 11-19 (SEQ ID NO: 223-230). These 8 phage clones specifically bound to T2 cells loaded with HPV16-E7 11-19, and T2 cells loaded with P20 peptide pool in the HLA-A ^* 02 ^: 01 situation were also associated with peptide loading. None of the T2 cells were recognized.

(Example 11)
Characterization of bispecific antibody molecules based on anti-HPV16-E7 affinity matured variants Generation of bispecific antibodies Bispecific antibodies (BsAbs) were prepared using the method described in Example 4 The affinity matured HPV16-E7 / HLA-A ^* 02 ^: 01 specific phage clone scFv sequence (SEQ ID NO: 223-232) isolated at 10 was used. The resulting single chain bispecific antibody contained the scFv sequence of HPV16-E7 / HLA-A ^* 02 ^: 01 specific phage clone at the N-terminus and anti-human CD3ε mouse monoclonal scFv at the C-terminus.

^{HPV16-E7 / HLA-A *} 02:01 determination of binding affinities of bispecific antibodies against recombinant ^{HPV16-E7 / HLA-A *} 02:01 7 kinds of HPV16-E7 BsAb antibody to complex (SEQ Of affinity matured clones 7-1, 7-3, 7-6, 7-7, 7-8 and 7-9 corresponding to numbers 223, 225 and 227-230, and the parent clone US-7) Binding affinity was measured by Surface Plasma Resonance (BiaCore). Multicycle using binding parameters between HPV16-E7 BsAb and HPV16-E7 / HLA-A ^* 02 ^: 01 complex using Biotin CAPture Kit (GE Healthcare, catalog number 28920233) in Biacore X100 (GE Healthcare). Measured according to manufacturer's protocol for kinetics measurement. All proteins used in the assay were diluted using HBS-EP running buffer. 5 μg / mL biotinylated HPV16-E7 11-19 / HLA-A ^* 02: 01 / β2M complex was pre-functionalized with streptavidin (approximately 3,800 RU of captured streptavidin). The solution was immobilized on the sensor chip CAP by flowing it through the flow cell at 5 μL per minute for 75 seconds (approximately 120 RU of MHC complex was captured per cycle). Binding to HPV16-E7 BsAb was analyzed at 150 nM, 75 nM, 37.5 nM, 18.8 nM and 9.4 nM, each run consisting of 30 μl per minute for 2 minutes of association and 10 minutes of dissociation. At the end of the cycle, the surface was regenerated using regeneration buffer from Biotin CAPture kit. Data was analyzed using BiaCore X-100 evaluation software and using a 1: 1 binding site mode. Next, binding parameters (association rate constant k _a , dissociation constant k _d , and equilibrium dissociation constant K _d ) were calculated and are shown in Table 9. BsAbs from clones 7-1, 7-3, 7-6, 7-7 and 7-9 showed increased binding affinity compared to BsAbs from parent clone US-7, BsAb from 7-8 showed reduced binding affinity.

Cross-reactivity and binding affinity of HPV16-E7 bispecific antibodies to multiple HLA-A02 alleles As described above in Example 5, the different subtypes of HLA-A02 groups are highly conserved, Cross-reactivity to the HLA-A02 subtype is highly desirable. Therefore, to determine whether the bispecific antibody (BsAb) generated from the parent clone US-7 and the affinity matured variant cross-reacts with the non-HLA-A ^* 02 ^: 01 subtype of the HLA-A02 group Experiments were performed. Specifically, HPV16-E7 11-19 / MHC class I complexes with various subtypes of the HLA-A02 allele are generated and HPV16-E7 11-19 / HLA-A ^* 02 ^: 01 specific antibodies Their binding affinities to were determined using Octet® QKe System by Pall ForteBio LLC (Menlo Park, Calif.) Utilizing Biolayer Interferometry (BLI) technology. BsAbs tested include parental clone US-7 and affinity matured variant clones 7-1, 7-3, 7-6, 7-7, 7-8 and 7-9. 5 μg / mL of biotinylated HPV16-E7 11-19 peptide / HLA-A02 complex with various subtypes of HLA-A02 was loaded onto a streptavidin biosensor. After washing away excess antigen (HPV16-E7 peptide / HLA-A02 complex), BsAb was tested for association and dissociation kinetics at 10 μg / mL. The binding parameters were calculated using a 1: 1 binding site, partial fitting model. The results of the BLI assay are shown in Table 10 below.

The results show that each of the tested antibody clones specifically bound to at least 4 of the 7 HLA-A02 subtypes tested. Several affinity matured variant antibodies bind to many HLA-A02 subtype than the parent clones, many of affinity matured variant antibodies, indicated by a lower K _D values, higher affinity than the parent clone To HPV16-E7 11-19 / HLA-A02.

Peptide Binding Specificity Assay To confirm the specificity of the peptides recognized by affinity matured variant antibodies, T2 cells loaded with HPV16-E7 11-19 peptide, T2 cells loaded with P20 peptide pool, or peptide loading FACS analysis was performed using unaccompanied T2 cells. The results show that all BsAbs tested, including parental clone US-7 and affinity matured variant clones 7-1, 7-3, 7-6, 7-7, 7-8 and 7-9, were HPV16-E7 11 It specifically bound to T2 cells loaded with the -19 peptide, but did not bind to T2 cells loaded with the P20 peptide pool nor to T2 cells loaded with any peptide. The FACS data is shown in Table 12 herein.

Epitope mapping by alanine walking 9 residues substituted with alanine to accurately investigate the sensitive residues of the HPV16-E7 11-19 peptide for recognition by BsAb clone number US-7 and its affinity matured variants A series of mutant peptides were designed and synthesized to have each of these (shown in Table 11).

BsAb clones were then tested by FACS analysis for binding to T2 cells loaded with the peptides of Table 11, T2 cells loaded with the negative control P20 peptide pool, or T2 cells without peptide loading. Peptide binding to MHC on T2 cells was assessed by BB7.2 mouse antibody staining. All peptides except HPV16-E7 11-19mut8 showed higher BB7.2 antibody binding (data not shown) compared to control T2 cells (cel) without peptide, HPV16-E7 11-19mut8. All but the peptides were shown to successfully bind to MHC on T2 cells. For BsAb analysis, cells were stained with 10 μg / mL BsAb, followed by 20 × dilution of APC anti-His antibody (R & D System # IC050A). The FACS mean fluorescence intensity (MFI) values for each FACS assay are shown in Table 12. Parental US-7 and affinity matured scFv are expected to bind to the C-terminal half of the HPV16-E7 peptide, as alanine substitution at positions 5-8 generally reduced binding to peptide loaded T2 cells. The The results of individual FACS binding assays are shown in FIGS. 10A and 10B.

(Example 12)
Production of T cell (CAR-T) presenting chimeric antigen receptor having anti-HPV16-E7 affinity matured scFv variant Construction of chimeric antigen receptor expressing affinity matured HPV16-E7 scFv and transduction of T cells did. CAR vectors for the production of lentiviruses encoding HPV16-E7 / HLA-A ^* 02 ^: 01-specific CARs were produced using the affinity matured scFv isolated in Example 10 (SEQ ID NO: 223, 225 and 227- 7-1, 7-3, 7-6, 7-7, 7-8 and 7-9) corresponding to 230 or the parent US-7 scFv, a polypeptide having the CD28 and CD3ζ signaling domains (SEQ ID NO: 256) ) Or a polypeptide having the 4-1BB and CD3ζ signaling domains (SEQ ID NO: 257).

A lentivirus containing the HPV16-E7 / HLA-A ^* 02 ^: 01 specific chimeric antigen receptor was generated by transfecting 293T cells with the CAR vector. Human T cells were used for transduction after 1 day stimulation with CD3 / CD28 beads (Dynabeads®, Invitrogen) in the presence of 100 U / ml interleukin-2. Concentrated lentivirus was applied to T cells for 72 hours in 6 well plates coated with Retronectin (Takara). Five days after transduction, HPV16-E7 CAR-T cells and mock-transduced cells were treated with biotinylated HPV16-E7 11-19 peptide / HLA-A ^* 02 ^: 01 tetramer and CD4 or CD8 antibody. Co-stained and analyzed by flow cytometry. FIGS. 11A-11C show flow cytometry results of HPV16-E7 CAR-T cells and mock-transduced cells. The results are summarized in Table 13. Each of the tested CARs with affinity matured scFv had a higher transduction efficiency than the CAR with the parent scFv.

(Example 13)
Killing assay by T cells using CAR-T with anti-HPV16-E7 affinity maturation variants using cancer cell lines In vitro cytotoxicity studies of HPV16-E7 CAR-T cells Affinity mature scFv (each T cells expressing CAR with 7-1, 7-3, 7-6, 7-7, 7-8 and 7-9) or parental US-7 scFv corresponding to SEQ ID NOS 223, 225 and 227-230 Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega). Human T cells purchased from AllCells were activated using CD3 / CD28 Dynabeads (Invitrogen) according to the manufacturer's protocol and then transduced and expanded with CAR. Activated CAR-T cells were cultured and maintained in RPMI 1640 medium with 10% FBS plus 100 U / ml IL-2 and used on days 7-14. Activated CAR-T cells (effector cells) and target cancer cells were co-cultured at a 5: 1 ratio for 16 hours. Cytotoxicity was then determined by measuring LDH activity in the culture supernatant.

The target cancer cells tested were cervical cancer cell line CaSki (ATCC CRL-1550; HLA-A2 ⁺ , HPV16 ⁺ ), cervical cancer cell line MS-751 (ATCC HTB-34; HLA-A2 ⁺ , HPV16). ^-), and mantle cell lymphoma cell line JeKo-1 (ATCC CRL-3006 ; HLA-A2 +, HPV16 -) it contained. As shown in FIG. 12, affinity matured clones (7-1, 7-3, 7-6, 7-7, 7-8 and 7-9) are compared to the parent clone US-7, Significantly increased killing activity was demonstrated in both the CD28 / CD3ζ format and the 4-1BB / CD3ζ format.

Additional target cancer cells that can be tested include head and neck squamous cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2 ⁺ , HPV16 ⁺ ), liver adenocarcinoma cell line SK-HEP-1 (ATCC HTB- 52; HLA-A2 ⁺ , HPV16 ⁻ ), cervical squamous cell carcinoma cell line SiHa (ATCC HTB-35; HLA-A2 ⁻ , HPV16 ⁺ ), cervical cancer cell line C33A (ATCC HTB-31; HLA -A2 ^+, HPV16 ^-), cervical adenocarcinoma cell line ^{HeLa (ATCC CCL-2; HLA} -A2 -, HPV16 -), hepatocellular carcinoma cell line ^{Hep3B (ATCC HB-8064; HLA} -A2 -, HPV16 - ), hepatocellular carcinoma cell line ^{HepG2 (ATCC HB-8065; HLA} -A2 -, HPV16 -), phosphorus Pas lymphoma cell lines ^{Raji (ATCC CCL-86; HLA} -A2 -, HPV16 -), T cell leukemia cell line ^{Jurkat (ATCC TIB-152; HLA} -A2 -, HPV16 -), lymphoma cell lines Daudi (ATCC CCL-213 ^{; HPA-A2 -, HPV16 -} ), and leukemia cell line ^{K562 (ATCC CCL-243; HLA} -A2 -, HPV16 -) are included.

Claims (31)

  Contains an antibody portion that specifically binds to a complex (HPV16-E7 / MHC class I complex, or E7MC) comprising a human papillomavirus subtype 16 (HPV16) E7 peptide and a major histocompatibility complex (MHC) class I protein Isolated anti-E7MC construct. The MHC Class I ^{proteins, HLA-A * 02: 01} , HLA-A * 02: 02, HLA-A * 02: 06, the group consisting of ^HLA-A * 02:07 and ^HLA-A * 02:11 2. An isolated anti-E7MC construct according to claim 1 which is selected. 3. The isolated anti-E7MC construct of claim 2, wherein the MHC class I protein is HLA-A ^* 02 ^: 01.   4. The isolated anti-E7MC construct according to any one of claims 1 to 3, wherein the HPV16-E7 peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-14.   5. The isolated anti-E7MC construct of claim 4, wherein the HPV16-E7 peptide has an amino acid sequence of YMLDLQPET (SEQ ID NO: 4).   6. The isolated anti-E7MC construct according to any one of claims 1 to 5, wherein the antibody portion is a full length antibody, Fab, Fab ′, (Fab ′) 2, Fv or single chain Fv (scFv). . 7. The isolated anti-E7MC construct according to any one of claims 1 to 6, which binds to the HPV16-E7 / MHC class I complex with a _Kd of about 0.1 pM to about 500 nM. The antibody portion is
i) heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of GF / G / YSS / TFSS / TSYA / G (SEQ ID NO: 183), Or a variant thereof containing up to about 3 amino acid substitutions, I / N / I-X-X-G-G / T / IT / A / P or I-S-X-S / D-G HC-CDR2 comprising the amino acid sequence of / NG / SNT / I / K (SEQ ID NO: 184 or 185), or variants thereof containing up to about 3 amino acid substitutions, and ARS / RY / S / G-Y / V-Y / W-G-X-Y-D, A-R-G-X-X-X-Y-Y / G / S or A-R-G- HC-CDR3 comprising any one amino acid sequence of XXXYQ / WWSXDD (SEQ ID NO: 186 to 188), or about 3 at most A heavy chain variable domain comprising a variant thereof containing a substitution of noic acid; and ii) an amino acid of NIGSGN / K or LRS / NXY (SEQ ID NO: 189 or 190) A light chain complementarity determining region (LC-CDR) 1 comprising a sequence, or variant thereof comprising substitutions of up to about 3 amino acids, and A / Q / NS / A / VW / Y / RD- Light chain variable comprising LC-CDR3 comprising the amino acid sequence of S / DSL / S / GXXXXV (SEQ ID NO: 191), or variants thereof containing substitutions of up to about 3 amino acids 8. The isolated anti-E7MC construct according to any one of claims 1 to 7, comprising a domain, wherein X can be any amino acid. The antibody portion is
i) HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 57-77, or variants thereof containing substitutions of up to about 5 amino acids, HC-CDR2 comprising any one amino acid sequence of SEQ ID NOs: 78-98 Or a variant thereof comprising a substitution of up to about 5 amino acids, and an HC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 99-119, 244 and 245; or a variant thereof comprising a substitution of up to about 5 amino acids A heavy chain variable domain comprising; and ii) any LC-CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or variants thereof comprising substitutions of up to about 5 amino acids, SEQ ID NOs: 141-161 LC-CDR2 containing one amino acid sequence, or substitution of up to about 3 amino acids And a light chain variable domain comprising a variant thereof comprising LC-CDR3 comprising any one amino acid sequence of SEQ ID NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5 amino acid substitutions. Item 8. The isolated anti-E7MC construct according to any one of Items 1 to 7.   The antibody portion is a) a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or at least about one of any one of SEQ ID NOs: 15-35 and 233-237; Variants thereof having 95% sequence identity; and b) a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or any of SEQ ID NOs: 36-56 and 238-243 10. The isolated anti-E7MC construct of claim 9, comprising a variant thereof having at least about 95% sequence identity to one.   11. The isolated anti-E7MC construct according to any one of claims 1 to 10, which is multispecific.   Tandem scFv, diabody (Db), single chain diabody (scDb), dual affinity retargeting (DART) antibody, dual variable domain (DVD) antibody, knob-into-hole (KiH) antibody, dock and 12. The isolated anti-E7MC construct of claim 11, which is a lock (DNL) antibody, a chemically cross-linked antibody, a heteromultimeric antibody or a heteroconjugate antibody.   13. The isolated anti-E7MC construct of claim 12, which is a tandem scFv comprising two scFvs linked by a peptide linker.   14. The isolated anti-E7MC construct of any one of claims 11-13, further comprising a second antibody portion that specifically binds to a second antigen.   15. The isolated anti-E7MC construct of claim 14, wherein the second antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L and HVEM.   The second antigen is CD3ε and the isolated anti-E7MC construct comprises a tandem scFv comprising an N-terminal scFv specific for the HPV16-E7 / MHC class I complex and a C-terminal scFv specific for CD3ε. 15. The isolated anti-E7MC construct of claim 14, wherein   11. A chimeric antigen receptor comprising an extracellular domain comprising said antibody portion, a transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence and a CD28 intracellular signaling sequence. An isolated anti-E7MC construct according to claim 1.   The isolated anti-E7MC construct is an immunoconjugate comprising the antibody portion and an effector molecule, wherein the effector molecule is selected from the group consisting of drugs, toxins, radioisotopes, proteins, peptides and nucleic acids 11. The isolated anti-E7MC construct according to any one of claims 1 to 10, which is an agent.   11. The isolated anti-E7MC construct according to any one of claims 1 to 10, which is an immunoconjugate comprising the antibody moiety and a label.   A pharmaceutical composition comprising the isolated anti-E7MC construct according to any one of claims 1-18.   A host cell expressing the isolated anti-E7MC construct according to any one of claims 1-19.   20. Nucleic acid encoding a polypeptide component of the isolated anti-E7MC construct according to any one of claims 1-19.   An effector cell expressing the isolated anti-E7MC construct of claim 17.   24. The effector cell of claim 23, which is a T cell.   20. A method of detecting a cell presenting on its surface a complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the cell is contacted with the isolated anti-E7MC construct of claim 19. And detecting the presence of the label on the cell. A method of treating an individual having an HPV16-E7 positive disease comprising:
A method comprising administering a) an effective amount of a pharmaceutical composition according to claim 20, or b) an effective amount of an effector cell according to claim 23 or 24. A method for diagnosing an individual having an HPV16-E7 positive disease, comprising:
a) administering to said individual an effective amount of an isolated anti-E7MC construct according to claim 19; and b) determining the level of said label in said individual, said label being above a threshold level Wherein the level indicates that the individual has the HPV16-E7 positive disease. A method for diagnosing an individual having an HPV16-E7 positive disease, comprising:
a) contacting a sample from the individual with an isolated anti-E7MC construct according to claim 19; and b) the number of cells bound to the isolated anti-E7MC construct in the sample. Wherein the value of the number of cells associated with the isolated anti-E7MC construct above a threshold level indicates that the individual has the HPV16-E7 positive disease .   29. The method according to any one of claims 26 to 28, wherein the HPV16-E7 positive disease is an HPV16-E7 positive cancer.   30. The method of claim 29, wherein the HPV16-E7 positive cancer is squamous cell carcinoma.   30. The method of claim 29, wherein the HPV16-E7 positive cancer is cervical cancer, anogenital cancer, head and neck cancer or oropharyngeal cancer.

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