EA044060B1 - ANTIGEN-BINDING PROTEINS AGAINST HUMAN PAPILLOMA VIRUS (HPV) AND METHODS OF THEIR APPLICATION - Google Patents

Description

Related Applications

This application claims the benefit of U.S. Provisional Application No. 62/525937, filed June 2017, the entire contents of which are expressly incorporated herein by reference in their entirety.

List of sequences

This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy in question, created on June 27, 2018, is called 10355WO01_seqlisting.txt and is 253600 bytes in size.

Field of technology

The present invention relates to antigen binding proteins that specifically bind to the HLA-presented peptide of human papillomavirus (HPV), as well as therapeutic and diagnostic methods for using these binding proteins.

State of the art

Human papillomavirus (HPV) belongs to a group of small, non-enveloped DNA viruses that are extremely common throughout the world. HPV is primarily transmitted through sexual contact, and most people become infected with HPV soon after becoming sexually active.

There are more than 170 types of HPV, some of which can cause warts or benign papillomas, and others, at least 13 of which cause malignancy (also known as high-risk HPV), including cervical cancer, anogenital cancer (cancer of the anus, penis , vagina and vulva), head/neck cancer and cancer of the oropharynx, including the back of the throat, base of the tongue and tonsils. In fact, HPV is present in 20-40% of all head and neck squamous cell carcinomas (HNSCC) and 100% of cervical cancers.

Cervical cancer is the second most common malignancy among women living in less developed regions, with an estimated 445,000 new cases in 2012 (84% of new cases worldwide). In 2012, approximately 270,000 women died from cervical cancer; more than 85% of these deaths occur in low- and middle-income countries.

Two types of HPV (16 and 18) cause approximately 70% of all cases of cervical cancer and precancerous lesions of the cervix. The development of cancer during persistent infection with high-risk HPV subtypes, such as HPV 16 or 18, is mainly due to the expression of two viral oncoproteins, E6 and E7, which are persistently expressed in lesions and are present on the cell surface of MHC class I, but are not expressed in normal cells. E6 and E7 promote genomic instability and cellular transformation by degrading the tumor suppressors p53 and Rb in a proteasome-dependent manner. Tumors arise several years after the initial immortalization processes in the cell, and continued expression of E6 and E7 is required to maintain the transformed phenotype and prevent cell growth arrest and/or apoptosis (McLaughlin-Drubin M.E. & Miinger K., Virology (2009) 384: 335- 344).

Although vaccines against HPV L1 and L2, the major capsid proteins of the HPV-6, -11, -16, and -18 subtypes, have been developed to prevent infection, such vaccines cannot treat patients with established lesions. Thus, treatment for patients suffering from cervical cancer still uses traditional highly invasive and painful approaches such as surgery, radiation therapy and chemotherapy. In addition, although such treatments may be beneficial for patients with early-stage cervical cancer, they are of limited value for patients with advanced or recurrent cervical cancer.

Accordingly, there is an unmet need in the field for new therapeutic strategies to target HPV with high specificity and to treat cervical cancer and other cancers caused by HPV.

Summary of the invention

The present invention relates to antigen-binding proteins that specifically bind to the conformational epitope of the HLA-presented E7 peptide of human papillomavirus (HPV) 16 (HLAA2:HPV16E7). The antigen-binding proteins of the present invention bind with a high degree of specificity to HLA-presented HPV16E7 and do not bind to HLA-presented peptides that differ by 1, 2, 3, 4, 5 or more amino acids. The antigen-binding proteins of the invention provide specific targeting of cells presenting the HPV16E7 peptide (i.e., cells presenting on their surface the HPV16E7 peptide associated with an MHC molecule, e.g., HLA-A2), such as tumor cells expressing HPV16E7, and in some in embodiments, promoting T cell activation, for example, to promote T cell-mediated killing of such cells. In addition, when fused to a detectable fragment, the antigen binding proteins of the present invention allow the diagnosis and prognosis of HPV16E7-positive diseases or disorders with high sensitivity to changes in the number and distribution of cells presenting the HPV16E7 peptide, which is a more relevant measure of disease progression than the level of HPV16E7 in blood.

The antigen binding proteins of the invention may be antibodies, such as full length (e.g. IgG1 or IgG4 antibodies), or may contain only an antigen binding portion

- 1 044060 antibodies (eg Fab, F(ab')2 or scFv fragment) and can be modified to affect functionality, for example to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164: 1925 -1933). In some embodiments, the antigen binding proteins of the invention may be antibodies or antigen binding fragments thereof. In some embodiments, the antigen binding proteins may be bispecific.

In a first aspect, the present invention provides isolated recombinant antigen-binding proteins that specifically bind to a conformational epitope of the HLA-presented E7 peptide of human papillomavirus (HPV) 16, such as the HLA-presented peptide containing amino acid residues 11-19 or 82-90 of HPV16E7. In some embodiments, the antigen binding proteins are antibodies. In some embodiments, the antibodies are fully human.

Representative antigen binding proteins against HLA-A2:HPV16E7 of the present invention are listed in table. 1 and 2 in the present description. In table 1 shows the amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of typical antibodies against HLA-A2:HPV16E7. In table Figure 2 shows the nucleic acid sequence identifiers HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of representative antibodies against HLA-A2:HPV16E7.

The present invention relates to antigen binding proteins containing HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to antigen binding proteins containing LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least , 99% sequence identity with them.

The present invention also relates to antigen binding proteins containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences HCVR listed in table. 1, in combination with any of the LCVR amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antigen binding proteins comprising the HCVR/LCVR amino acid sequence pair contained in any of the exemplary anti-HLA-A2:HPV16E7 antigen binding proteins listed in Table. 1. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/ 306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498 506 /514 and 522/530. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NO: 2/10 (e.g., H4sH17364N), 34/42 (e.g., H4sH17670P), 82/90 (e.g., H4sH17675P), 194/202 (e.g. , H4sH17930N2), 282/290 (for example, H4sH21064P) and 506/514 (for example, H4sH17363N).

In some embodiments, the present invention relates to antigen binding proteins against HLA-A2:HPV16E7, containing HCVR and LCVR, and the specified HCVR contains the amino acid sequence specified in table. 1, having no more than five amino acid substitutions, and the specified LCVR contains the amino acid sequence specified in table. 1, having no more than five amino acid substitutions. For example, the present invention provides antigen binding proteins against HLA-A2:HPV16E7 comprising HCVR and LCVR, wherein said HCVR comprises the amino acid sequence SEQ ID NO: 194 having no more than five amino acid substitutions, and said LCVR comprises the amino acid sequence SEQ ID NO: 194. 202, having no more than five amino acid substitutions. In another exemplary embodiment, the present invention provides anti-HLA-A2:HPV16E7 antigen binding proteins comprising HCVR and LCVR, wherein said HCVR comprises the amino acid sequence of SEQ ID NO: 194 having at least one amino acid substitution, and said LCVR comprises the amino acid sequence of SEQ ID NO: 202, having at least one amino acid substitution.

The present invention also relates to antigen binding proteins containing a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in table. 1, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity.

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The present invention also relates to antigen binding proteins containing a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in table. 1, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity.

The present invention also relates to antigen binding proteins containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in table. 1, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity.

The present invention also relates to antigen binding proteins containing a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in table. 1, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity.

The present invention also relates to antigen binding proteins containing a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in table. 1, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antigen binding proteins containing light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in table. 1, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antigen binding proteins containing a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), containing any of the amino acid sequences of HCDR3 listed in table. 1, in combination with any of the LCDR3 amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antigen binding proteins comprising the HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary anti-HLA-A2:HPV16E7 antigen binding proteins listed in Table. 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8/16 (eg, H4sH17364N), 40/48 (eg, H4sH17670P), 88/96 (eg, H4sH17675P), 200 /208 (eg H4sH17930N2), 288/296 (eg H4sH21064P) and 512/520 (eg H4sH17363N).

The present invention also relates to antigen binding proteins containing HCVR and LCVR, wherein said HCVR contains HCDR1 containing an amino acid sequence different from the amino acid sequence shown in table. 1, per 1 amino acid, HCDR2 contains an amino acid sequence different from the amino acid sequence shown in table. 1, per 1 amino acid, and HCDR3 contains an amino acid sequence that differs from the amino acid sequence indicated in table. 1, per 1 amino acid. In some embodiments, the present invention provides antigen binding proteins comprising HCVR and LCVR, wherein said LCVR comprises LCDR1 containing an amino acid sequence different from the amino acid sequence set forth in Table. 1, per 1 amino acid, LCDR2 containing an amino acid sequence different from the amino acid sequence shown in table. 1, per 1 amino acid, and LCDR3 containing an amino acid sequence different from the amino acid sequence shown in table. 1, per 1 amino acid. For example, the present invention relates to antigen binding proteins against HLA-A2:HPV16E7 containing HCVR and LCVR, wherein said HCVR contains HCDR1 containing the amino acid sequence of SEQ ID NO: 196 or an amino acid sequence different from SEQ ID NO: 196 by 1 amino acid, HCDR2 contains the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence different from SEQ ID NO: 198 by 1 amino acid, and HCDR3 contains the amino acid sequence of SEQ ID NO: 200 or an amino acid sequence different from SEQ ID NO: 200 by 1 amino acid. In another illustrative embodiment, the present invention provides antigen binding proteins comprising HCVR and LCVR, wherein said LCVR contains LCDR1 containing the amino acid sequence of SEQ ID NO: 204 or an amino acid sequence different from SEQ ID NO: 204 by 1 amino acid, LCDR2 containing the amino acid sequence SEQ

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ID NO: 206 or an amino acid sequence different from SEQ ID NO: 206 by 1 amino acid, and LCDR3 containing the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence different from SEQ ID NO: 208 by 1 amino acid.

The present invention also provides antigen binding proteins comprising a set of six CDRs (ie, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary antigen binding proteins listed in table. 1. In some embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NO: 4-6-8-12-14-16 (for example, H4sH17364N), 36-38 -40-44-46-48 (for example, H4sH17670P), 84-86-88-92-94-96 (for example, H4sH17675P), 196-198-200-204-206-208 (for example, H4sH17930N2), 284 - 286-288-292-294-296 (for example, H4sH21064P) and 508-510-512-516-518-520 (for example, H4sH17363N).

In a related embodiment, the present invention provides antigen binding proteins comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the amino acid sequence pair HCVR/LCVR, as defined in any of the exemplary antigen binding proteins listed in table. 1. For example, the present invention includes antigen binding proteins containing the amino acid sequences HCDR1HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10 (for example, H4sH17364N) , 34/42 (eg H4sH17670P), 82/90 (eg H4sH17675P), 194/202 (eg H4sH17930N2), 282/290 (eg H4sH21064P) and 506/514 (eg H4sH17363N).

Methods and methods for identifying CDRs in HCVR amino acid sequences. and LCVR. are well known in the art and can be used to identify CDRs in specified HCVR amino acid sequences. and/or LCVR disclosed herein. Exemplary conventions that may be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches; see, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). Public databases are also available to identify CDR sequences in the antigen binding protein.

The present invention includes antigen binding proteins against HLA-A2:HPV16E7 having a modified glycosylation profile. In some embodiments, modification to remove unwanted glycosylation sites, or an antibody that lacks a fucose moiety present in the oligosaccharide chain, may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modification can be made to modify complement dependent cytotoxicity (CDC).

In some embodiments, the antigen-binding proteins of the invention are monoclonal antibodies containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences HCVR listed in table. 1, in combination with any of the LCVR amino acid sequences listed in table. 1. In some embodiments, the monoclonal antibodies comprise an Fc domain of an isotype selected from the group consisting of IgA, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM, and a variant thereof.

The present invention relates to antigen binding proteins or antigen binding fragments thereof containing a heavy chain containing an amino acid sequence selected from any of the HC amino acid sequences listed in table. 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to antigen binding proteins or antigen binding fragments thereof containing a light chain containing an amino acid sequence selected from any of the LC amino acid sequences listed in table. 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also relates to antigen binding proteins or antigen binding fragments thereof containing a pair of HC amino acid sequences (LC/LC) (HC/LC) containing any of the HC amino acid sequences listed in table. 3, in combination with any of the LC amino acid sequences listed in table. 3. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HC/LC amino acid sequence pair contained in any of the exemplary anti-PD-1 antibodies listed in Table. 3. In some embodiments, the HC/LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 578/579, 580/581, 582/583, 584/585, 586/587, 588/589, 590/591, and 592/593.

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In one aspect, the present invention relates to antigen binding proteins or antigen binding fragments thereof that bind to an HLA peptide complex, wherein the antigen binding protein or antigen binding fragment thereof contacts at least 60%, at least 70%, at least 80% or at least 90% of the amino acid residues of the peptide that is part of the HLA-peptide complex. In some embodiments, the antigen binding protein or antigen binding fragment thereof covers or contacts all amino acid residues of a peptide included in the HLA peptide complex. In some embodiments, the antigen binding protein or antigen binding fragment thereof binds to the HLA peptide complex with high affinity and specificity, wherein the antigen binding protein or antigen binding fragment thereof contacts along the entire length of the presented peptide. As used herein, the term contact includes direct or water-mediated hydrogen bonds, charge-charge interactions, or hydrophobic/van der Waals interactions. In one embodiment, the antigen binding protein or antigen binding fragment thereof binds to the HLA-A2-HPV16E7 11-19 peptide complex, wherein the antigen binding protein binds to at least 6 of the 10 amino acid residues of peptide 11-19 (SEQ ID NO: 538) and HLA- A2 in such a way that it completely overlaps the HLA-A2 peptide complex. In some embodiments, the antigen binding protein or antigen binding fragment thereof comprises an HCVR CDR and an LCVR CDR, where each HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table. 1. In one embodiment, the antigen binding protein is entirely human. In some embodiments, fully human antigen-binding proteins are produced using methods and technologies other than phage display. In one embodiment, the antigen binding proteins comprise a light chain variable region of the IGKV1-39 subtype.

In some embodiments, the present invention provides antigen binding proteins or antigen binding fragments thereof that bind to the HLAA2:HPV16E7 11-19 peptide complex, wherein the antigen binding protein binds to one or more amino acids of SEQ ID NO: 538. In one embodiment, the antigen binding protein binds to with at least 6 amino acids of SEQ ID NO: 538. In one embodiment, the antigen binding protein binds to one or more amino acids selected from the group consisting of Y11, D14, L15, P17 and E18 of SEQ ID NO: 538.

In some embodiments, the present invention provides an antigen binding protein that specifically binds to a conformational epitope of the HLA-A2-presented human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), wherein the conformational epitope comprises one or more amino acids of SEQ ID NO: 538 In some embodiments, the conformational epitope comprises one or more amino acids selected from the group consisting of Y11, D14, L15, P17, and E18 of SEQ ID NO: 538.

The present invention also provides antigen binding proteins that compete for specific binding to HLA-A2:HPV16E7 with an antigen binding protein comprising a HCVR CDR and an LCVR CDR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table . 1.

The present invention also provides antigen binding proteins that cross-compete for binding to HLA-A2:HPV16E7 with a reference antigen binding protein comprising a HCVR CDR and an LCVR CDR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in table 1.

The present invention also provides antigen binding proteins that bind to the same epitope as a reference antigen binding protein comprising a HCVR CDR and an LCVR CDR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table. 1. In some embodiments, the present invention provides antigen binding proteins that bind to the same epitope as a reference antigen binding protein comprising an HCVR CDR and an LCVR CDR, wherein the HCVR is selected from the group consisting of SEQ ID NO: 2, 34, 82 , 194, 282 and 504, and the LCVR is selected from the group consisting of SeQ ID NOs: 10, 42, 90, 202, 290 and 514.

In one embodiment, the invention provides a recombinant isolated antigen binding protein that specifically binds to a conformational epitope HEA-A2 of the human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), wherein the antigen binding protein has a property selected from the group consisting of the following: ( a) binds to the monomeric HLA-A2:HPV16E7 11-19 peptide with a binding dissociation equilibrium constant (KD) of less than about 20 nM, as measured in a surface plasmon resonance assay at 25°C; (b) binds to the monomeric HLA-A2:HPV16E7 82-90 peptide with a binding dissociation equilibrium constant (KD) of less than about 25 nM, as measured in a surface plasmon resonance assay at 25°C; (c) binds to cells expressing the HLA-A2:HPV16E7 peptide 11-19, with EC ₅₀

- 5044060 is less than about 6 nM and does not bind to cells expressing predicted non-target peptides, as determined by luminescent assay; (d) binds to cells expressing the HLA-A2:HPV16E7 82-90 peptide with an EC ₅₀ of less than about 1 nM and substantially does not bind to cells expressing predicted non-target peptides as determined by luminescence assay; (e) binds to cells expressing the HLA-A2:HPV16E7 11-19 peptide with an EC ₅₀ of less than about 30 nM, as determined by flow cytometry; (f) binds to cells expressing the HPV16E7 82-90 peptide with an EC ₅₀ of less than about 75 nM, as determined by flow cytometry; and (g) the conformational epitope contains one or more amino acids of SEQ ID NO: 538. As disclosed elsewhere herein, a non-target peptide refers to a peptide that differs by 1, 2, 3, 4, 5 or more amino acids from the target peptide (for example, HPV16 E7 peptide 11-19).

In a second aspect, the present invention relates to nucleic acid molecules encoding antigen binding proteins against HLA-A2:HPV16E7. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule includes a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least at least 99% sequence identity with them.

The present invention also provides nucleic acid molecules encoding HCVR, wherein HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as defined by any of the exemplary antigen binding proteins against HLA-A2:HPV16E7 listed in table. 1.

The present invention also provides nucleic acid molecules encoding LCVR, wherein LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as defined by any of the exemplary antigen binding proteins against HLA-A2:HPV16E7 listed in table. 1.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains the amino acid sequence of any of the HCVR amino acid sequences listed in table. 1, and where LCVR contains the amino acid sequence of any of the LCVR amino acid sequences listed in table. 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them. In some embodiments, according to this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from the same antigen binding protein against HLA-A2:HPV16E7 listed in table. 1.

The present invention relates to nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in table. 3. The present invention also relates to nucleic acid molecules encoding any of the light chain amino acid sequences listed in table. 3.

The present invention also relates to nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), where the HC contains the amino acid sequence of any of the amino acid sequences of the HC listed in table. 3, and where LC contains the amino acid sequence of any of the LC amino acid sequences listed in table. 3.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy and/or light chain variable region of an anti-HLA-A2:HPV16E7 antigen binding protein. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, i.e. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences, as indicated in table. 1. The present invention also relates to recombinant expression vectors capable of expressing a polypeptide containing a heavy and/or light chain of an anti-HLA-A2:HPV16E7 antigen binding protein. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, i.e. nucleic acid molecules encoding any of the heavy chain or light chain sequences as specified in table. 2. Also included within the scope of the present invention are the host cells into which such vectors have been introduced, as well as methods for producing antigen binding proteins by culturing the host cells under conditions allowing the production of antigen binding proteins and recovering the antigen binding proteins thus obtained.

In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an isolated recombinant antigen binding protein that specifically binds to a conformational epitope of an HLA-A2 presented HPV16E7 peptide (e.g., a peptide containing amino acid residues 11-19 or 82-90 of HPV16E7) and pharmaceutically acceptable carrier. In a related aspect, the invention provides a composition that is a combination of an anti-HLA-A2:HPV16E7 antigen binding protein and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-HLAA2:HPV16E7 antigen-binding protein. Exemplary agents that may advantageously be combined with anti-HLA-A2:HPV16E7 antigen binding protein include, but are not limited to, other agents that bind and/or modulate HPV replication or infection (including other antibodies or antigen binding fragments thereof, etc.) and /or agents that modulate immune cell activation. Additional treatments that can be used in combination with the anti-HLA-A2:HPV16E7 antigen binding proteins of the present invention are disclosed elsewhere herein.

In a fourth aspect, the invention relates to methods of treating a patient having an HPV-associated disease or disorder, such as an HPV16E7-positive cancer. The methods include administering a therapeutically effective amount of an anti-HLA-A2:HPV16E7 antigen binding protein of the invention or a pharmaceutical composition of the invention to a patient in need thereof. The disorder being treated is any disease or condition that is improved, neutralized, inhibited or prevented by the antigen binding proteins and compositions provided herein. In some embodiments, an antigen binding protein (or pharmaceutical composition) of the invention is administered in combination with a second therapeutic agent to a patient in need thereof. The second therapeutic agent may be selected from the group consisting of an antibody to a T cell coinhibitor, an antibody to a tumor cell antigen, an antibody to a T cell receptor, an antibody to an epitope on a cell infected with a virus, a cytotoxic agent, an antitumor drug, an antiviral drug , an anti-inflammatory drug (eg, corticosteroids), a chemotherapy agent, surgery, radiation therapy, an immunosuppressant, and any other drug or therapy known in the art. In some embodiments, the second therapeutic agent may be an agent that helps neutralize or reduce any potential side effect(s) associated with the antigen binding protein of the invention if such side effect(s) are to occur.

In some embodiments, the present invention provides methods for inhibiting the growth of an HPV-associated malignancy. For example, the present invention relates to methods of suppressing tumor growth of a primary tumor or metastatic tumor in a patient. In some embodiments, the present invention provides methods for improving survival (eg, progression-free survival or overall survival) in a patient with an HPV-associated malignancy. Examples of malignancy include, but are not limited to, squamous cell carcinoma, such as squamous cell carcinoma of the head and neck, cervical cancer, anogenital cancer, oropharyngeal cancer.

In some embodiments, the present invention provides methods for inhibiting or suppressing the growth of identified tumors. The methods include administering to a patient in need thereof a pharmaceutical composition containing a therapeutically effective amount of an antigen binding protein of the present invention. In some embodiments, the antigen binding protein is administered in combination with a second therapeutic agent.

The antigen binding protein, such as an antibody or an antigen binding fragment thereof, can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

An antigen binding protein, such as an antibody or an antigen binding fragment thereof, can be administered at a dose of from about 0.1 mg/kg body weight to about 100 mg/kg body weight of the patient.

In a fifth aspect, the present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR). The CAR may include an extracellular binding domain that specifically binds to a conformational epitope of the HLA-A2-presented human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), e.g., amino acid residues 11-19 or 82-90 of HPV16E7, a transmembrane domain, and an intracellular signaling domain domain. In one embodiment, the extracellular binding domain is an anti-HLA-A2:HPV16E7 antigen binding protein or an antigen binding fragment thereof. Exemplary anti-HLA-A2:HPV16E7 antigen binding proteins of the present invention are any of the antigen binding proteins described herein.

For example, in some embodiments, an antigen binding protein suitable for use in a CAR of the invention comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in any one of the heavy chain variable region (HCVR) sequences, listed in table. 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the light chain variable region (LCVR) sequences listed in table. 1.

In other embodiments, an antigen binding protein suitable for use in a CAR of the invention includes an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table. 1; and/or LCVR having an amino acid sequence selected from the group consisting of LCVR sequences listed in table. 1.

In some embodiments, an antigen binding protein suitable for use in a CAR of the invention includes (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table. 1; and (b) LCVR,

- 8 044060 having an amino acid sequence selected from the group consisting of LCVR sequences listed in table. 1.

In one embodiment, an antigen binding protein suitable for use in a CAR of the invention comprises (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SeQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 4 92, 508 and 524; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430, 446, 462, 478, 494, 510 and 526; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512 and 528; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 228, 244,260 , 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516 and 532; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518 and 534; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248 , 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520 and 536.

In a further embodiment, an antigen binding protein suitable for use in a CAR of the invention comprises a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66 /74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258 , 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418, 426/434, 442/450, 458 /466, 474/482, 490/498, 506/514 and 522/530, such as the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 34/42, 82/ 90, 194/202, 282/290 and 506/514.

In some embodiments, the isolated antigen binding protein for use in the CAR of the present invention is a scFv.

In other aspects, the present invention provides vectors containing isolated CAR nucleic acid molecules; and immune effector cells containing such vectors.

In other aspects, the present invention provides methods for treating a patient having an HPV-associated disease or disorder, such as an HPV16E7-positive malignancy, such as squamous cell cancer, such as cervical cancer, small cell head and neck cancer, anogenital cancer, and oropharyngeal cancer. The methods include administering to a patient a population of immune effector cells containing the CARs of the invention.

In some aspects, the present invention relates to methods for detecting HPV16E7 positive cells, for example, in a patient or in a sample obtained from a patient. The methods include contacting a cell, such as a cell sample obtained from a patient, or administering to the patient an antigen binding protein of the invention containing a detectable fragment, and detecting the presence of the detectable fragment.

Other embodiments will become apparent from a review of the following detailed description.

Detailed description

Before the methods of the present invention are described, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is necessary only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as would be given to them by one of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials will be described below. All publications mentioned in this description are incorporated herein by reference in their entirety.

The term human papillomavirus (HPV) refers to a small, non-enveloped deoxyribonucleic acid (DNA) virus that infects skin or mucosal cells. The closed, double-stranded viral genome is approximately 8 kb in length. The genome encodes 6 early proteins responsible for viral replication and 2 late proteins, L1 and L2, which are viral structural proteins. There are over 170 types of HPV that have been identified, and they are designated by numbers. Some types of HPV, such as HPV-5, can cause infections that persist throughout a person's life without any clinical symptoms. HPV types 1 and 2 can cause

- 9 044060 common warts in some infected people. HPV types 6 and 11 can cause genital warts and respiratory papillomatosis. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 82 are considered carcinogenic.

The term HPV16E7 refers to the early HPV type 16 gene, designated E7, and the protein translated from the gene.

The amino acid sequence of full-length HPV16E7 is provided in GenBank under accession number NP_041326.1 (SEQ ID NO: 537). The term HPV16E7 includes recombinant HPV16E7 or a fragment thereof. The term also includes HPV16E7 or a fragment thereof linked to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1. In some embodiments, the term includes HPV16E7 or a fragment thereof in the context of HLA-A2, associated with HLA-A2, or presented by HLA-A2.

The term HLA refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex that encodes major histocompatibility complex (MHC) proteins in humans. These cell surface proteins are responsible for regulating the immune system in humans. HLAs corresponding to MHC class I (A, B and C) present peptides inside the cell.

The term HLA-A refers to a group of human leukocyte antigens (HLA) that are encoded by the HLA-A locus. HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer and consists of a heavy α chain and a smaller β chain. The α chain is encoded by a variant of the HLA-A gene, and the β chain (β2-microglobulin) is an invariant β2-microglobulin molecule.

The term HLA-A2 refers to one specific group of major histocompatibility complex class I (MHC) alleles at the HLA-A locus; The α chain is encoded by the HLA-A*02 gene, and the β chain is encoded by the B2-microglobulin or B2M locus.

As used herein, the term antigen binding protein, binding protein, or binding molecule includes molecules that contain at least one antigen binding site that specifically binds to a molecule of interest, such as a conformational epitope of the HLA-A2 presented E7 peptide of human papillomavirus (HPV) 16 (peptide HPV16E7), for example, an HLA-A2-presented peptide containing amino acid residues 11-19 or 82-90. The binding protein may be an antibody, such as a full-length antibody, or an antigen binding fragment of an antibody, or a chimeric antigen receptor (CAR), or any other polypeptide, such as a receptor-antibody (Rab) protein.

The term HLA-A2:HPV16E7 antigen binding protein or HLAA2:HPV16E7 antigen binding protein or the like refers to an antigen binding protein, such as an antibody, or an antigen binding portion thereof, that specifically binds to a conformational epitope by presenting a peptide fragment of HPV16E7, for example amino acid residues 11-19 or amino acid residues 82-90 by HLA-A2. In some embodiments, a conformational epitope is created on the cell surface by an HLA-A2-presented HPV16E7 peptide.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antigen-binding protein, known as a paratope. One antigen can have more than one epitope. Thus, different antigen-binding proteins may bind to different sites on the antigen and may have different biological effects. The term epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to the region of an antigen that is bound by antigen binding protein. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. consist of nonlinear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface moieties of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in some embodiments, may have specific three-dimensional structural characteristics and/or specific charge.

In some embodiments, the binding protein is an antibody or an antigen-binding fragment thereof, such as a full-length antibody or an antigen-binding fragment thereof.

As used herein, the term antibody is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds (i.e., complete antibody molecules), as well as their multimers (for example, IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (consisting of CH1, _CH2 and CH3 domains). Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region (CL). The VH and _VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), alternating with regions that are more conserved, called framework regions (FR). Each VH and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

Replacing one or more CDR residues or omitting one or more CDRs is also possible. Antigen-binding proteins, such as antibodies, have been described in the scientific literature in which one or two CDRs for binding can be dispensed with. Padlan et al. (1995 FASEB J. 9: 133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only one-fifth to one-third of the CDR residues actually bind the antigen. Padlan also found many antibodies in which one or two CDRs did not have amino acids in contact with the antigen (see also Vajdos et al. 2002 J MolBiol 320: 415-428).

Non-antigen-binding CDR residues can be identified based on previous studies (eg, residues H60-H65 in CDRH2 are often not required) from Kabat CDR regions outside the Chothia CDR, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is typically replaced by an amino acid occupying the corresponding position in another human antibody sequence, or a consensus of such sequences. The positions to be replaced in the CDR and the amino acids to be replaced can also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

Antigen-binding proteins against HLA-A2:HPV16E7, such as fully human monoclonal antibodies against HLA-A2:HPV16E7 or antigen-binding fragments thereof, or CARs, disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention includes antigen-binding proteins, such as antibodies or antigen-binding fragments, or CARs thereof, that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and/or CDR region are mutated to a corresponding residue (residues) of the germline sequence from which the antigen binding protein was derived, or into the corresponding residue(s) of another human germline sequence, or into a residue by a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein together as germline mutations). One of ordinary skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antigen binding proteins, such as antibodies or antigen binding fragments thereof, or CARs, that contain one or more individual germline mutations or combinations thereof. In some embodiments, all of the framework residues and/or CDRs in the V _H and/or V _L domains are mutated back to residues found in the original germline sequence from which the antigen binding protein, such as an antibody, was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework residues and/or CDRs are mutated to the corresponding residue(s) of a different germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, antigen-binding proteins, such as antibodies or antigen-binding fragments or CARs thereof, of the present invention may contain any combination of two or more germline mutations in framework and/or CDR regions, for example, in which some individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated into the corresponding residue of another germline sequence. Once produced, antigen-binding proteins, such as antibodies and antigen-binding fragments, that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (in depending on the circumstances), reduced immunogenicity, etc. Antigen-binding proteins, eg antibodies or antigen-binding fragments thereof, or CARs, produced by this general method are included in the present invention.

The present invention also includes antigen binding proteins, such as fully human monoclonal antibodies against HLA-A2:HPV16E7 or antigen binding fragments thereof, or CARs, containing variants of any of the amino acid sequences HCVR, LCVR and/or CDR disclosed herein, having one or more conserved replacement For example, the present invention includes anti-HLA-A2:HPV16E7 antigen binding proteins having amino acid sequences HCVR, LCVR and/or CDRs, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

The term human antibody as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies (mAbs) of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR and, in particular, CDR3 . However, as used herein, the term human antibody is not intended to include mAbs in which CDR sequences derived from the germ line of another mammalian species (eg, mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated or generated in humans.

As used herein, the term recombinant refers to antigen binding proteins, such as antibodies or antigen binding fragments thereof, of the invention generated, expressed, isolated or produced by technologies or methods known in the art, such as recombinant DNA technology, which includes , such as DNA splicing and transgene expression. The term refers to antigen-binding proteins, for example antibodies expressed in a non-human mammal (including transgenic non-human mammals, such as transgenic mice), or in a cell expression system (for example, CHO cells), or isolated from recombinant combinatorial library of human antibodies.

As used herein, the terms chimeric antigen receptor or CAR, used interchangeably herein, refer to a recombinant fusion protein containing an extracellular domain capable of binding to an antigen (e.g., a conformational epitope of an HLA-A2-presented HPV16E7 peptide, e.g., a peptide containing amino acid residues 11-19 or 82-90 HPV16E7), transmembrane domain and at least one intracellular signaling domain.

As used herein, the term immune effector cell refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, cytokine secretion, induction of ADCC and/or CDC). In one embodiment, the immune effector cells used with CARs as described herein are T lymphocytes, particularly cytotoxic T cells (CTL; CD8+ T cells) and helper T cells (HTL; CD4+ T cells). Other T cell populations are also useful here, such as naïve T cells and memory T cells. As one skilled in the art will appreciate, other cells may also be used as immune effector cells with CARs as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include effector cell precursors, where such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in this regard, an immune effector cell includes progenitor effector cells, such as hematopoietic stem cells (HSCs), contained in a population of CD34+ cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which, when administered to a patient, differentiate into mature immune cells. effector cells or which can be induced in vitro to differentiate into mature immune effector cells.

As disclosed herein, the term non-target peptide refers to a peptide that differs by 1, 2, 3, 4, 5 or more amino acids from the target peptide (eg, HPV16 E7 1119 peptide). In some embodiments, the term includes a peptide that differs by 3 or less amino acids compared to the target peptide. For example, for a 9-mer peptide, if 1, 2 or 3 amino acids are not identical to the target peptide, it is considered a non-target peptide. In some embodiments, amino acid identity is expressed in terms of degree of similarity (DoS). If 6 or more amino acids in a 9-mer peptide are identical, the DoS is 6. In some embodiments, a peptide with a DoS < 6 is considered a non-target peptide. The term non-target peptide also refers to a peptide that is similar to a target peptide based on sequence homology, is predicted to bind HLA-A2, and is contained in a protein that is expressed in major normal tissues.

The term specifically binds or specifically binds to or the like means that an antigen binding protein, such as an antibody or antigen binding fragments or CARs thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may be characterized by an equilibrium dissociation constant of at least about 1 x 10'8 M or less (eg, a smaller K _D indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antigen-binding proteins, such as antibodies, have been identified using surface plasmon resonance, such as BIACORE™, that specifically bind to the HLA-A2 conformational epitope of human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), e.g. a peptide containing amino acid residues 11-19 or 82-90 of HPV16E7.

The term high-affinity antigen-binding protein, e.g., antibody, refers to those antigen-binding proteins, e.g., mAbs, that have binding affinity to the conformational epitope of an HLA-A2-presented HPV16E7 peptide, e.g., a peptide containing amino acid residues 11-19 or 82-90 of HPV16E7 expressed in the form of K _D at least 10' ⁸ M; preferably 10'9 M; more preferably ^10'10 M, even more preferably ^10'11 M, even more preferably ^10'12 M, as measured by surface plasmon resonance, eg, BIACORE™ or solution-affinity ELISA.

By the term Koff rate or kd is meant an antigen binding protein that dissociates from HLA-A2:HPV16E7, with a rate constant of 1x10'3 ^s'1 or less, preferably ^{1x10'4 s'1} or less, as determined by surface plasmon resonance, e.g. BIACORE™.

The terms antigen-binding portion of an antigen-binding protein (e.g., antibodies), antigen-binding fragment of an antigen-binding protein (e.g., antibodies), and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds with antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to bind to the HLA-A2 conformational epitope of an HPV16E7 presented peptide, e.g., a peptide containing HPV16E7 HLA-bound amino acid residues 11-19 or 82-90 -A2.

In specific embodiments, antigen binding proteins, such as an antibody or antibody or CAR fragments, of the invention may be conjugated to a fragment, such as a ligand, detectable fragment, or therapeutic fragment (immunoconjugate), such as a cytotoxin, a second antigen binding protein against HLA-A2:HPV16E7, an antibody to a tumor-specific antigen, an antitumor drug, or any other therapeutic component useful for treating a disease or condition, including an HPV-associated disease or disorder, such as an HPV16E7-positive malignancy or HPV infection, including chronic HPV infection.

The term isolated antigen-binding protein, e.g., isolated antibody, as used herein, is intended to mean an antigen-binding protein, e.g., an antibody, that is substantially free of other antigen-binding proteins, e.g., antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that binds specifically to HLA-A2:HPV16E7 or a fragment thereof, and is substantially free of antigen-binding proteins, such as antibodies, that specifically bind to antigens other than the conformational epitope of the HLA-A2-presented HPV16E7 peptide.

As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detecting changes in protein concentrations in a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

As used herein, the term K _D is intended to refer to the dissociation equilibrium constant of a particular antigen-binding protein-antigen interaction.

As used herein, the term cross-competes means that an antigen binding protein, such as an antibody or an antigen binding fragment thereof, binds to an antigen and inhibits or blocks the binding of another antigen binding protein, such as an antibody or an antigen binding fragment thereof. The term also includes competition between two antigen-binding proteins, such as antibodies, in both orientations, i.e. a first antigen binding protein, such as an antibody, that binds and blocks the binding of a second antigen binding protein, such as an antibody, and vice versa. In some embodiments, a first antigen binding protein, such as an antibody, and a second antigen binding protein, such as an antibody, can bind to the same epitope. Alternatively, the first and second antigen-binding proteins, eg antibodies, may bind to different but overlapping epitopes such that binding of one inhibits or blocks binding of the second, eg, through steric hindrance. Cross-competition between antigen-binding proteins, such as antibodies, can be measured by methods known in the art, for example, using label-free real-time biolayer interferometry analysis. Cross-competition between two antigen binding proteins, such as antibodies, can be expressed as the binding of a second antigen binding protein, such as an antibody, that is less than the background signal due to self-binding (where the first and second antigen binding proteins, such as antibodies, are the same antigen binding protein , for example an antibody). Cross-competition between 2 antigen-binding proteins, such as antibodies, can be expressed, for example, as the % binding of a second antigen-binding protein, such as an antibody, that is less than the baseline self-binding (where the first and second antigen-binding proteins, such as antibodies, are the same aka antigen-binding protein, such as an antibody).

The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least approximately nucleotide sequence identity 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any known sequence identity measurement algorithm, as. discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

Sequence identity can be calculated using an algorithm such as the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment or the Smith Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195-197) for local alignment. Another preferred algorithm is described by Dufresne et al. In Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and used in GenePAST software (GQ Life Sciences, Inc. Boston, MA).

As applied to polypeptides, the term substantially similar or substantially similar means that two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap weights, have at least 90% sequence identity, even more preferably less at least 95%, 96%, 97%, 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for effecting this regulation are well known to those skilled in the art; see, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that have side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acid side chains: aspartate and glutamate and 7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity scores assigned to different substitutions,

- 14 044060 deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein; see for example GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; program in GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity of regions of best overlap between query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

The expression therapeutically effective amount means an amount that provides the desired effect for which it is administered. The exact amount will depend on the purpose of treatment and will be determined by one skilled in the art using known methods (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term patient refers to an animal, preferably a mammal, in need of amelioration, prevention and/or treatment of a disease or disorder, such as HPV infection, or an HPV-associated disease or disorder, such as an HPV-associated malignancy (eg, HPV16E7 -positive malignant neoplasm). The term includes people who have or are at risk of HPV-associated disease, such as HPV-associated malignancy, metastatic HPV-associated malignancy, or HPV infection.

As used herein, the term antineoplastic drug means any agent useful for treating or attenuating or inhibiting cancer, including, but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea , asparaginase, corticosteroids, cyclophosphamide, mitotane (O,P'-(DDD)), biological agents (eg, antibodies and interferons), and radioactive agents. As used herein, the term cytotoxin or cytotoxic agent also refers to a chemotherapeutic agent and means any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracine dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.

As used herein, the term antiviral drug refers to any drug or therapy used to treat, prevent, or attenuate a viral infection in a host patient. The term antiviral drug includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids.

An immunogen containing any of the following can be used to generate antigen-binding proteins, for example antibodies, to a conformational epitope of an HLA-A2-presented HPV16E7 peptide, for example, a peptide containing amino acid residues 11-19 or residues 82-90 of HPV16E7 associated with HLA-A2 . In some embodiments, antigen binding proteins, such as antibodies, of the invention are produced using mice immunized with full-length native HPV16E7 protein (see NCBI accession number NP_041326.1) (SEQ ID NO: 537) or a recombinant HPV16E7 peptide, such as a peptide containing either amino acid residues 11-19 (YMLDLQPET; SEQ ID NO: 538) from GenBank accession number NP_041326.1 (SEQ ID NO: 537), or amino acid residues 82-90 (LLMGTLGIV; SEQ ID NO: 539) GenBank accession number NP041326. 1 (SEQ ID NO: 537) associated with HLA-A2.

Alternatively, HPV16E7 or a fragment thereof can be produced using standard biochemical methods and modified in the context of HLA-A2 and used as an immunogen.

In some embodiments, the immunogen may be a recombinant HPV16E7 peptide expressed in E. coli or any other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells.

In some embodiments, antigen binding proteins that specifically bind to a conformational epitope of the HLA-A2-presented HPV16E7 peptide can be produced using fragments of the above regions or peptides that extend beyond the designated regions by about 5-20 amino acid residues on one or both sides N- or C-terminus as described herein. In some embodiments, any combination of the above regions or fragments thereof can be used in the production of specific HLA-A2:HPV16E7 antigen binding proteins, such as antibodies.

Peptides can be modified to include or replace certain residues for labeling or for conjugation purposes with carrier molecules such as KLH. For example, a cysteine may be added at either the N-terminus or the C-terminus of the peptide, or a linker sequence may be added to prepare the peptide for conjugation, for example, with KLH for immunization.

Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in the examples herein. In Example 4, the binding affinities and kinetic constants of specific human antigen-binding proteins against HLA-A2:HPV16E7, eg antibodies, were determined using surface plasmon resonance, and measurements were carried out on a Biacore 4000 or T200 instrument. Examples 6 and 7 describe antibody binding to cells overexpressing HPV16E7 fragments.

Antigen-binding proteins, such as antibodies specific for HLA-A2:HPV16E7, may not contain additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the localization of the tag (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if a surface is coated with avidin, the peptide containing N-terminal biotin will be oriented such that the C-terminal part of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or an MRI detectable label. In some embodiments, such labeled antigen binding proteins can be used in diagnostic assays, including imaging assays.

Antigen-binding proteins.

The present invention relates to antigen binding proteins, which include antibodies or antigen binding fragments thereof, and CARs (eg, nucleic acid molecules encoding the CARs of the invention) (described below). Unless specifically stated otherwise, the term antibody as used herein is to be understood to include antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (ie, complete antibody molecules), as well as antigen-binding fragments thereof. The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to specifically bind to a conformational epitope of an HLA-A2 presented HPV16E7 peptide. An antigen binding protein, such as an antibody fragment, may include a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, a dAb fragment, a CDR containing fragment, or isolated CDRs. Antigen-binding proteins, such as antigen-binding antibody fragments, can be produced, for example, from complete antibody molecules using any suitable standard methods, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding variable and (optionally) constant domains antibodies. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries) or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to place one or more variable and/or constant domains in a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, and so on.

Non-limiting examples of antigen binding antibody fragments include: (i) Fab fragments; (ii) F(ab') ₂ -fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide) or the restricted peptide FR3-CDR3-FR4. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc.) , small molecule modulating immunopharmaceuticals (SMTPs) and shark IgNAR variable domains are also included in the expression of the antigen binding fragment as used herein.

An antigen-binding fragment of an antigen-binding protein (eg, an antibody) will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent or in frame with one or more framework sequences. In antigen-binding

- 16 044060 proteins having a V _H domain linked to a V _L domain, the V H and V _L domains can be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric _VH or _VL domain.

In some embodiments, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen binding fragment of the antigen binding protein of the present invention include: (i) V _H -C _H 1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) V _h -C _h 1Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) VlC _h 1-C _h 2-C _h 3; (xiii) V _l -C _h 2-C _h 3; and (xiv) V _l -C _l . In any variable and constant domain configuration, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other, and/or with one or more monomeric V _H or V _L domains (eg, disulfide bond(s)).

As with full antibody molecules, antigen-binding proteins, eg antigen-binding antibody fragments, can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine techniques available in the art.

Preparation of antigen-binding proteins.

Methods for generating antigen-binding proteins, such as human antibodies, in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to produce human antibodies that specifically bind to the conformational epitope of the HLA-A2-presented E7 peptide of human papillomavirus (HPV) 16 (HPV16E7 peptide).

Use of VELOCIMMIINE® technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing antigen binding proteins, for example monoclonal antibodies, high affinity antigen binding proteins, for example chimeric antibodies to a conformational epitope of an HLA-A2-resident peptide HPV16E7 is initially isolated with a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antigen binding protein, such as an antibody, containing the human variable region and the mouse constant region in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and functionally linked to DNA encoding the constant regions of the human heavy and light chain. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is stimulated with an antigen of interest, and lymphatic cells (such as B cells) are isolated from mice that express antigen-binding proteins, such as antibodies. Lymph cells can be fused with a myeloma cell line to generate immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotype heavy chain and light chain constant regions. Such an antigen binding protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific antigen-binding proteins, such as chimeric antibodies, or light and heavy chain variable domains, can be isolated directly from antigen-specific lymphocytes.

First, high-affinity antigen-binding proteins are isolated, such as chimeric antibodies having a human variable region and a mouse constant region. As in the experimental section below, antigen binding proteins are characterized and selected for desired characteristics,

- 17 044060 including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region to generate antigen binding proteins, for example the fully human antibodies of the invention, for example wild type or modified IgG1 or IgG4. While the selected constant region may vary depending on the particular application, the characteristics of high antigen binding affinity and target specificity are found in the variable region.

Bioequivalents.

Antigen-binding proteins against HLA-A2:HPV16E7 of the present invention include proteins having amino acid sequences that differ from those of described antigen-binding proteins, such as antibodies, but which retain the ability to bind the conformational epitope of the HLA-A2-presented HPV16E7 peptide. Such variants of antigen binding proteins contain one or more insertions, deletions or substitutions of amino acids relative to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antigen binding proteins. Likewise, DNA sequences encoding an antigen binding protein of the present invention include sequences that include one or more insertions, deletions or substitutions of nucleotides relative to the disclosed sequence, but which encode an antigen binding protein that is substantially bioequivalent to the antigen binding protein of the invention.

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, whether a single dose or multiple doses. doses Certain antigen-binding proteins or antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may nevertheless be considered bioequivalent because such differences in rate of absorption are intentional and reflected in the labeling, and are not essential for achieving effective concentrations of the drug in the body, for example, when used on a chronic basis, and are considered medically insignificant for the specific drug product being studied.

In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if there are no clinically significant differences in their safety, purity or potency.

In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if the patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduction in efficacy compared to continuing therapy without such switching

In one embodiment, two antigen binding proteins (or antibodies) are bioequivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antigen-binding protein or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with in vivo human bioavailability data and is reasonably predictive; (c) an in vivo test in humans or other mammals in which the relevant immediate pharmacological effect of the antigen binding protein (or its target) is measured as a function of time; and (d) a well-controlled clinical study that establishes the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

Bioequivalent variants of the antigen binding proteins (or antibodies) of the invention can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen binding proteins may include variant antigen binding proteins containing amino acid substitutions that modify the glycosylation characteristics of the antigen binding proteins, such as mutations that eliminate or remove glycosylation. Antigen binding proteins against HLA-A2:HPV16E7 containing Fc variants

In accordance with some embodiments of the present invention, antigen binding proteins against HLA-A2:HPV16E7 are provided, for example, antibodies comprising an Fc domain containing one or more mutations that increase or decrease antigen binding

- 18 044060 protein with FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention includes anti-HLA-A2:HPV16E7 antigen binding proteins containing a mutation in the C _H 2 or C _H 3 region of the Fc domain, where the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome, where the pH ranges from about 5.5 to about 6). Such mutations may result in an increase in the serum half-life of the antigen-binding protein when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T); or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K) and/or 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modification at position 250 and/or 428; or modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification comprises modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, Н433К) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or 308P). In yet another embodiment, the modification comprises modification 265A (eg, D265A) and/or 297A (eg, N297A).

For example, the present invention includes antigen binding proteins against HLA-A2:HPV16E7 containing an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); 257I and 311I (for example, P257I and Q311I); 257I and 434H (for example, P257I and n434H); 376V and 434H (for example, D376V and N434h); 307A, 380A and 4z4a (for example, T307A, E380A and N434A); and 433K and 434F (for example, Н433К and N434F). In one embodiment, the present invention includes anti-HLA-A2:HPV16E7 antigen binding proteins comprising an Fc domain containing the S108P mutation in the IgG4 hinge region to promote dimer stabilization. All possible combinations of the above Fc domain mutations and other mutations in the variable domains of the antigen binding protein disclosed herein are contemplated within the scope of the present invention.

The present invention also includes anti-HLA-A2:HPV16E7 antigen binding proteins comprising a chimeric heavy chain constant region ( _CH ), wherein the chimeric _CH region contains segments derived from the _CH regions of immunoglobulins of more than one isotype. For example, the antigen binding proteins of the invention may comprise a chimeric CH region containing part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with part or all of a CH3 domain derived from human IgG1, human IgG2 or human IgG4. In certain embodiments, the antigen binding proteins of the invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from the human IgG1, human IgG2, or human IgG4 region in combination with a lower hinge sequence (amino acid residues corresponding to positions 228 to 236 according to EU numbering) derived from the loop region of human IgG1, human IgG2 or human IgG4. In some embodiments, the chimeric hinge region comprises amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. An antigen binding protein containing a chimeric CH region as described herein can, in some embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antigen binding protein; (See, for example, US Patent Publication No. 20140243504, the disclosure of which is incorporated herein by reference in its entirety).

Biological characteristics of antigen-binding proteins.

In general, the antigen-binding proteins of the present invention function by binding to the conformational epitope of the HLA-A2-presented E7 peptide of human papillomavirus (HPV) 16 (HPV16E7).

The present invention includes anti-HLA-A2:HPV16E7 antigen binding proteins that bind to the HPV16E7 peptide in the context of HLA-A2 with high specificity. Antigen-binding proteins against HLA-A2:HPV16E7 do not bind to the HPV16E7 peptide in the absence of HLA-A2. In addition, antigen binding proteins against HLA-A2:HPV16E7 do not bind to the non-target peptide in the context of HLA-A2.

The present invention includes anti-HLA-A2:HPV16E7 antigen-binding proteins that bind to the HLA-A2:HPV16E7 monomeric peptide 11-19 with high affinity. For example, the present invention includes antigen binding proteins that bind to monomeric HLA-A2:HPV16E7 11-19 peptide (eg, at 25°C or 37°C) with a KD of less than about 20 nM, as

- 19 044060 measured by surface plasmon resonance, for example, using the analysis format defined in example 4 in the present description. In some embodiments, the antigen binding proteins bind to the HLA-A2:HPV16E7 11-19 monomeric peptide with a K _D of less than about 15 nM, less than about 12 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM , less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.04 nM, as measured by surface plasmon resonance, for example, using the format assay as defined in Example 4 herein, or using a substantially similar assay.

The present invention includes antigen binding proteins that bind to a monomeric HLA-A2:HPV16E7 82-90 peptide (e.g., at 25° C. or 37° C.) with a K _D of less than about 25 nM, as measured by surface plasmon resonance, e.g. using an assay format as defined in Example 4 herein, or using a substantially similar assay. In some embodiments, the antigen binding proteins bind to the HLA-A2:HPV16E7 82-90 monomeric peptide with a K _D of less than about 20 nM, less than about 15 nM, less than about 12 nM, less than about 10 nM, less than about 5 nM , less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.04 nM, as measured by surface plasmon resonance, for example, using assay format as defined in Example 4 herein, or using a substantially similar assay.

The present invention also includes antigen binding proteins that bind to a cell expressing the HLA-A2:HPV16E7 peptide 11-19 with an EC ₅₀ of less than about 6 nM and do not bind to cells expressing predicted non-target peptides as determined by a luminescence assay as determined in Example 6 herein, or using a substantially similar analysis. In some embodiments, the antigen binding proteins bind to a cell expressing the HLA-A2:HPV16E7 11-19 peptide with an EC ₅₀ of less than about less than 6 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM and does not bind to cells expressing predicted non-target peptides, as determined by a luminescence assay as defined in Example 6 herein, or using a substantially similar assay, for example, using the assay format of Example 6 of the present disclosure, or using substantially similar analysis.

The present invention also includes antigen binding proteins that bind to a cell expressing the HLA-A2:HPV16E7 peptide 82-90 with an EC ₅₀ of less than about 1 nM and do not bind to cells expressing predicted non-target peptides as determined by a luminescence assay as determined in Example 6 herein, or using a substantially similar analysis. In some embodiments, the antigen binding proteins bind to a cell expressing the HLA-A2:HPV16E7 peptide 82-90 with an EC ₅₀ of less than about less than 1 nM, less than about 0.5 nM, less than about 0.2 nM, or less than approximately 0.01 nM and does not bind to cells expressing predicted non-target peptides, as determined by a luminescence assay, as determined in Example 6 herein, or using a substantially similar assay, for example, using the assay format of Example 6 herein, or using a substantially similar analysis.

The present invention also includes antigen binding proteins that bind to a cell expressing the HLA-A2:HPV16E7 peptide 11-19 with an EC ₅₀ of less than about 30 nM, as measured by flow cytometry analysis as defined in Example 7 herein, or using essentially similar analysis. In some embodiments, the antigen binding proteins bind to a cell expressing HLA-A2:HPV16E7 peptide 11-19 with an EC ₅₀ of less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by a flow cytometry assay, for example, using the assay format of Example 7 herein, or using a substantially similar assay .

The present invention also includes antigen binding proteins that bind to a cell expressing the HLA-A2:HPV16E7 82-90 peptide with an EC ₅₀ of less than about 75 nM, as measured by flow cytometry analysis as determined in Example 7 herein, or with using essentially similar analysis. In some embodiments, the antigen binding proteins bind to a cell expressing the HLA-A2:HPV16E7 82-90 peptide with an EC ₅₀ of less than about 75 nM, less than about 70 nM, less than about 65 nM, less than about 60 nM, less than less than about 55 nM, less than about 50 nM, less than about 45 nM, less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by flow cytometry analysis, for example, using ana format - 20 044060 lyse of Example 7 of the present description, or using a substantially similar analysis.

In some embodiments, the antigen binding proteins of the present invention are useful for inhibiting tumor growth or delaying the progression of a cancer when administered prophylactically to a patient in need thereof, and may increase patient survival. For example, administration of the antigen binding protein of the present invention may result in shrinkage of the primary tumor and may prevent metastasis or development of secondary tumors. In some embodiments, the antigen-binding proteins of the present invention are useful for inhibiting tumor growth when administered therapeutically to a patient in need thereof and may increase patient survival. For example, administering to a patient a therapeutically effective amount of an antigen binding protein of the invention may result in shrinkage and disappearance of an established tumor in the patient.

In one embodiment, the invention relates to an isolated recombinant antigen binding protein thereof that binds to a conformational epitope of an HLA-A2 presented peptide of HPV16E7, wherein the antigen binding protein exhibits one or more of the following characteristics: (i) contains an HCVR having an amino acid sequence selected from the group , consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 218, 234, 250, 266, 282, 298, 314, 330 , 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506 and 522, or a substantially similar sequence thereto having at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (ii) contains an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514 and 530, or a substantially similar sequence having at least the same 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) contains an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224 , 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512 and 528, or a substantially similar sequence having therewith at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520 and 536, or substantially similar sequences having at least 90% similarity thereto , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (iv) contains an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220 , 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508 and 524, or a substantially similar sequence having thereto at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 228, 244, 260, 276 , 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516 and 532, or substantially similar sequences having at least 90% similarity thereto, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262, 278 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502 518 and 534, or substantially similar sequences having at least 90% similarity thereto at least 95%, with at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (v) binds monomeric HLA-A2:HPV16E7 11-19 with a dissociation equilibrium constant (KD) of less than about 20 nM, as measured in a surface plasmon resonance assay at 25°C; (vi) binds the monomeric HLA-A2:HPV16E7 82-90 peptide with a binding dissociation equilibrium constant ( _KD ) of less than about 25 nM, as measured in a surface plasmon resonance assay at 25°C; (vii) binds to cells expressing the HLA-A2:HPV16E7 peptide 11-19 with an EC ₅₀ of less than about 6 nM and does not bind to cells expressing predicted non-target peptides as determined by luminescence assay; (viii) binds to cells expressing the HLA-A2:HPV16E7 82-90 peptide with an EC ₅₀ of less than 1 nM and substantially does not bind to cells expressing predicted non-target peptides as determined by luminescence assay; (ix) binds to cells expressing the HLA-A2:HPV16E7 11-19 peptide with an _EC50 of less than about 30 nM, as determined by flow cytometry analysis; (x) binds to cells expressing the HLA-A2:HPV16E7 82-90 peptide with an EC ₅₀ of less than about 75 nM, as determined

- 21 044060 flow cytometry analysis; (xi) does not bind to an HLA-A2-presented non-target peptide where the peptide differs by 1, 2, 3, 4, 5 or more amino acids from SEQ ID NO: 538; and (xii) does not contact

An HLA-A2-presented non-target peptide, wherein the peptide differs by 1, 2, 3, 4, 5 or more amino acids from SEQ ID NO: 539.

The antigen binding proteins of the present invention may have one or more of the above characteristics or any combination thereof. Other biological characteristics of the antigen binding proteins of the present invention will be apparent to one skilled in the art from a review of the present disclosure, including the worked examples provided herein.

Epitope mapping and related technologies.

The present invention includes anti-HLA-A2:HPV16E7 antigen-binding proteins that interact with one or more amino acids found in one or more domains of the HLAA2-presented HPV16E7 peptide. An epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within one or both of the above domains of the HPV16E7 molecule (eg, a conformational epitope).

Various methods known to those skilled in the art can be used to determine whether an antigen binding protein interacts with one or more amino acids within a polypeptide or protein. Exemplary methods include, for example, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide digestion assay, crystallographic studies and NMR analysis. In addition, methods such as epitope removal, epitope isolation, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids in a polypeptide with which an antigen binding protein interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves labeling a protein of interest with deuterium, followed by coupling of the antigen-binding protein to the deuterium-labeled protein. The protein/antigen-binding protein complex is then transferred to water, and the exchangeable protons in the amino acids that are protected by the antigen-binding protein complex undergo back-deuterium-hydrogen exchange at a slower rate than the exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antigen-binding protein interface can retain deuterium and therefore exhibit relatively greater mass compared to amino acids not included in the interface. After dissociation of the antigen binding protein, the target protein is subjected to protease digestion and mass spectrometry analysis, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antigen binding protein interacts; see, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term epitope refers to the site on an antigen to which B and/or T cells respond. Cellular epitopes can be formed from both contiguous amino acids and non-contiguous amino acids that become contiguous during tertiary protein folding. Epitopes formed from contiguous amino acids are typically retained when exposed to denaturing solvents, whereas epitopes formed from tertiary folding are typically lost when exposed to denaturing solvents. An epitope typically comprises at least 3, and more often at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method that classifies a large number of monoclonal antigen-binding proteins, such as antibodies (mAbs), directed against the same antigen, according to the similarity of the binding profile of each antibody to chemically or enzymatically modified antigenic surfaces (see US 2004/0101920, incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is clearly different from or overlapping with the epitope represented by another category. This technology allows for rapid filtering of genetically identical antigen-binding proteins so that characterization can be focused on genetically distinct antigen-binding proteins. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce antigen-binding proteins that have desired characteristics. MAP can be used to sort the antigen binding proteins of the invention into groups of antigen binding proteins that bind different epitopes.

The present invention includes antigen binding proteins against HLA-A2:HPV16E7 that bind to the same epitope or portion of an epitope as any of the specific exemplary antigen binding proteins described herein in Table. 1, or an antigen binding protein having the CDR sequences of any of the exemplary antigen binding proteins described in table. 1. Likewise, the present invention also includes antigen binding proteins against

- 22 044060

HLA-A2:HPV16E7 that compete for binding to HLA-A2:HPV16E7 or a fragment thereof with any of the specific exemplary antigen-binding proteins described herein in Table. 1, or an antigen binding protein having the CDR sequences of any of the exemplary antigen binding proteins described in table. 1.

Whether an antigen binding protein binds to the same epitope, or competes for binding with a reference antigen binding protein against HLA-A2:HPV16E7, can be readily determined using routine methods known in the art. For example, to determine whether a test antigen binding protein binds to the same epitope as an anti-HLA-A2:HPV16E7 reference antigen binding protein of the invention, the reference antigen binding protein is allowed to bind to the HLA-A2:HPV16E7 protein or peptide under saturation conditions. The ability of the test antigen binding protein to bind to the HLA-A2:HPV16E7 molecule is then assessed. If the test antigen binding protein is able to bind to HLA-A2:HPV16E7 after saturated binding to the anti-HLA-A2:HPV16E7 reference antigen binding protein, it can be concluded that the test antigen binding protein binds to a different epitope than the anti-HLA-A2:HPV16E7 reference antigen binding protein. On the other hand, if the test antigen binding protein is unable to bind to the HLA-A2:HPV16E7 protein after saturated binding to the reference antigen binding protein against HLA-A2:HPV16E7, then the test antigen binding protein may bind to the same epitope as the epitope associated with the antigen binding protein. protein against HLA-A2:HPV16E7 according to the invention.

To determine whether an antigen binding protein competes for binding to a reference antigen binding protein against HLA-A2:HPV16E7, the binding technique described above is performed in two orientations: in the first orientation, the reference antigen binding protein is allowed to bind to the HLA-A2:HPV16E7 protein under saturation conditions, followed by evaluation binding of the test antigen-binding protein to the HLA-A2:HPV16E7 molecule. In the second orientation, the test antigen binding protein binds to the HLA-A2:HPV16E7 molecule under saturated conditions, followed by assessment of the binding of the reference antigen binding protein to the HLA-A2:HPV16E7 molecule. If in both orientations only the first (saturating) antigen binding protein is able to bind to the HLA-A2:HPV16E7 molecule, then it is concluded that the test antigen binding protein and the reference antigen binding protein are competing for HLA-A2:HPV16E7 binding. As one skilled in the art will appreciate, an antigen binding protein that competes for binding to a reference antigen binding protein may not necessarily bind to an identical epitope as the reference antigen binding protein, but may sterically block binding of the reference antigen binding protein by binding to an overlapping or adjacent epitope.

Two antigen-binding proteins bind to the same or overlapping epitope if each competitively inhibits (blocks) the other from binding to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen binding protein inhibits the binding of another by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competitive binding assay (See, for example, Junghans et al., Cancer Res. 1990, 50: 1495-1502). Alternatively, two antigen binding proteins have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antigen binding protein reduce or eliminate the binding of the other. Two antigen binding proteins have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antigen binding protein reduce or eliminate the binding of the other.

Additional routine experiments (e.g., peptide mutations and binding assays) can then be performed to confirm whether the observed lack of binding of the test antigen-binding protein is due to binding to the same epitope as the reference antigen-binding protein or steric blocking (or other phenomenon) is the reason for the lack of observed binding. Experiments of this nature can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antigen binding protein binding assay available in the art.

Immunoconjugates.

The invention includes antigen-binding proteins against HLA-A2:HPV16E7 conjugated to a therapeutic moiety (immunoconjugate), such as a cytotoxin or a chemotherapeutic agent for the treatment of cancer. As used herein, the term immunoconjugate refers to an antigen-binding protein that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, such as a detection fragment, enzyme, toxin, peptide or protein, or therapeutic agent. An antigen binding protein can be associated with a cytotoxin, radioactive agent, cytokine, interferon, target or reporter fragment, enzyme, toxin, peptide or therapeutic agent anywhere in the molecule as long as it is capable of binding its target. Examples of immunoconjugates include antigen binding protein-drug conjugates and

- 23 044060 antigen-binding protein-toxin fusion proteins. In one embodiment, the agent may be a second different antibody to HPV16E7 or HLA-A2:HPV16E7. In some embodiments, the antigen binding protein may be conjugated to an agent specific for a tumor cell or a cell infected with a virus, i.e. cells infected with HPV. The type of therapeutic moiety that can be conjugated to the anti-HLA-A2:HPV16E7 antigen binding protein will take into account the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for the formation of immunoconjugates are known in the art; see, for example, PCT publication No. WO 05/103081.

Chimeric antigen receptors (CARs).

Chimeric antigen receptors (CARs) redirect T cell specificity to antibody-recognized antigens expressed on the surface of tumor cells, whereas T cell receptors (TCRs) expand the range of targets to include intracellular tumor antigens. CAR-redirected T cells specific for the B-cell differentiation antigen CD19 have shown significant efficacy in the treatment of B-cell malignancies, while TCR-redirected T cells have demonstrated benefits in patients suffering from solid cancer. Stauss et al. describe strategies for modifying therapeutic CARs and TCRs for use in the treatment of malignancy, for example, to enhance antigen-specific effector function and limit the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24: 113-118).

One aspect of the invention includes a chimeric antigen receptor (CAR) that is specific for an HPV16E7 peptide presented on the surface of tumor cells by HLA-A2, such as a peptide containing amino acid residues 11-19 or 82-90 of HPV16E7. In one embodiment of the present invention, a CAR as described herein comprises an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a CD3zeta- or FcR-gamma-derived signaling domain), and/or one or more co-stimulatory signaling domains. domains derived from a costimulatory molecule such as, but not limited to, CD28, CD137, CD134 or CD278. In one embodiment, the CAR includes a hinge or spacer region between the extracellular binding domain and the transmembrane domain, such as the CD8 alpha hinge. In another embodiment of the present invention, a CAR as described herein comprises an extracellular target-specific binding domain and a T cell receptor constant domain (T-body construct).

It should be understood that for use in any of the CARs described herein, the extracellular target-specific binding domain may comprise a Fab, Fab', (Fab') ₂ , Fv or single chain Fv (scFv) antigen binding protein of the invention.

As used herein, the binding domain or extracellular domain of a CAR provides the CAR with the ability to bind to a target antigen of interest. The binding domain can be any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biological molecule (eg, a cell surface receptor or tumor protein or component thereof). A binding domain includes any naturally occurring synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, and as further described herein, the binding domain may be the light chain and heavy chain variable regions of an antibody, or the light and heavy chain variable regions may be linked together into a single chain and in any orientation (e.g., VL-VH or VH-VL). Numerous assays are known for identifying binding domains of the present invention that specifically bind to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (eg, using the BIACORE assay), and are described herein. The target can be any antigen of clinical interest against which it would be desirable to initiate an effector immune response that leads to tumor destruction. In one embodiment, the target antigen of the chimeric antigen receptor binding domain is a conformational epitope of an HLA-A2-presented HPV16E7 peptide on the surface of tumor cells, such as a peptide containing amino acid residues 11-19 or 82-90 of HPV16E7.

Exemplary binding domains include antigen binding proteins, such as antigen binding antibody fragments such as scFv, scTCR, extracellular domains of receptors, ligands for cell surface molecules/receptors or receptor binding domains thereof, and tumor binding proteins. In some embodiments, the antigen binding domains included in the CAR of the invention may be a variable region (Fv), CDR, Fab, scFv, VH, VL, variant antibody domain (dAb), camel antibody (VHH), variant fibronectin domain 3, an ankyrin repeat variant and a different antigen-specific binding domain derived from other protein scaffolds.

In one embodiment, the CAR binding domain is a single chain antibody against HLA-A2:HPV16E7 (scFv) and may be murine, human, or human

- 24 044060 manized scFv. Single chain antibodies can be cloned from hybridoma V region genes specific for the desired target. A method that can be used for cloning a heavy chain variable region (VH) and a light chain variable region (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. Thus, in some embodiments, the binding domain comprises a binding domain derived from an antibody, but may be a binding domain not derived from an antibody. An antibody-derived binding domain may be an antibody fragment or a genetically engineered product of one or more antibody fragments, the fragment being involved in binding to an antigen.

In some embodiments, the CARs of the present invention may contain a linker between different domains added to suit the spacing and conformation of the molecule. For example, in one embodiment, a linker may exist between the VH or VL binding domain, which may be 1-10 amino acids in length. In other embodiments, the linker between any of the domains of the chimeric antigen receptor may be from 1 to 20 or 20 amino acids in length. Accordingly, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In other embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges including numbers described herein are also included herein, for example, a linker of 10-30 amino acids in length.

In some embodiments, linkers suitable for use in the CAR described herein are flexible linkers. Suitable linkers can be easily selected and can be of any suitable varying length, such as 1 amino acid (eg Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids , 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Typical flexible linkers include glycine polymers (G)n, glycine-serine polymers, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as a neutral tether between the domains of fusion proteins such as the CARs described herein. Glycine gains access to significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of ordinary skill in the art will appreciate that the CAR construct may include linkers that are fully or partially flexible, such that the linker may include a flexible linker as well as one or more portions that impart a less flexible structure to provide the desired CAR structure .

The CAR binding domain may be followed by a spacer or hinge, which refers to the region that pushes the antigen binding domain away from the surface of effector cells to allow proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6:412 -419). The hinge region in CAR is typically located between the transmembrane (TM) and binding domains. In some embodiments, the hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8alpha, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or can be changed. In one embodiment, the hinge region comprises a CD8 alpha hinge.

The transmembrane region or domain is the part of the CAR that anchors the extracellular binding site to the plasma membrane of the immune effector cell and facilitates the binding of the binding domain to the target antigen. The transmembrane domain may be a CD3zeta transmembrane domain, however other transmembrane domains that may be used include those derived from CD8alpha, Cd4, CD28, CD45, CD9, Cd16, CD22, CD33, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain is the CD137 transmembrane domain. In some embodiments, the transmembrane domain is synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine.

The intracellular signaling domain refers to the portion of the mercury antigen receptor protein that is involved in relaying the message of effective binding of a CAR to a target antigen to the interior of an immune effector cell to induce cell effector function, such as cytokine activation, production, proliferation, and cytotoxic activity, including release of cytotoxic factors into the CAR-bound target cell, or other cellular responses caused by antigen binding to the extracellular CAR domain. The term effector function refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or assistance or activity including secretion

- 25 044060 cytokine. Thus, the term intracellular signaling domain refers to the part of the protein that transduces the effector function signal and that directs the cell to perform a specialized function. Although it is generally possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the entire domain as long as it signals the effector function. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit a signal to effector function. The intracellular signaling domain is also known as the signal transduction domain and is typically derived from parts of the human CD3 or FcRy chains.

It is known that signals generated through the T cell receptor alone are not sufficient to fully activate T cells and that a secondary or co-stimulatory signal is also required. Thus, it can be said that T cell activation is mediated by two different classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation through the T cell receptor (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner. providing a secondary or costimulatory signal (secondary cytoplasmic signal sequences). Primary cytoplasmic signal sequences regulate the primary activation of the T cell receptor complex by either inhibition or inhibition. Primary cytoplasmic signal sequences that act in a costimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motif or ITAM.

Examples of ITAMs containing primary cytoplasmic signal sequences that are specifically used in the invention include sequences derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b and CD66d. In some specific embodiments, the intracellular signaling domain of the anti-HLA-A2:HPV16E7 CARs described herein is of CD3 zeta or FcR gamma origin.

As used herein, the term costimulatory signaling domain or costimulatory domain refers to the portion of the CAR containing the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for efficient T cell activation and function upon binding to antigen. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds to CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD3-zeta and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. Inclusion of one or more co-stimulatory signaling domains can enhance the efficacy and expansion of T cells expressing CAR receptors. Intracellular signaling and co-stimulatory signaling domains can be linked in any order in tandem with the carboxyl terminus of the transmembrane domain.

Although scFv-based CARs engineered to contain a signaling domain from CD3 or FcR-gamma have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to induce signals that promote survival and expansion T cells in the absence of a concomitant co-stimulatory signal. Other CARs containing a binding, hinge, transmembrane, and signaling domain derived from CD3 zeta or FcR gamma together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278) may more effectively target antitumor activity, as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106: 3360-3365).

In one embodiment, the HLA-A2:HPV16E7 CARs of the invention comprise (a) an anti-HLA-A2:HPV16E7 scFv as a binding domain (e.g., a scFv having binding regions (e.g., CDRs or variable domains) from any one or more HLA antibodies -A2:HPV16E7 described in Table 1) (b) human CD8 alpha-derived hinge region, (c) human CD8 alpha transmembrane domain, and (d) human T cell receptor CD3 zeta chain intracellular signaling domain (CD3) and optionally one or more co-stimulatory signaling domains derived from CD28, CD137, CD134 and CD278. In one embodiment, the different protein domains are arranged from the amino to the carboxyl terminus in the following order: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and optional co-stimulatory signaling domains are linked to the transmembrane carboxy terminus in any order in tandem to form a single-chain chimeric polypeptide. In one embodiment, it is implemented

- 26 044060 The design of the nucleic acid encoding HLA-A2:HPV16E7 CAR is a chimeric nucleic acid molecule containing a nucleic acid molecule containing different coding sequences, for example (5' to 3') coding sequences of the human scFv against HLA-A2 :HPV16E7, human CD8alpha hinge, human CD8alpha transmembrane domain, and CD3-zeta intracellular signaling domain. In another embodiment, the nucleic acid construct encoding the HLA-A2:HPV16E7 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule containing different coding sequences, for example (5' to 3') coding sequences of a human scFv against HLA- A2:HPV16E7, human CD8-alpha hinge, human CD8-alpha transmembrane domain, CD137 costimulatory domain, and CD3-zeta costimulatory domain. In some embodiments, the nucleic acid construct encoding the HLA-A2:HPV16E7 CAR is a chimeric nucleic acid molecule comprising a nucleic acid molecule comprising different coding sequences, for example (5' to 3') coding sequences of a human scFv anti-HLA- A2:HPV16E7, human CD8-alpha hinge, human CD8-alpha transmembrane domain, CD137 costimulatory domain, and CD3-zeta costimulatory domain, wherein the anti-HLA-A2:HPV16E7 scFv contains a VH selected from the group consisting of SEQ ID NO: 2. 34, 82, 194, 282 and 506, and VL selected from the group consisting of SeQ ID NOs: 10, 42, 90, 202, 290 and 514. In some embodiments, the present invention includes a nucleic acid molecule that encodes HLA- A2:HPV16E7 CAR selected from the group consisting of SEQ ID NOs: 540, 541, 542, 543, 544 and 545.

In some embodiments, a polynucleotide encoding a CAR described herein is inserted into a vector. As used herein, the term vector refers to a carrier into which a polynucleotide encoding a protein can be covalently inserted for expression of the protein and/or cloning of the polynucleotide. Such vectors may also be referred to as expression vectors. The isolated polynucleotide can be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector can be digested using appropriate restriction enzymes and then ligated to the isolated polynucleotide having matching restriction ends. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences encoding at least a portion of a gene product capable of being transcribed in a cell. In most cases, the RNA molecules are then translated into protein. Expression vectors may contain a variety of control sequences, which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that direct transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also perform other functions and are discussed below. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, allowing it to be maintained in two organisms, for example for expression in human cells and for cloning and amplification in a prokaryotic host.

The expression vector may have necessary regulatory elements located in the 5' region and in the 3' region, such as promoter sequences such as CMV, PGK and EF1 alpha, promoters, TATA box, ribosome recognition and binding block, and termination sequence transcription of the 3'-UTR of AAUAAA for efficient transcription and translation of the gene in the appropriate host cell. Other suitable promoters include the constitutive early promoter of simian virus 40 (SV40), mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters may also be used, including, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In some embodiments, inducible promoters are also contemplated as part of the chimeric antigen receptor expression vectors. This provides a molecular switch capable of turning on or off the expression of a polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, the metallothioneine promoter, the glucocorticoid promoter, the progesterone promoter, or the tetracycline promoter.

The expression vector may have additional sequence, such as a 6xhistidine tag, c-Myc and FLAG, which are included in the expressed CARs. Thus, the expression vector can be designed to contain 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions and/or terminator sequences that can facilitate or enhance efficient transcription of the target of interest. nucleic acid(s) that is inserted into the expression vector. An expression vector can also be designed to

- 27 044060 replication and/or expression functionality (eg, transcription and translation) in a particular cell type, cell location or tissue type. Expression vectors may include a selection marker to maintain the vector in a host or recipient cell.

Examples of vectors are a plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as phage lambda or phage M13 and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40). Examples of expression vectors are Lenti-XTM bicistronic expression system (Neo) vectors (Clontrch), pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST.TM., pLenti6/V5DEST.TM. and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The CAR coding sequences disclosed herein can be ligated into such expression vectors to express the chimeric protein in mammalian cells.

In some embodiments, the nucleic acids encoding the CARs of the present invention are presented in viral vectors. The viral vector may be a vector derived from a retrovirus, lentivirus, or foam virus. As used herein, the term viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector may contain the coding sequence for the various chimeric proteins described herein in place of nonessential viral genes. The vector and/or particle can be used to transfer DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

In some embodiments, the viral vector containing the coding sequence for a CAR described herein is a retroviral vector or a lentiviral vector. The term retroviral vector refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus. The term lentiviral vector refers to a vector containing structural and functional genetic elements outside the LTR, which are primarily derived from a lentivirus.

Retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), leukemia virus gibbon virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV)). The retroviruses of the invention also include the human T-cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as human immunodeficiency virus, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus ( FIV), equine immunodeficiency virus (EIV) and other classes of retroviruses.

A lentiviral vector as used herein refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that cause a slowly developing disease. Viruses included in this group include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi; goat arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). Preparation of recombinant lentivirus can be achieved using methods according to Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72: 8463-8471 and Zufferey et al., J. Virol. 1998; 72: 9873-9880).

Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be prepared using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel , FM et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85:6460-6464 Wilson et al (1988) Proc Natl Acad Sci USA 85:3014-3018 Armentano et al (1990) Proc Natl Acad Sci USA USA 87:6141-6145 Huber et al (1991) Proc Natl Acad Sci USA 88:8039-8043 Ferry et al (1991) Proc Natl Acad Sci USA 88:8377 8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3 :641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J Immunol 150:4104–4115; US Patent No. 4868116; US Patent No. 4980286; PCT application WO 89/07136; PCT application WO 89/02468; PCT application WO 89/05345 and PCT application WO

- 28 044060

92/07573).

Suitable sources for obtaining retroviral (ie, both lentiviral and nonlentiviral) sequences for use in vector formation include, for example, genomic RNAs and cDNAs available from commercial sources, including Type Culture Collection (ATCC), Rockville, Md. The sequences can also be synthesized chemically.

For expression of HLA-A2:HPV16E7 CAR, the vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. Expression vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be selected accordingly by one skilled in the art, as described above. For example, promoter sequences may be selected to promote transcription of a polynucleotide in a vector. Suitable promoter sequences include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, beta actin promoter, EF1a promoter, CMV promoter and SV40 promoter. Enhancer sequences can be selected to enhance transcription of a polynucleotide. Selectable markers may be selected to allow selection of host cells into which the vector is inserted from those into which the vector is not inserted, for example, selectable markers may be genes that confer resistance to an antibiotic. Signal sequences can be selected so that the expressed polypeptide can be transported outside the host cell.

To clone a polynucleotide, a vector can be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify copies of the polynucleotide it contains. Cloning vectors may contain sequence components, generally without limitation, including origin of replication sequences, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be selected accordingly by one skilled in the art. For example, the origin of replication sequence may be selected to promote autonomous replication of the vector in the host cell.

In some embodiments, the present disclosure relates to isolated host cells containing the vectors provided herein. Host cells containing the vector may be useful for expressing or cloning the polynucleotide contained in the vector. Suitable host cells may include, without limitation, prokaryotic cells, fungal cells, yeast cells or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia , for example Serratia marcescans and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

The CARs of the present invention are introduced into a host cell using transfection and/or transduction techniques known in the art. As used herein, the terms transfection and transduction refer to the processes by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the DNA of the host cell or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be administered stably. Transfection can be accomplished by a variety of methods known in the art, including, but not limited to, calcium phosphate co-precipitation with DNA, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of gene(s) using a viral or retroviral vector through viral infection rather than transfection. In some embodiments, retroviral vectors are transduced by packaging the vectors into virions prior to contact with the cell. For example, a nucleic acid encoding a CAR. against HLA-A2:HPV16E7 carried by a retroviral vector can be transduced into the cell through infection and viral integration.

As used herein, the term genetically engineered or genetically modified refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell.

The terms genetically modified cells, engineered cells, and redirected cells are used interchangeably.

In particular, the CARs of the present invention are introduced and expressed in immune effector cells to redirect their specificity to a target antigen of interest, for example a conformational epitope of the HLA-A2 presented HPV16E7 peptide, for example amino acid residues 11-19 or 82-90.

The present invention relates to methods for producing immune effector cells that express CAR, as described herein. In one embodiment, the method

- 29044060 involves transfecting or transducing immune effector cells isolated from a patient, such as a patient having a disease or disorder associated with HPV16E7, such that the immune effector cells express one or more CARs, as described herein. In some embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells can then be reintroduced directly to the individual. In other embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before they are genetically modified to express CARs. In this regard, immune effector cells can be cultured before or after genetic modification (ie, transduced or transfected to express CARs, as described herein).

Prior to in vitro manipulation or genetic modification of immune effector cells described herein, the cell source may be obtained from a patient. In particular, immune effector cells for use with CARs, as described herein, contain T cells. Such recombinant T cells are referred to herein as T bodies.

In one embodiment of the present invention, the T body includes a CAR of the invention comprising an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a CD3-zeta or FcRgamma-derived signaling domain), and/or one or more co-stimulatory domains. signaling domains derived from a co-stimulatory molecule such as, but not limited to, CD28, CD137, CD134 or CD278. In another embodiment of the present invention, the T body includes a CAR of the invention including an extracellular target-specific binding domain, a transmembrane domain, a hinge or spacer region between the extracellular binding domain and the transmembrane domain, an intracellular signaling domain (such as a CD3-derived signaling domain, zeta or FcR-gamma), and/or one or more costimulatory signaling domains derived from a costimulatory molecule. In yet another embodiment of the present invention, the T body includes a Tbody CAR construct comprising an extracellular target-specific binding domain and a T cell receptor constant domain. An extracellular target-specific binding domain suitable for use in a T body containing any of the CARs described herein may include a Fab, Fab', (Fab') ₂ , Fv or single chain Fv (scFv) of the antigen binding protein of the invention.

T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, splenic tissue, and tumors. In some embodiments, T cells can be obtained from a blood sample collected from a patient using any number of techniques known to one skilled in the art, such as FICOLL separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. Alternatively, the wash solution does not contain calcium and may not contain magnesium or may not contain many, if not all, divalent cations. As will be appreciated by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as the use of a semi-automated flow centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solution with or without buffer. In some embodiments, unwanted components of the apheresis sample can be removed directly in the resuspended culture medium.

In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysis of red blood cells and depletion of monocytes, for example, by PERCOLL gradient centrifugation. A specific subset of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be additionally isolated by positive or negative selection methods. For example, enrichment of the T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which uses a panel of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

PBMCs can be used directly for genetic modification with CARs using methods as described herein. In some embodiments, after

- 30 044060 isolation PBMCs further release T lymphocytes, and in some embodiments, cytotoxic and helper T lymphocytes can be sorted into subpopulations of naïve T cells, memory T cells, and effector T cells, either before or after genetic modification and/ or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory cells, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+ CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of SCI central memory phenotypic markers includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127 and positive for granzyme B and perforin. In some embodiments, naïve CD8+ T lymphocytes are characterized by the expression of naïve T cell phenotypic markers, including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

In some embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained using standard methods. In some embodiments, the naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, the central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, the effector CD4+ cells are negative for CD62L and CD45RO.

Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro before being genetically modified. In another embodiment, immune effector cells, such as T cells, are genetically modified using chimeric antigen receptors described herein (eg, transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and propagated in vitro. Methods for activating and propagating T cells are known in the art and are described, for example, in US Pat. No. 6,905,874; US Patent No. 6867041; US patent 6797514; WO2012079000, US 2016/0175358. Typically, such methods involve contacting PBMCs or isolated T cells with a stimulatory agent and a co-stimulatory agent, such as anti-CD3 and anti-CD28 antibodies, typically attached to a microsphere or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same microsphere serve as a surrogate antigen presenting cell (APC). In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US Pat. No. 6,040,177; US Patent No. 5827642; and WO2012129514.

The invention relates to a population of modified immune effector cells for the treatment of an HPV-associated disease or disorder, such as cancer, wherein the modified immune effector cells include HLA-A2:HPV16E7 CAR, as described herein.

CAR-expressing immune effector cells obtained as described herein can be used in methods and compositions for adoptive immunotherapy in accordance with known techniques or variations thereof that will be apparent to those skilled in the art based on the present disclosure; see, for example, US Patent Application Publication No. 2003/0170238 issued to Gruenberg et al; see also US Patent No. 4690915 Rosenberg.

In some embodiments, the cells are prepared by first collecting them from their culture medium and then washing and concentrating the cells in a medium and container system suitable for administration (a pharmaceutically acceptable carrier) in a treatment-effective amount. Suitable infusion media can be isotonic media of any composition, usually normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's can also be used. The infusion medium may be supplemented with human serum albumin.

The treatment-effective number of cells in the composition is at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell) or more often greater than 10 ² cells and up to 10 ⁶ , up to and including 108 or 109 cells and may be more than 10 ¹⁰ cells. The number of cells will depend on the end use for which the composition is intended, as well as the type of cells included therein.

- 31 044060

The cells may be autologous or heterologous to the patient undergoing therapy.

If desired, treatment may also include administration of mitogens (eg, PHA) or lymphokines, cytokines and/or chemokines (eg, IFN-γ, IL-2, IL-12, TNF-α, IL-18 and TNF-β, GM- CSF, IL-4, IL-13,

Flt3-L, RANTES, MIP1a, etc.), as described herein, to enhance the induction of an immune response.

The CAR-expressing immune effector cell populations of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components, such as IL-2, or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may contain a CAR-expressing population of immune effector cells, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

The antitumor immune response induced in a patient by administration of T cells expressing the CARs described herein, using the methods described herein or other methods known in the art, may include a cellular immune response mediated by cytotoxic T cells , capable of killing infected cells, and the response of regulatory T cells and helper T cells. Humoral immune responses, mediated primarily by helper T cells, can also be induced, capable of activating B cells, resulting in the production of antibodies. To analyze the type of immune responses induced by the compositions of the present invention, various techniques can be used that are well described in the art; for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

Thus, the present invention provides methods for treating an individual who is diagnosed or suspected of having, or is at risk of developing, an HPV-associated disease or disorder, such as an HPV16E7-positive malignancy, the methods comprising administering to the individual a therapeutically effective amount of CAR-expressing immune effector cells , as described in this document.

In one embodiment, the invention relates to a method of treating a patient diagnosed with an HPV16E7-positive cancer, comprising removing immune effector cells from a patient diagnosed with an HPV16E7-positive cancer, genetically modifying said immune effector cells with a vector containing a nucleic acid encoding a chimeric antigen receptor of the present invention, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same patient. In one embodiment, the immune effector cells comprise T cells.

Methods of administering the cellular compositions described herein include any method that is effective to result in the ex vivo reintroduction of genetically modified immune effector cells that either directly express the CAR of the invention into a patient or the reintroduction of genetically modified immune effector cell precursors , which, when administered to a patient, differentiate into mature immune effector cells that express CAR. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct in accordance with the invention and returning the transduced cells to the patient.

Therapeutic administration and formulations.

The invention relates to therapeutic compositions containing antigen binding proteins against HLA-A2:HPV16E7, for example antibodies or antigen binding fragments or CARs of the present invention. Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients and other agents that are included in the formulations to provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in textbooks familiar to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions and water-in-water emulsions. in-oil, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax; see also Powell et al. Compendium of excipients for

- 32 044060 parenteral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

The dosage of an antigen-binding protein, such as an antibody or antigen-binding fragments thereof, may vary depending on the age and size of the patient to be administered, the target disease, conditions, route of administration, and the like. When an antigen binding protein of the present invention is used to treat or prevent a disease or disorder in an adult patient, it is advantageous to administer the antigen binding protein, such as an antibody or antigen binding fragments thereof, of the present invention, typically in a single dose of about 0.1 to about 60 mg/day. kg body weight, more preferably from about 5 to about 60, from about 20 to about 50, from about 10 to about 50, from about 1 to about 10, or from about 0.8 to about 11 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. In some embodiments, an antigen binding protein, such as an antibody or antigen binding fragments thereof, of the invention may be administered in an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, up to about 100 mg, or up to about 50 mg. In some embodiments, the initial dose may be followed by the administration of a second or multiple subsequent doses of an antigen-binding protein, such as an antibody or antigen-binding fragments thereof, in an amount that may be approximately the same as or less than the initial dose, with subsequent doses spaced apart by at least , from 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, for encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) ) J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelial or mucous membrane (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration may be systemic or local. The pharmaceutical composition can also be delivered in a vesicle, in particular in a liposome (see, for example, Langer (1990) Science 249: 1527-1533).

The use of nanoparticles for the delivery of antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, of the present invention is also discussed herein. Antigen-binding protein conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antigen-binding protein conjugated nanoparticles and methods for their preparation and use are described in detail by Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), which is incorporated herein by reference. Nanoparticles can be formulated and conjugated to antigen-binding proteins contained in pharmaceutical compositions to target tumor cells or autoimmune tissue cells or virus-infected cells. Nanoparticles for drug delivery have also been described, for example, in US Pat. No. 8,257,740 or US Pat. No. 8,246,995, each of which is incorporated herein by reference in its entirety.

In some situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. Alternatively, polymeric materials may be used. In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition, resulting in only a fraction of the systemic dose being required.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antigen binding protein or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyalcohol ( e.g. propylene glycol, polyethylene glycol), nonionic surfactant [e.g. polysorbate 80, HCO-50 (poly- 33 044060 oxyethylene (50 mol) hydrogenated castor oil adduct)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, with regard to subcutaneous delivery, a pen-type delivery device can easily be used in delivering the pharmaceutical composition of the present invention. A delivery device such as a pen syringe can be reusable or disposable. A reusable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. Rather, the disposable pen delivery device comes pre-filled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and auto-injector delivery devices are used in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, pen HUMALOG™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark) , BD™ pen (Becton Dickinson, Franklin Lake, New Jersey, OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany) are just a few. Examples of disposable devices for pen delivery devices having use in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR™ pen (SanofiAventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK auto-injector ™ (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dose dosage forms suitable for the dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antigen binding protein contained is typically from about 5 to about 500 mg per dosage form per dosage unit; particularly in injection form, it is preferred that the antigen binding protein be contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic applications of antigen-binding proteins.

The antibodies of the invention are useful, among other things, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by HPVI6. For example, the present invention relates to methods of treating an HPV-associated disease or disorder, such as an HPV-associated malignancy (eg, HPV16E7-positive malignancy) (tumor growth inhibition) and/or HPV infections, by administering an anti-HLA-A2 antigen binding protein: HPV16E7 (or a pharmaceutical composition containing anti-HLA-A2:HPV16E7 antigen-binding protein) as described herein, for a patient in need of such treatment, and anti-HLA-A2:HPV16E7 antigen-binding proteins (or pharmaceutical compositions containing anti-HPV16E7 antigen-binding protein) HLA-A2:HPV16E7) for use in the treatment of HPV-associated malignancy (tumor growth inhibition) and/or HPV infections. The antigen-binding proteins of the present invention are useful for treating, preventing and/or neutralizing a disease or disorder or condition, such as HPV-associated cancer or HPV infection, and/or for neutralizing at least one symptom associated with such disease, disorder or condition. In the context of the treatment methods described herein, anti-HLA-A2:HPV16E7 antigen binding protein can be administered as monotherapy (ie, as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere place of this description).

In some embodiments, the antibodies described herein are useful for treating patients having a primary or recurrent cancer, including, but not limited to, HPV-associated cancer, such as squamous cell carcinoma, such as head and neck squamous cell carcinoma, cervical cancer, cancer

- 34 044060 anogenital region, oropharyngeal cancer.

Antigen-binding proteins can be used to treat early or late-stage symptoms of HPV-associated cancer. In one embodiment, an antibody or fragment thereof of the invention may be used to treat advanced or metastatic cancer. Antigen binding proteins are useful for reducing or inhibiting or shortening tumor growth. In some embodiments, treatment using an antigen binding protein of the invention results in greater than 40% regression, greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of the tumor in patient. In some embodiments, antigen binding proteins can be used to prevent tumor recurrence. In some embodiments, antigen binding proteins are useful for increasing progression-free survival or overall survival in a patient with HPV-associated cancer. In some embodiments, antibodies are useful for reducing toxicity due to chemotherapy or radiation therapy while maintaining long-term survival in a patient having HPV-associated cancer.

In some embodiments, the antigen binding proteins of the invention are useful for treating patients suffering from chronic HPV infection. In some embodiments, the antigen binding proteins of the invention are useful for reducing viral titers in a host.

One or more antibodies of the present invention can be administered to alleviate or prevent or reduce the severity of one or more symptoms or conditions of a disease or disorder.

Also contemplated herein is the prophylactic use of one or more antibodies of the present invention for patients at risk of developing a disease or disorder, such as an HPV-associated disease or disorder, such as HPV-associated malignancy and HPV infection.

In a further embodiment of the invention, the present antibodies are used to prepare a pharmaceutical composition for the treatment of patients suffering from an HPV-associated disease or disorder, such as an HPV-associated malignancy or HPV infection. In another embodiment, the antibodies of the present invention are used as adjunctive therapy to any other agent or any other therapy known to those skilled in the art useful for treating HPV-associated cancer or HPV infection.

Combination therapy and formulations.

The combination therapy may include an anti-HLA-A2:HPV16E7 antigen binding protein of the invention, such as a CAR of the invention (e.g., an immune effector cell containing a CAR of the invention) or a pharmaceutical composition of the invention, and any additional therapeutic agent that can be advantageously combined with antigen binding protein according to the invention. The antigen-binding proteins of the present invention can be synergistically combined with one or more anticancer drugs or therapies used to treat or inhibit an HPV16E7-associated disease or disorder, such as an HPV-positive malignancy, e.g., squamous cell carcinoma, cervical cancer, anogenital cancer, head and neck cancer or oropharyngeal cancer.

The present disclosure contemplates the use of anti-HLAA2:HPV16E7 antigen-binding proteins of the invention in combination with immunostimulatory and/or immunosuppressive therapy to inhibit tumor growth and/or improve survival of cancer patients. Immune-stimulating therapy involves direct immunostimulating therapy to increase immune cell activity by releasing the brake on suppressed immune cells or stepping on the gas to activate an immune response. Examples include targeting other key receptors, vaccination, and adjuvants. Immune-sustaining conditions may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that stimulate an antitumor immune response. Examples include radiation, chemotherapy, antiangiogenic agents, and surgery.

In various embodiments, one or more antigen binding proteins of the present invention can be used in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), a PD-L1 inhibitor ( e.g., anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105 or REGN3504), CTLA-4 inhibitor (eg, ipilimumab), TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, GITR inhibitor, other antagonist T cell co-inhibitor or ligand (eg, anti-CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), indoleamine 2,3-dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist [eg , a VEGF-Trap such as aflibercept or another VEGF inhibitory fusion protein as described in US 7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (at

- 35 044060 example, bevacizumab or ranibizumab) or small molecule VEGF receptor kinase inhibitor (eg sunitinib, sorafenib or pazopanib)], Ang2 inhibitor (eg nesvacumab), transforming growth factor beta (TGFP) inhibitor, epidermal growth factor receptor (EGFR) inhibitor ) (eg, erlotinib, cetuximab), CD20 inhibitor (eg, anti-CD20 antibody such as rituximab), tumor-specific antigen antibody [eg, CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin , tumorM2-PK, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9], vaccine (eg, Bacillus Calmette-Guerin tumor vaccine), adjuvant to enhance antigen presentation (eg, granulocyte-macrophage colony-stimulating Factor), bispeicenic antibodies (for example, CD3XCD20 bispeicentic antibodies or PSMaxcd3 bispecycytic antibody), cytotoxin, chemotherapeutic agent (for example, Dakarbazin, Temozolomide, Cyclophosfamide, Docesel, Doxorubicin, Daunorubicin, Cisplatin, Carboplatin, Hem, hem quotabin, methotrexate, mitoxantron, oxaliflatin, paclitaksel and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitor (eg, sarilumab), IL-4R inhibitor (eg, dupilumab), IL-10 inhibitor, cytokine such as IL-2, IL-7, IL-21 and IL -15, an antibody-drug conjugate (ADC) (eg, anti-CD19-DM4 ADC and anti-DS6-DM4 ADC), an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs), a dietary supplement such as antioxidants, or with any other therapy for the treatment of malignant neoplasm. In some embodiments, the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention can be used in combination with an HPV vaccine. Representative HPV vaccines include Gardasil, Gardasil 9 and Cervarix, Lm-LLo-E7 (ADXS11-001; ADXS-HPV; Advaxis, Inc.); GLBLlOlc (GENOLAC BL Corp.); TA-HPV (European Organization for Research and Treatment of Cancer (EORTC)); TG4001 (Transgene/Roche); MVA E2 (Institute Mexicano del Seguro Social); HPV16-SLP (ISA Pharmaceuticals); GL-0810 (Gliknik Inc.); Pepcan+Candin (University of Arkansas); GTL001 (ProCervix; Genticel); TA-CIN (Xenova Research Limited); TA-CIN+TA-HPV (Celtic Pharma); pNGVL4a-sig/E7 (detox)/H8P70+TA-NRU (Sidney Kimmel Comprehensive Cancer Center); pNGVL4a-CRT/E7 (detox) (Sidney Kimmel Comprehensive Cancer Center); GX-188E (Genexine, Inc.); VGX-3100 (Inovio Pharmaceuticals); Dendritic cells stimulated with HPV-16 and HPV-18 E7 and keyhole limpet hemocyanin (National Institutes of Health); DC stimulated with HPV + tumor lysate (Department of Biotechnology (DBT, Govt. India)); PDS0101 (PDS Biotechnology Corp.); ProCervix (Genticel); GX-188E (Genexine, Inc.); pNGVL4a-CRT/E7 (detox) (Sidney Kimmel Comprehensive Cancer Center); pNGVL4a-sig/E7 (detox)/HSP70+TA-HPV (Sidney Kimmel Comprehensive Cancer Center); TVGV-1+GPI-0100 (THEVAX Genetics Vaccine Co.); Pepcan+Candin (University of Arkansas); ISA101 (SLP-HPV-01; HPV16-SLP; ISA Pharmaceuticals); ADXS 11-001 (Lm-LLo-E7; Advaxis, Inc.); ISA101 (SLPHPV-01; HPV16-SLP; ISA Pharmaceuticals); DPX-E7 (Dana-Farber Cancer Institute); ADXS11-001 (LmLLo-E7; Advaxis, Inc.); INO-3112 (VGX-3100+INO-9012; Inovio Pharmaceuticals); ADXS 11-001 (Lm-LLoE7; Advaxis, Inc.); INO-3112 (VGX-3100+INO-9012; Inovio Pharmaceuticals); ISA101 (SLP-HPV-01; HPV16-SLP; ISA Pharmaceuticals); and TA-CIN+GPI-0100 (Sidney Kimmel Comprehensive Cancer Center). In some embodiments, the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention can be used in combination with anti-tumor vaccines, including dendritic cell vaccines, oncolytic viruses, anti-tumor vaccines, etc., to enhance the anti-tumor response. Examples of tumor vaccines that can be used in combination with the HLA-A2:HPV16E7 antigen binding proteins of the present invention include MAGE3 vaccine for melanoma and bladder cancer, MUC1 vaccine for breast cancer, EGFRv3 (eg, Rindopepimut) for brain cancer (including glioblastoma multiforme) or ALVAC-CEA (for CEA+ malignancies).

In some embodiments, anti-HLA-A2:HPV16E7 antigen-binding proteins of the invention can be administered in combination with radiation therapy in ways to generate long-lasting antitumor responses and/or improve survival of cancer patients. In some embodiments, anti-HLA-A2:HPV16E7 antigen-binding proteins of the invention may be administered before, simultaneously with, or after the administration of radiation therapy to a cancer patient. For example, radiation therapy can be administered in one or more doses to tumor sites followed by one or more doses of anti-HLA-A2:HPV16E7 antigen binding proteins of the invention. In some embodiments, radiation therapy may be administered locally to tumor lesions to enhance the local immunogenicity of the patient's tumor (adjuvant radiation therapy) and/or to kill tumor cells (ablative radiation), followed by systemic administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the invention. For example, intracranial irradiation can be administered to a patient with brain cancer (eg, glioblastoma multiforme) in combination with systemic administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the invention. In some embodiments, the anti-HLAA2:HPV16E7 antigen-binding proteins of the invention may be administered in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or a VEGF antagonist (eg, aflibercept).

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In some embodiments, the anti-HLA-A2:HPV16E7 antigen-binding proteins of the invention can be administered in combination with one or more antiviral drugs to treat chronic HPV infection. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine and corticosteroids.

Additional therapeutically active agent(s)/component(s) may be administered before, simultaneously with, or after administration of the anti-HLA-A2:HPV16E7 antigen binding proteins of the present invention. For purposes of the present invention, such administration regimens are considered to be administration of an anti-HLA-A2:HPV16E7 antigen binding protein in combination with a second therapeutically active component.

Additional therapeutically active component(s) may be administered to the patient prior to administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention. For example, the first component may be considered administered before the second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before , 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before the introduction of the second component. In other embodiments, additional therapeutically active component(s) may be administered to the patient following administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention. For example, the first component may be considered administered after the second component if the first component is administered after 1 minute, after 5 minutes, after 10 minutes, after 15 minutes, after 30 minutes, after 1 hour, after 2 hours, after 3 hours, after 4 hours, after 5 hours, after 6 hours, after 12 hours, after 24 hours, after 36 hours, after 48 hours, after 60 hours, after 72 hours after administration of the second component. In yet other embodiments, additional therapeutically active component(s) may be administered to a patient concurrently with administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention. Co-administration for purposes of the present invention includes, for example, administering anti-HLA-A2:HPV16E7 antigen binding protein and an additional therapeutically active component to a patient in a single dosage form (e.g., a co-formulated form) or in separate dosage forms administered to the patient over a period of about 30 minutes or less relative to each other. When administered in separate dosage forms, each dosage form can be administered by the same route (eg, the antigen binding protein against HLAA2:HPV16E7 and the additional therapeutically active component can be administered intravenously, subcutaneously, etc.); alternatively, each dosage form can be administered by a different route (eg, the antigen binding protein against HLA-A2:HPV16E7 can be administered intravenously, and the additional therapeutically active component can be administered subcutaneously). In either case, administration of the components in the same dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered simultaneous administration for purposes of this disclosure. For purposes of this disclosure, administration of an anti-HLA-A2:HPV16E7 antigen-binding protein before, simultaneously with, or after (as those terms are defined above) administration of an additional therapeutically active component is considered to be administration of an anti-HLA-A2:HPV16E7 antigen-binding protein in combination with an additional therapeutically active component).

The present invention includes pharmaceutical compositions in which the anti-HLA-A2:HPV16E7 antigen binding protein of the present invention is formulated in conjunction with one or more additional therapeutic component(s) as described elsewhere herein, using various dosage combinations.

Administration schemes.

In accordance with some embodiments of the present invention, multiple doses of anti-HLA-A2:HPV16E7 antigen-binding protein (or a pharmaceutical composition comprising a combination of anti-HLA-A2:HPV16E7 antigen-binding protein and any of the additional therapeutically active agents mentioned herein) can be administered to a patient over a certain period of time. Methods according to this aspect of the invention involve sequentially administering to a patient multiple doses of the anti-HLAA2:HPV16E7 antigen binding protein of the invention. As used herein, the term sequential administration means that each dose of anti-HLA-A2:HPV16E7 antigen binding protein is administered to a patient at a different point in time, for example, on different days separated by a predetermined interval (eg, hours, days, weeks or months). The present invention includes methods that include sequentially administering to a patient one initial dose of anti-HLAA2:HPV16E7 antigen-binding protein, followed by one or more secondary doses of anti-HLA-A2:HPV16E7 antigen-binding protein, and optionally followed by one or more tertiary doses of anti-HLA-A2:HPV16E7 antigen-binding protein. A2:HPV16E7. Antigen binding protein against HLA-A2:HPV16E7 can be administered at a dose of 0.1 to 100 mg/kg of patient body weight.

The terms initial dose, secondary doses and tertiary doses refer to the timing sequence of administration of the anti-HLA-A2:HPV16E7 antigen binding protein of the invention. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may all contain the same amount of antigen binding protein against HLA-A2:HPV16E7, but generally may differ from each other in the frequency of administration. In some embodiments, the amount of anti-HLA-A2:HPV16E7 antigen binding protein contained in the initial, secondary, and/or tertiary dose varies from each other (eg, increases or decreases as needed) during treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by doses that are administered less frequently (eg, maintenance doses).

In some embodiments, the amount of anti-HLAA2:HPV16E7 antigen binding protein contained in the initial, secondary and/or tertiary doses may be suboptimal or subtherapeutic. As used herein, the terms subtherapeutic or suboptimal refer to a dose of antibody administered at a level too low to produce a therapeutic effect or below the level required to treat a disease such as a cancer.

In some illustrative embodiments of the present invention, each secondary and/or tertiary dose is administered at 1-26 weeks (eg, 1.1 ^1/2 , 2.2 ^1/2 , 3, 3 ^1/2 , 4, 4 ^{1/2 weeks). 2} , 5, 5 ^1/2 , 6, 6 ^1/2 , 7, 7 ^1/2 , 8, 8 ^1/2 , 9, 9 ^1/2 , 10, 10 ^1/2 , 11, 11 ^1/2 , 12, 12 ^1/2 , 13, 13 ^1/2 , 14, 14 ^1/2 , 15, 15 ^1/2 , 16, 16 ^1/2 , 17, 17 ^1/2 , 18, 18 ^1/2 , 19, 19 ^1/2 , 20, 20 ^1/2 , 21, 21 ^1/2 , 22, 22 ^1/2 , 23, 23 ^1/2 , 24, 24 ^1/2 , 25, 25 ^1/2 , 26 , 26 ^1/2 or more) weeks after the immediately preceding dose. The expression immediately preceding dose as used herein means, in a multiple dosing sequence, the dose of anti-HLA-A2:HPV16E7 antigen binding protein that is administered to a patient before the next dose in the sequence without intervening doses.

The methods of this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of anti-HLA-A2:HPV16E7 antigen binding protein. For example, in some embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in some embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Likewise, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In some embodiments, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of treatment. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical examination.

Diagnostic use of antigen-binding proteins.

The anti-HLA-A2:HPV16E7 antigen-binding proteins of the present invention can be used to detect and/or measure HPV16E7 in a sample, for example, for diagnostic purposes. Some embodiments provide for the use of one or more antigen-binding proteins of the present invention in assays for detecting a disease or disorder, such as an HPV-associated disease or disorder, such as an HPV16E7-positive malignancy or HPV infection. Typical diagnostic assays for HPV16E7 may include, for example, contacting a sample obtained from an individual (eg, a patient) with an anti-HLA-A2:HPV16E7 antigen binding protein of the invention, wherein the anti-HLA-A2:HPV16E7 antigen binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand for the selective isolation of HPV16E7 from test samples. Alternatively, an unlabeled antigen binding protein against HLA-A2:HPV16E7 can be used for diagnostic purposes in combination with a secondary antigen binding protein, for example an antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific common assays that can be used to detect or measure HPV16E7 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

- 38 044060

Samples that can be used in HPV16E7 diagnostic assays in accordance with the present invention include any tissue or fluid sample obtained from a patient containing detectable amounts of HPV16E7 protein or fragments thereof under normal or pathological conditions. Typically, the levels of HPV16E7 in a specific sample obtained from a healthy patient (eg, a patient who does not have a disease or disorder associated with HPV16E7, such as an HPV16E7-positive malignancy) will be measured to initially establish a baseline or standard level of HPV16E7. This baseline level of HPV16E7 can then be compared with levels of HPV16E7 measured in samples obtained from individuals suspected of having a disease associated with a malignancy or symptoms associated with such a condition.

Antigen binding proteins specific for HPV16E7 may not contain additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the localization of the tag (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if a surface is coated with avidin, the peptide containing N-terminal biotin will be oriented such that the C-terminal part of the peptide is distal to the surface.

Aspects of the invention relate to the use of the disclosed antigen-binding proteins as markers to predict the prognosis of HPV16E7-positive cancer or HPV infection in patients. The antigen binding proteins of the present invention can be used in diagnostic assays to assess the prognosis of a patient's cancer and to predict survival.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of methods for making and using the invention, and are not intended to limit the scope of what the inventors consider their invention to be. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure.

Example 1. Production of human antibodies to HLA-A2:HPV16E7.

Human antibodies to HLA-A2:HPV16E7 were generated using peptide fragments of HPV16E7 that include either amino acids 11-19 (YMLDLQPET; SEQ ID NO: 538) from GenBank accession number NP_041326.1 (SEQ ID NO: 537) or amino acid residues 82-90 (LLMGTLGIV; SEQ ID NO: 539) GenBank NP_041326.1 (SEQ ID NO: 537) linked to HLA-A2. The immunogen was administered directly, with an adjuvant to stimulate the immune response of a mouse VELOCIMMUNE® (ie, an engineered mouse containing DNA encoding the variable regions of the heavy chain and light chain of human immunoglobulin), for example, as described in US Patent No. 8502018. The immune response of antibodies was monitored with using an HLA-A2:HPV16E7 specific immunoassay. Once the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce HLA-A2:HPV16E7-specific antibodies. Using this technique and the immunogen described above, several chimeric antibodies against HPV16E7 (ie, antibodies having human variable domains and mouse constant domains) have been generated. Exemplary antibodies thus produced were designated as follows: H4sH17364N; H4sH17368N2; H4sH17930N; H4sH17930N2; H4sH17363N and H4sH17368N3.

Antibodies against HLA-A2:HPV16E7 were also isolated directly from antigen-positive B cells (from any of the immunized mice) without fusion with myeloma cells, as described in US Patent 7,582,298, which is incorporated herein by reference in its entirety. Using this method, several fully human antibodies against HLAA2:HPV16E7 (ie, antibodies having human variable domains and human constant domains) have been produced.

Exemplary antibodies produced in accordance with the above methods were designated as follows: H4sH17670P; H4sH17672P; H4sH17673P; H4sH17675P; H4sH17680P; H4sH17697P; H4sH17707P; H4sH17715P; H4sH17726P; H4sH17730P; H4sH21051P; H4sH21054P;

H4sH21055P; H4sH21058P; H4sH21064P; H4sH21073P; H4sH21077P; H4sH21079P; H4sH21080P;

H4sH21083P; H4sH21086P; H4sH21090P; H4sH21091P; H4sH21093P; H4sH21099P; H4sH21100P;

H4sH21103P; and H4sH21104P.

Biological properties of exemplary antibodies prepared in accordance with methods thereof

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Examples are described in detail in the examples below.

Example 2. Amino acid and nucleotide sequences of the variable regions of the heavy and light chains.

In table 1 shows identifiers of the amino acid sequences of the variable regions of the heavy and light chains and CDRs of selected antibodies against HLA-A2:HPV16E7 according to the invention. The corresponding nucleic acid sequence identifiers are given in Table. 2.

Table 1

Amino acid sequence identifiers

SEQ ID NO: Antibody designation HCV R HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 L.C. D.R. 3 H4sH17364N 2 4 6 8 10 12 14 16 H4sH17368N2 18 20 22 24 26 28 thirty 32 H4sH17670P 34 36 38 40 42 44 46 48 H4sH17672P 50 52 54 56 58 60 62 64 H4sH17673P 66 68 70 72 74 76 78 80 H4sH17675P 82 84 86 88 90 92 94 96 H4sH17680P 98 100 102 104 106 108 110 112 H4sH17697P 114 116 118 120 122 124 126 128 H4sH17707P 130 132 134 136 138 140 142 144 H4sH17715P 146 148 150 152 154 156 158 160 H4sH17726P 162 164 166 168 170 172 174 176 H4sH17730P 178 180 182 184 186 188 190 192 H4sH17930N 210 212 214 216 202 204 206 208 H4sH17930N2 194 196 198 200 202 204 206 208 H4sH21051P 218 220 222 224 226 228 230 232 H4sH21054P 234 236 238 240 242 244 246 248 H4sH21055P 250 252 254 256 258 260 262 264 H4sH21058P 266 268 270 272 274 276 278 280 H4sH21064P 282 284 286 288 290 292 294 296 H4sH21073P 298 300 302 304 306 308 310 312 H4sH21077P 314 316 318 320 322 324 326 328 H4sH21079P 330 332 334 336 338 340 342 344 H4sH21080P 346 348 350 352 354 356 358 360 H4sH21083P 362 364 366 368 370 372 374 376 H4sH21086P 378 380 382 384 386 388 390 392 H4sH21090P 394 396 398 400 402 404 406 408 H4sH21091P 410 412 414 416 418 420 422 424 H4sH21093P 426 428 430 432 434 436 438 440 H4sH21099P 442 444 446 448 450 452 454 456 H4sH21100P 458 460 462 464 466 468 470 472 H4sH21103P 474 476 478 480 482 484 486 488 H4sH21104P 490 492 494 496 498 500 502 504 H4sH17363N 506 508 510 512 514 516 518 520 H4sH17368N3 522 524 526 528 530 532 534 536

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table 2

Nucleic acid sequence identifiers

SEQ ID NO: Designation antibodies HCV R HCDR1 HCDR 2 HCDR3 LCVR LCDR 1 LCDR2 LCD R3 H4sH17364N 1 3 5 7 9 eleven 13 15 H4sH17368N 2 17 19 21 23 25 27 29 31 H4sH17670P 33 35 37 39 41 43 45 47 H4sH17672P 49 51 53 55 57 59 61 63 H4sH17673P 65 67 69 71 73 75 77 79 H4sH17675P 81 83 85 87 89 91 93 95 H4sH17680P 97 99 101 103 105 107 109 111 H4sH17697P 113 115 117 119 121 123 125 127 H4sH17707P 129 131 133 135 137 139 141 143 H4sH17715P 145 147 149 151 153 155 157 159 H4sH17726P 161 163 165 167 169 171 173 175 H4sH17730P 177 179 181 183 185 187 189 191 H4sH17930N 209 211 213 215 201 203 205 207 H4sH17930N 2 193 195 197 199 201 203 205 207 H4sH21051P 217 219 221 223 225 227 229 231 H4sH21054P 233 235 237 239 241 243 245 247 H4sH21055P 249 251 253 255 257 259 261 263 H4sH21058P 265 267 269 271 273 275 277 279 H4sH21064P 281 283 285 287 289 291 293 295 H4sH21073P 297 299 301 303 305 307 309 311 H4sH21077P 313 315 317 319 321 323 325 327 H4sH21079P 329 331 333 335 337 339 341 343 H4sH21080P 345 347 349 351 353 355 357 359

H4sH21083P 361 363 365 367 369 371 373 375 H4sH21086P 377 379 381 383 385 387 389 391 H4sH21090P 393 395 397 399 401 403 405 407 H4sH21091P 409 411 413 415 417 419 421 423 H4sH21093P 425 427 429 431 433 435 437 439 H4sH21099P 441 443 445 447 449 451 453 455 H4sH21100P 457 459 461 463 465 467 469 471 H4sH21103P 473 475 477 479 481 483 485 487 H4sH21104P 489 491 493 495 497 499 501 503 H4sH17363N 505 507 509 511 513 515 517 519 H4sH17368N 3 521 523 525 527 529 531 533 535

Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (eg, H1M, H4sH, H4H, etc.) followed by a numerical identifier (eg, 17670, 17930, etc., as shown in Table . 1), followed by the suffix P, N or N2. Thus, according to this nomenclature, the antibody may be referred to herein as, for example, H4sH17670P, H4sH17930N, H4sH17368N2, etc. The prefixes H4sH and H4H in the antibody designations used herein indicate the specific isotype of the Fc region of the antibody. For example, antibody H4sH has a human IgG4 Fc with 2 or more amino acid substitutions as described in US Patent Publication No. 20140243504 (incorporated herein in its entirety), antibody H4H has a human IgG4 Fc with a serine to proline mutation in the hinge region (S108P) , the H1M antibody has a mouse IgG1 Fc and the H2M antibody has a mouse IgG2 Fc (all variable regions are fully human, as indicated by the first H in the antibody designation). As one skilled in the art will appreciate, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4 Fc, etc.), but in in any case, variable domains (including CDRs), which are designated by the numeric identifiers shown in table. 1 will remain the same and the antigen binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

In some embodiments, the selected murine IgG1 Fc antibodies have been converted to human IgG4 Fc antibodies. In some embodiments, the antibody comprises a human IgG4 Fc with 2 or more amino acid substitutions, as described in US Patent Publication No. 20100331527 (incorporated herein in its entirety). In one embodiment, the IgG4 Fc domain contains a serine to proline mutation in the hinge region (S108P) to help stabilize the dimer.

In table 3 shows the amino acid sequence identifiers of the heavy and light chain sequences of selected antibodies according to the invention.

Table 3

Heavy chain and light chain sequence identifiers

SEQ ID NO: Antibody designation Heavy chain Light chain H4sH17363N 578 579 H4sH17364N 580 581 H4sH17670P 582 583 H4sH17675P 584 585 H4sH17930N2 586 587 H4sH21058P 588 589 H4sH21064P 590 591 H4sH21104P 592 593

Example 3 Variable gene preference analysis.

To analyze the structure of the resulting antibodies, the nucleic acids encoding the variable regions of the antibodies were cloned and sequenced. From the nucleic acid sequence and the predicted amino acid sequence of the antibodies, a gene preference for each heavy chain variable region (HCVR) and light chain variable region (LCVR) was identified (Table 4).

Table 4

HCVR (HPV) LCVR (HPV) Antibody designation VH D.H. JH VH JH H4sH17363N V3-23 D6-6 J6 Vl-39 J5 H4sH17364N V3-23 D6-6 J6 Vl-39 J5 H4sH17368N2 V3-23 D3-9 J4 Vl-39 J5 H4sH17368N3 V3-23 D3-9 J4 Vl-39 J5 H4sH17670P V3-64 DI-26 J6 Vl-39 J5 H4sH17672P V3-64 DI-26 J6 Vl-39 J5 H4sH17673P V3-23 D4-11 J6 Vl-39 J5 H4sH17675P V3-64 DI-26 J6 Vl-39 J5 H4sH17680P V3-23 D4-23 J6 Vl-39 J5 H4sH17697P V3-11 D6-13 J4 Vl-39 J2 H4sH17707P V3-23 DI-20 J4 Vl-39 J5 H4sH17715P V6-1 Dl-7 J3 Vl-39 J2 H4sH17726P Vl-18 Dl-7 J4 V3-15 J4 H4sH17730P V3-11 Dl-7 J4 Vl-17 J2

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H4sH17930N V3-64 D2-2 J6 Vl-39 J5 H4sH17930N2 V3-64 D2-2 J6 Vl-39 J5 H4sH21051P V3-23 D7-27 J4 Vl-39 J5 H4sH21054P V3-23 Dl-7 J4 Vl-39 J5 H4sH21055P V3-11 D7-27 J2 Vl-39 J2 H4sH21058P V3-20 D2-2 J5 Vl-39 J2 H4sH21064P V3-64 D6-6 J6 Vl-39 J5 H4sH21073P V3-43 D6-19 J3 Vl-39 J2 H4sH21077P V3-23 D6-19 J3 Vl-39 J2 H4sH21079P V3-15 Dl-7 J4 Vl-39 J2 H4sH21080P V3-23 Dl-7 J6 V2-28 L H4sH21083P V3-23 Dl-7 J2 V3-15 J5 H4sH21086P V3-33 D2-21 J6 V4-1 J5 H4sH21090P V3-23 DI-20 J4 V3-15 J4 H4sH21091P V3-15 D6-19 J6 Vl-17 J4 H4sH21093P V3-33 D3-3 J3 Vl-6 J2 H4sH21099P V3-9 Dl-1 J6 Vl-39 J5 H4sH21100P V3-9 Dl-7 J3 Vl-39 J5 H4sH21103P V3-15 Dl-7 J4 Vl-39 J5 H4sH21104P V3-11 D3-10 J3 Vl-39 J5

Example 4: Surface Plasmon Resonance Binding Affinities and Kinetic Constants of Human Anti-HLA-A2:HPV16E7 Monoclonal Monospecific Antibodies.

The binding affinity and kinetic constants of human anti-HLAA2/HPV16E7 antibodies were determined by real-time surface plasmon resonance (SPR; Biacore 4000 or Biacore T-200, GE Healthcare Life Sciences, Pittsburgh, PA) at 25°C. Antibodies were captured on a CM5 Biacore touch surface (GE Healthcare Life Sciences) derivatized by amino condensation with an anti-human Fc monoclonal antibody (GE, #BR-1008-39). Various concentrations of the monomeric HLA-A2:HPV16E7 peptide complex containing either the E7:11-19 peptide (SEQ ID NO:538) or the E7:82-90 peptide (SEQ ID NO:539) were injected over the anti-HLA-A2 antibody: HPV16E7 with a flow rate of 50 μl/min (Biacore T-200) or 30 μl/min (Biacore 4000). The reagent-antibody association was monitored for 4-5 minutes. and dissociation was monitored for 10 min. All binding studies were performed in HBS-ET buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v/v surfactant P20).

Kinetic association (ka) and dissociation (kd) rate constants were determined by fitting real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constants (KD) and dissociation half-periods (t ^1/2 ) were calculated from the kinetic rate constants as kd Ke (M) =

AND

1п(2) t4 (min) = bTy

Binding kinetic parameters for monospecific anti-HLA-A2:HPV16E7 antibodies to the monomeric HLA-A2/HPV16E7 peptide complex are shown in Table 1 below. 5 and 6.

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Table 5

Binding affinities based on Biacore antibodies against HLA-A2/HPV16E7 (11-19) at 25°C____________

HLA-A2:HPV16E7( 11-19)

Antibody ka (1/Mc) kd(1/c) KD(M) tl/2 (min) H4sH17670P 8D6E+04 l,43E-03 l,75E-08 8D H4sH17672P l,29E+05 8D9E-04 6.37E-09 14.1 H4sH17673P N.B. N.B. N.B. N.B. H4sH17675P 5.99E+04 l,38E-03 2.31E-08 8.4 H4sH17680P N.B. N.B. N.B. N.B. H4sH17697P N.B. N.B. N.B. N.B. H4sH17707P N.B. N.B. N.B. N.B. H4sH17715P N.B. N.B. N.B. N.B. H4sH17726P N.B. N.B. N.B. N.B. H4sH17730P N.B. N.B. N.B. N.B. H4sH17363N 8.72E+04 l,54E-03 l,76E-08 7.5 H4sH17364N 8.56E+04 l,57E-03 l,83E-08 7.4 H4sH17368N2 N.B. N.B. N.B. N.B. H4sH17368N3 N.B. N.B. N.B. N.B. H4sH17930N 7.84E+04 7.96E-04 l,02E-08 14.5 H4sH17930N2 8.28E+04 7.92E-04 9.57E-09 14.6 H4sH21051P N.B. N.B. N.B. N.B. H4sH21054P N.B. N.B. N.B. N.B. H4sH21055P N.B. N.B. N.B. N.B. H4sH21058P N.B. N.B. N.B. N.B. H4sH21064P 5.47E+04 7.91E-04 l,44E-08 14.6 H4sH21073P N.B. N.B. N.B. N.B. H4sH21077P N.B. N.B. N.B. N.B. H4sH21079P 3.74E+04 l,09E-02 2.90E-07 1D H4sH21080P l,79E+05 3.90E-02 2D8E-07 0.3 H4sH21083P N.B. N.B. N.B. N.B. H4sH21086P N.B. N.B. N.B. N.B. H4sH21090P N.B. N.B. N.B. N.B. H4sH21091P N.B. N.B. N.B. N.B. H4sH21093P N.B. N.B. N.B. N.B. H4sH21099P N.B. N.B. N.B. N.B. H4sH21100P N.B. N.B. N.B. N.B. H4sH21103P N.B. N.B. N.B. N.B. H4sH21104P N.B. N.B. N.B. N.B.

*NB indicates that under experimental conditions the peptide reagent HLAA2:HPV16E7(11-19) does not bind to captured anti-HLA-A2:HPV16E7 mAb.

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Table 6

Biacore binding assays for anti-HLA-A2/HPV16E7 (82-90) antibodies at 25°C

HLA-A2:HPV 16E7(82-90)

Antibody ka (1/Mc) kd(l/c) KD(M) tl/2 (min) H4sH17670P N.B. N.B. N.B. N.B. H4sH17672P N.B. N.B. N.B. N.B. H4sH17673P N.B. N.B. N.B. N.B. H4sH17675P N.B. N.B. N.B. N.B. H4sH17680P N.B. N.B. N.B. N.B. H4sH17697P N.B. N.B. N.B. N.B. H4sH17707P 7D5E+04 3.61E-04 5.05E-09 32.0 H4sH17715P 4.58E+04 5.68E-04 l,24E-08 20.3 H4sH17726P 5D7E+04 4D9E-04 8D0E-09 27.6 H4sH17730P N.B. N.B. N.B. N.B. H4sH17363N N.B. N.B. N.B. N.B. H4sH17364N N.B. N.B. N.B. N.B. H4sH17368N2 8.31E+O5 l,92E-03 2.30E-09 6.0 H4sH17368N3 7D2E+05 l,22E-03 l,71E-09 9.5 H4sH17930N N.B. N.B. N.B. N.B. H4sH17930N2 N.B. N.B. N.B. N.B. H4sH21051P l,37E+04 3.31E-04 2.41E-08 34.9 H4sH21054P l.98E+05 7.65E-04 3.86E-09 15.1 H4sH21055P l.56E+05 l,21E-03 7.76E-09 9.6 H4sH21058P 2.46E+05 2.60E-04 l,06E-09 44.5 H4sH21064P N.B. N.B. N.B. N.B. H4sH21073P 5.77E+05 1D5E-04 2.00E-10 100.3 H4sH21077P N.B. N.B. N.B. N.B. H4sH21079P N.B. N.B. N.B. N.B. H4sH21080P N.B. N.B. N.B. N.B. H4sH21083P 5.38E+04 2D2E-04 3.94E-09 54.5 H4sH21086P 6.97E+04 1D4E-03 l,63E-08 10.2 H4sH21090P 8D1E+O4 l,91E-04 2.35E-09 60.6 H4sH21091P l,74E+05 l,46E-04 8.42E-10 79.1 H4sH21093P 1D8E+O5 l,92E-03 l,63E-08 6.0 H4sH21099P l,24E+05 9.79E-05 7.88E-10 118.0 H4sH21100P 2.90E+05 l,82E-04 6.26E-10 63.5 H4sH21103P 8.35E+O5 3.22E-03 3.86E-09 3.6 H4sH21104P 4.36E+04 2D5E-04 4.94E-09 53.7

*NB indicates that under experimental conditions the peptide reagent HLAA2:HPV16E7(82-90) does not bind to captured anti-HLA-A2:HPV16E7 mAb.

The data demonstrate that the majority of the anti-HLA-A2/HPV16E7 antibodies of the present invention bound to the soluble HLA-A2/HPV16E7 peptide complex, with some demonstrating sub-nanomolar affinity. Some antibodies, however, showed a lack of binding to the HLA-A2/HPV16E7 complex.

Example 5: Prediction of potential non-target peptides.

Given a target 9-mer peptide-HLA-A2 complex, an associated potential off-target peptide is identified based on three criteria: A) the peptide is 9-mer and predicted to bind HLA-A2, B) the peptide is similar to the target peptide on based on sequence homology, and C) the peptide is derived from a gene that is expressed in major normal tissues. Thus, the following methodology was used to predict potential off-target peptides associated with YMLDLQPET (HPV16 E711-19; SEQ ID NO: 538) and LLMGTLGIV (HPV16 E782-90; SEQ ID NO: 539) (generally, see Dhanik, Ankur, et al (2016) BMC Bioinformatics 17(1): 286).

As a first step, canonical human protein sequences were downloaded from the UniprotKB database (version September 2014) (Magrane, Michele, and UniProt Consortium. Database 2011 (2011): bar009) and all 9-mers were isolated. This resulted in 11,118,076 peptides from 20,014 protein sequences.

HLA-A2 peptide binding affinities were then calculated using the NetMHCstab web server (version 1.0) (Jnrgensen, Kasper W., et al. (2014) Immunology 141(1): 18-26). Peptides with an affinity value <500 nM were predicted to bind to HLA-A2 and the rest were discarded, resulting in a remaining 338,452 peptides.

The peptide sequences were then assessed for sequence homology to the target peptide. For each peptide, its degree of similarity (DoS) was calculated for the target peptide. Meaning

- 45 044060

DoS represents the number of identical amino acids at identical positions between two peptides. Peptides with a DoS value <6 were rejected, leaving the remaining 21 peptides in the case

HLA-A2/HPV16E7: 11-19 and 78 peptides in the case of HLA-A2/HPV16E7: 82-90.

The genes corresponding to the 21 peptides were screened for their expression in major normal tissues. Expression assessment was performed using gene expression data obtained from the GTEx (Tissue Gene Expression) and TCGA (Cancer Genome Atlas) databases provided by OmicSof (Hu, Jun, et al. Bioinformatics (2012) 28(14):1933- 1934). Data were available in RPKM (reads per thousand base pairs per million) values from 497 contiguous TCGA normal samples (across 15 major tissue types) and 2928 GTEx normal samples (across 22 major tissue types). Tissues other than breast, cervix, fallopian tube, testis, uterus, and vagina were considered essential. A gene was considered expressed in major normal tissues if the maximum 95% expression in each major normal tissue type in the GTEx and TCGA databases was^0.5 RPKM. For HLA-A2/HPV16E7: 11-19 (YMLDLQPET), of the 21 peptides, 10 peptides were derived from genes that are expressed in major normal tissues. For HLA-A2/HPV16E7: 82-90 (LLMGTLGIV), of the 78 peptides, 49 peptides were derived from genes that are expressed in major normal tissues.

These 10 peptides constitute the predicted off-target peptides associated with the YMLDLQPET-HLA-A2 target complex (Table 7). Of the 49 potential peptides predicted to constitute likely off-target peptides associated with the LLMGTLGIV-HLA-A2 complex, 13 were randomly selected for experimental validation and are listed in Table 1 . 8.

Table 7

Predicted non-target HLA-A2/HPV16E7-like peptides: 11-19 (YMLDLQPET: SEO ID NO: 538)

No Peptide sequence Peptide name Gene Predicted May IC ₅₀ (nM) 1 YMLDLQKQL (SEQ ID NO: 546) SH3GLB 1:244-252 SH3GLB1 9.2 2 KMLDKNPET (SEQ ID NO: 547) With AMKK1:388-396 CAMKK1 107.9 3 YMFDLLLET (SEQ ID NO: 548) USP47:691-699 USP47 3.5 4 YTLDLQLEA (SEQ ID NO: 549) CHPF:463:471 CHPF 132.8 5 MMLILQAET (SEQ ID NO: 550) PKD 1:2694-2702 PKD1 244.3 6 LMLPLQPCT (SEQ ID NO: 551) NBRl:357-365 NBR1 487.8 7 YILDLLPDT (SEQ ID NO: 552) CBL:83-91 CBL 145.9 8 YMEDLQELT (SEQ ID NO: 553) PPP4R4:20-28 PPP4R4 482.1 9 GLLDLDPET (SEQ ID NO: 554) SBK3:285-293 SBK3 91.6 10 VMKDLLPET (SEQ ID NO: 555) FNDC3B:921-929 FNDC3B 379.9

Table 8

Predicted non-target HLA-A2/HPV16E7-like peptides: 82-90 (LLMGTLGIV; SEO ID NO: 539)

No. Peptide sequence Peptide name Gene Predicted May IC50 (nM) 1 LLMGTFLSV (SEQ ID NO: 556) VPREB3:9-17 VPREB3 5.9 2 LLGGTLERV (SEQ ID NO: 557) B4GALT2:4-12 B4GALT2 93.6 3 LLMGSNTIV (SEQ ID NO: 558) GCAT:312-320 GOAT 13.2 4 LLQATLDIV (SEQ ID NO: 559) CYP39A1:246-254 CYP39A1 88.7 5 LLLTFLGIV (SEQ ID NO: 560) ALDH3A2:467-475 ALDH3A2 85.4 6 LLAGTLAGV (SEQ ID NO: 561) CLCN4:79-87 CLCN4 11.0 7 LLQDTLGHV (SEQ ID NO: 562) ZHX2:234-242 ZHX2 50.5 8 LLLAVLGIV (SEQ ID NO: 563) GRM6:590-598 GRM6 64.4 9 LVMETLCIV (SEQ ID NO: 564) IPO9:582-590 IPO9 18.8 10 LLNETLGEV (SEQ ID NO: 565) IPO4:163-171 IPO4 25.8 eleven KLMGHLGVV (SEQ ID NO: 566) SF3B1:969-977 SF3B1 11.2 12 LLMCYLYIV (SEQ ID NO: 567) DOCK11:1282-1290 DOCK11 2.7 13 LLNKVLGIV (SEQ ID NO: 568) Human CNOT1:1962-1970 CNOT1 247.8

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Example 6. Stimulation of T2 cells with peptide to determine the specificity of HLAA2/HPV16E7 M.

To determine the specificity of the anti-HLA-A2/HPV16E7 monoclonal antibody, peptide-stimulated T2 cells loaded with target or non-target peptides (identified in the previous Example) were used. Experiments were performed as follows: For exogenous loading of HPV16E7 target or non-target peptides, T2 cells were washed in AIM V® medium and counted using a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience). Approximately 6 million T2 cells per T-75 flask were cultured for 24 hours at 26°C in 9 ml of AIM V® medium containing 10 μg of human b2m and 100 μg of HPV16E7 peptide or non-target peptide (Tables 6 and 7). Peptide-loaded T2 cells were washed once with PBS without Ca2+/Mg2+ and counted. Approximately 10,000 cells per well loaded with T2 peptide or untreated T2 in cell wash buffer were seeded into 96-well carbon electrode plates (MULTI-ARRAY high binding, MSD) and incubated for 1 hour at 37°C to allow cells attach to the tablet. Nonspecific binding sites were blocked using 2% BSA (w/v) in PBS for 1 hour at room temperature. Serial dilutions of anti-HLA-A2/HPV16E7:11-19, anti-HLA-A2/HPV16E7:82-90, or control antibody were added to the cells bound to the plates at serial dilutions ranging from 1.7 μM to 100 nM, and also solutions without antibody. Plates were incubated for 1 hour at room temperature and then washed to remove unbound antibody using AquaMax2000 plate wash (MDS Analytical Technologies). Antibodies bound to the plate were detected using a goat polyclonal anti-human IgG conjugated to Fc-gamma fragment-specific SULFO-TAG antibody (Jackson Immunoresearch, Meso Scale Discovery) for 1 hour at room temperature. After washing, plates were developed using reading buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded using a SECTOR Imager 600 (Meso Scale Discovery). Luminescence intensity, measured in relative light units (RLU), was recorded to indicate the binding intensity of each antibody over a range of concentrations. The cell binding signal ratio for each anti-HLA-A2/HPV16E7 antibody compared to the isotype control at 11 nM is shown in Table 1. 8 and 9 and is an indicator of specificity. At a concentration of 11 nM, most antibodies showed minimal binding to untreated T2 cells. Not all antibodies have been tested with all relevant related non-target peptides. Those that have not been tested are marked as NT Not Tested. Antibodies with a binding coefficient greater than 15 are marked (+++), those with a binding coefficient equal to or less than 15, but greater than or equal to 10, are marked (++), with a coefficient less than 10, but greater than or equal to 3, are marked (+ ), and antibodies with a binding coefficient of less than 3 were classified as non-binding and designated as (-). In addition, direct binding signals (in RLU) were analyzed as a function of antibody concentration and data fitted to a sigmoidal (four-parameter logistic) dose-response model using GraphPad Prism™. EC ₅₀ values, defined as the antibody concentration at which 50% of the maximum binding signal on cells is detected, were determined where possible to indicate the activity of each antibody. EC ₅₀ values for cell surface binding to HLA-A2/HPV16E7:11-19 or HLA-A2/HPV16E7:82-90 alone are also shown in Table 1. 9 and 10.

Ten of the 13 anti-HLA-A2/HPV16E7:11-19 antibodies of the invention bind to the HLA-A2/peptide complex on the cell surface of T2 cells. Seven of these 10 antibodies (H4sH17670P; H4sH17675P; H4sH17363N; H4sH17364N; H4sH17930N; H4sH17930N2; and H4sH21064P are specific for the HLA-A2/HPV16E7:11-19 complex. Three antibodies (H4sH17 672P, H4sH21079P, H4sH21080P) showed higher activity with EC values ₅₀ below 1.1 nM. Three antibodies (H4sH17673P, H4sH17680P, H4sH17697P) did not bind to peptide-loaded T2 cells and are indicated (-) in the first column of Table 9.

Cell binding results on T2 cells loaded with the HPV16E7:82-90 target and predicted non-target peptides are summarized in Table 1. 9. Sixteen of the 21 anti-HLAA2/HPV16E7:82-90 mAbs of the invention bound to the HLA-A2/peptide complex on the cell surface of T2 cells. Only 2 mAbs from this group (H4sH17368N2, H4sH21086P) showed specificity for the HLA-A2/HPV16E7 complex 82-90. Five antibodies (H4sH17730P, H4sH21051P, H4sH21054P, H4sH21055P, H4sH21077P) did not bind to peptide-loaded T2 cells and are indicated (-) in the table. 10.

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Binding specificity of monoclonal antibodies against HLA-A2/HPV16E7:11-19

Table 9

Bound cell EC ₅₀ (M) T2+peptide cell binding specificity compared to the irrelevant h!gG4 isotype control at 11 nM AbPID Т2+Н PV16 E7 11-19 HPV 16E7 1119 SH3 GLB 1 244252 SA MK K1 388- 396 US P47 691 699 CH PF 463 :47 1 PK DI 269 4- 270 2 NB RI 357 365 CB L 8391 PPP 4R4 2028 S.B. KZ 285 293 T2 neo bra bot annay H4sH17670 Ρ TsE09 H4sH17672 Ρ 4.5E- 10 -|—|- + H4sH17673 Ρ H4sH17675 Ρ SHCHE09 + H4sH17680 Ρ H4sH17697 Ρ H4sH17363 Ν 2DE- 09 -|—|- H4sH17364 Ν 2.0E09 -|—|- H4sH17930 Ν ZDE09 -|—|- H4sH17930 N2 5.8E09 -|—|- H4sH21064 P 2.2E09 -1—1—1- NT NT NT - NT - - NT NT - H4sH21079 P SHCHE09 +++ NT NT NT + NT + NT NT H4sH21080 P 2.2E- 10 -|—|- NT NT NT + NT - + NT NT - Cell binding signal at 11 nM, RLU Isotype control - 1029 976 772 110 2 107 7 112 3 820 eleven 04 103 8 945 847

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Table 10

Binding specificity of monoclonal antibodies against HLA-A2/HPV16E7:82-90

Knitted okES cage

T2+peptide cell binding specificity compared to the irrelevant h!gG4 isotype control at 11 nM

(Μ) AbPID T2+ HPV 16E7 8290 HP VI 6E 7 8290 VP RE VZ 917 V 4 G A L T 2 4- 12 G C A T 31 232 0 CY РЗ 9А 1 24 625 4 A L D H 3 A 2 46 747 5 CL CN 4 7987 ZH X2 23 4- 24 2 G R M 6 59 0- 59 8 IP 09 58 2- 59 0 IP 04 16 3- 17 1 S F 3 V 1 96 997 7 D0 C K 11 12 82 12 90 C N 0 T 1 19 62 19 70 T 2 not CT and M UL and po va Η Η y e H4sHl 7707Р 2,ZE08 + - - - - - - + - - - - - - - H4sHl 7715P 2.9E- 10 ++ - - - - + - - + + - - + - + - H4sHl 7726P 4DE- 10 ++ + + + - ++ + ++ + + + + - + + + - H4sHl 7730P - H4sHl 7368N2 7.5E- 10 ++ - H4sHl 7368N3 1.8E- 10 ++ - - - - - + - - - - - - - - H4sH2 105 IP - - - - N T NT N T NT - N T NT NT N T N T N T - H4sH2 1054P - - - - N T NT N T NT - N T NT NT N T N T N T - H4sH2 1055P - - - - N T NT N T NT - N T NT NT N T N T N T - H4sH2 1077P - - - - N T NT N T NT - N T NT NT N T N T N T - H4sH2 1086P 5.0E10 + - - N T NT N T NT - N T NT NT N T N T N T - H4sH2 1090P 5.6E10 + - - N T NT N T NT + N T NT NT N T N T N T - H4sH2 1091P 1.7E- 10 + - - N T NT N T NT ++ + N T NT NT N T N T N T - H4sH2 1093P 3.0E10 ++ - - N T NT N T NT + N T NT NT N T N T N T - H4sH2 1058P 1.9E- 10 ++ - - - - - - - + - - + - - - H4sH2 1073P 9.0E11 ++ - - - - - - - + - - - - - - H4sH2 1O83P 3,ZE- 10 ++ - - - - + - - + - - + + - + - H4sH2 1099P 8.7E- eleven ++ - - - - + - - + - - + - + - H4sH2 HOOP 9.6E11 ++ - - - - - - - + - - + - - - H4sH2 1103P 4, SEII ++ - - - - + - - + ++ + + + + + + + + - H4sH2 1104P 4.8E- 10 ++ - - - + - - - - - - + - - - Cell binding signal at 11 nM, RLU Control 0 or isotype a - 83 5 87 1 92 6 87 2 56 2 85 6 72 5 79 7 80 7 76 8 10 67 70 2 77 6 78 0 86 0

As shown in table. 9 and 10, the anti-HLA-A2:HPV16E7 antigen binding proteins of the invention bind with high specificity only to a specific HPV peptide (SEQ ID NO: 538 in Table 9

- 49 044060 or with SEQ ID NO: 539 in table. 10), presented by HLA-A2, and does not bind to any non-target peptides not presented by HLA-A2.

Example 7 Binding Specificity Analysis Using Peptide-Stimulated T2 Cells and FACS Analysis.

The relative binding and specificity of antibodies to HPV16E7 were determined by flow cytometry on NIH3T3 cells expressing the HLA-A2 complex presenting either the HPV 11-19 peptide (3T3/HLA.A2/hB2M/HPV16E7:11-19) or the HPV 82-peptide 90 (3T3 /HLA.A2/hB2M/HPV16E7 (82-90). NIH3T3 cells expressing the HLA complex were obtained by transfecting human HLA.A2 (accession number P01892), human B2M (accession number NP_004039.1) and a peptide cassette ubiquitin (Levy F., et al. (1996) Proceedings of the National Academy of Sciences of the United States of America 93(10):4907-4912; Valmori D, et al. (1999) Journal of Experimental Medicine 189 (6 ): 895-906) containing either HPV16E7 amino acids 11-19 (SEQ ID NO: 538) or amino acids 82-90 (SEQ ID NO: 539) (accession number AK185233) using Lipofectomin 2000 (Invitrogen, Cat # 11668) followed by selection for at least 2 weeks using 1 μg/ml puromycin, 500 μg/ml G418 and 100 μg/ml hygromycin. For staining, cells were collected using cell dissociation buffer (Millipore, Cat # S-004-C ), and counted. Cells were seeded in staining buffer (PBS, without calcium and magnesium (Irving 9240) + 2% FBS (ATCC 30-2020) at a density of 200,000 cells per well in a 96-well V-bottom plate and stained with three-fold serial dilutions (1. 7 pM - 100 nM) primary antibodies for 30 min at 4° C. After primary antibody incubation, cells were washed once in staining buffer and stained with secondary antibody conjugated to Alexa-Flour 647 (Jackson ImmunoResearch, Cat # 109-606- 170), at 10 μg/ml for 30 minutes at 4°C. Cells were then washed and fixed using 50% BD Cytofix (BD, Cat # 554655) diluted in staining buffer. Samples were run and analyzed on an iQue flow cytometer intellicyt to calculate mean fluorescence intensity (MFI).MFI values were plotted in Graphpad Prism using a four-parameter logistic equation over a 12-point response curve to calculate _EC50 values.One secondary antibody (i.e., not a primary antibody) for each The dose-response curve is also included in the analysis as a continuation of the three-fold serial dilution and is presented as the lowest dose. EC ₅₀ (M) values and maximum fold binding (fold change from highest to lowest dose) are shown in Table. 11. Several antibodies specifically bound to either the 3T3/HLA.A2/hB2M/HPV16E7:11-19 or 3T3/HLA.A2/hB2M/HPV16E7:82-90 cell line. EC ₅₀ values ranged from 5-500 nM, and binding folds ranged from 1X to 43.8X.

- 50 044060

FACS binding of antibodies against HPV16E7

Table 11

3T3/HLA.A2/hB2M/H PV16E7 (11-19) 3T3/HLA.A2/hB2M/H PV16E7 (82-90) HEK293 Antibody designation ec ₅₀ Max magnification ec ₅₀ Max multiple ec ₅₀ Max multiple *H4sH17363N l,40E-08 11.4 ND 1.7 ND 1.3 *H4sH17364N 2.40E-08 11.4 2.91E-08 2.3 ND 1.2 H4sH17368N2 ND 1.8 l,94E-07 9.1 ND 1.8 H4sH17368N3 ND 1.1 3.26E-08 6.1 ND 1.7 *H4sH17670P 2.37E-08 6.7 ND 1.5 ND 0.8 H4sH17672P 3.72E-08 10.2 ND 1.4 ND 1.6 H4sH17673P ND 1.8 ND 1.3 ND 1.6 *H4sH17675P l,27E-08 5.1 ND 1.3 ND 0.65 H4sH17680P ND 1.5 ND 1.1 ND 1 H4sH17697P ND 1.2 ND 1.5 ND 1.1 H4sH17707P ND 1.5 ND 1.8 ND 2 H4sH17715P ND 1.7 6.60E-06 6.7 3.47E-08 3.1 H4sH17726P 5.20E-08 28.11 7D9E-08 43.8 ND 2.0 H4sH17730P ND 0.9 5.80E-08 2.9 ND 0.9 H4sH17930N l,73E-08 13.5 6.62E-08 10.3 l,53E-07 4.3 *H4sH17930N2 2.78E-08 11.4 7.48E-08 3 3.93E-08 3 H4sH21051P ND 1.2 ND 2.0 ND 1.2 H4sH21054P ND 3.8 ND 4.9 3.51E-08 3.6 H4sH21055P ND 1.4 ND 1.5 ND 1.9 H4sH21058P ND 1.3 l,01E-08 8.6 ND 0.9 *H4sH21064P 2.05E-08 12.3 6.60E-08 2.2 ND 0.7 H4sH21073P ND 0.9 4.09E-08 6.5 ND 0.9 H4sH21077P ND 2.0 ND 1.5 ND 1.6 H4sH21079P 4.02-08 26.6 5.40E-08 20.5 ND 1.3 H4sH21080P ZDOE-O8 11.7 2D1E-08 7.4 5D9E-08 4.4 H4sH21083P ND 1.5 3.31E-09 8.5 ND 1.4 H4sH21086P 5.537E- 14.6 3.49E-07 22 ND 1.3 H4sH21090P ND 1.6 l,85E-09 6.25 ND 1.1 H4sH21091P ND 1.5 3.74E-10 5.6 ND 1.6 H4sH21093P ND 1.9 2.95E-08 3.9 ND 1.4 H4sH21099P ND 1 1D9E-09 4.8 ND 2 H4sH21100P 3.55E-08 4.4 1D9E-08 10.3 ND 0.7 H4sH21103P ND 1.6 9.20E-09 6.3 ND 1.5 H4sH21104P ND 1.6 5.481E-09 8.5 ND 1 Control ND 1 ND 1 ND 1.2

Antibodies with (*) were run together in a separate experiment.

ND=EC5 ₀ He determined when the maximum fold binding was less than or equal to 2-fold.

The specificity of the six anti-HPV16E7:11-19 antibodies was further characterized by assessing binding to T2 cells (174 CEM.T2) stimulated with HPV16E7:11-19, HPV16E7:8290 or predicted non-target peptides (Table 7). For T2 stimulation (174 CEM.T2) was resuspended in AIM V medium at a density of 1x106 cells/ml (Gibco. Cat # 31035-025). Cells were stimulated by adding 10 μg/ml hB2M (EMD Millipore Cat # 475828) and 100 μg/ml of the indicated peptide. T2 cells were then incubated overnight at 26°C, washed in staining buffer, and stained with the indicated antibodies at a concentration of 10 μg/ml according to the protocol described above. MFI values were calculated and presented as fold change for unstained cells. The relative binding of six antibodies against HPV16E7:11-19 on T2 cells stimulated with HPV16E7:11-19 is 986-1200 times higher than unstained cells. No significant binding above the isotype control was observed on T2 cells stimulated with other peptides (Table 12).

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Table 12

FACS HPV16E7 antibody binding to stimulated T2 cells (fold change relative to unstained)

HPV16 E7:1119 YMLD LQPET SH3G LB1 244252 SAM KK1 388396 USP 47 691699 SNR F 463: 471 RK D1 269 4- 270 2 N.B. R1 357 365 CBL 8391 RR P4 R4 2028 SB KZ 285 293 Not stimulating the cells and H4sH1736 3N 1001.4 13.6 2.0 0.6 7.4 1.2 3.6 19.5 6.1 0.4 8.5 H4sH1736 4N 986.1 15.2 1.0 1.3 10.9 1.0 3.0 23.9 9.0 0.8 11.7 H4sH1767 OR 1005.5 3.7 2.9 5.3 3.6 3.5 2.2 2.0 2.9 2.6 5.0 H4sH1767 5P 1204.2 10.5 2.4 13.3 5.0 2.9 2.2 4.9 5.5 2.9 7.2 H4sH1793 0N2 1166.7 28.2 2.6 8.9 7.5 3.3 3.3 4.6 7.3 3.0 6.7 H4sH2106 4P 1204.2 10.5 2.4 13.3 5.0 2.9 2.2 4.9 5.5 2.9 7.2 Isotype control 17.1 8.5 6.3 11.5 9.9 9.8 8.7 9.0 8.0 6.8 10.2 Only 14.2 3.7 5.8 5.2 4.8 4.4 3.7 4.4 4.0 4.1 6.5 secondary Not painted 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Example 8 Epitope Analysis Using Alanine Scanning Peptides.

Alanine scanning was performed to determine which residues in the HPV16E7:11-19 peptide were critical for antibody binding. T2 cells were stimulated with alanine scanning peptides and stained with anti-HPV16E7:11-19 antibodies as described above. The following alanine scanning peptides were used (Table 13).

Table 13 Alanine scanning peptides used in the study

SEQ GO NO: Peptide Ala replacement 569 AMLDLQPET Y11A 570 YALDLQPET M12A 571 YMADLQPET L13A 572 YMLALQPET D14A 573 YMLDAQPET L15A 574 YMLDLAPET Q16A 575 YMLDLQAET Р17А 576 YMLDLQPAT E18A 577 YMLDLQPEA T19A

Conversion of aspartate 14 to alanine (D14A) and glutamine 16 to alanine (Q16A) significantly reduced antibody binding for all antibodies tested. Conversion of tyrosine 11 to alanine (Y11A) reduces the binding of H4sH17670P, H4sH17675P, H4sH21064P, and H4sH17930N2; but not H4sH17363N or H4sH17364N. Conversion of leucine 13 to alanine (L13A) and proline 17 to alanine (P17A) reduces overall antibody binding (Table 14).

To summarize, D14 and Q16 are critical residues for antibody binding.

Table 14

FACS Binding of HPV16E7 Antibodies to T2 Cells Stimulated with Alanine Scanning Peptides

AML DLQP ET (Y11 A) YAL DLQP ET (M12 A) YMA DLQP ET (L13A) YML ALQP ET (D14 A) YMLD AQPET (L15A) YMLD LAPE T (Q16A) YML DLQ AET (P17A) YML DLQP AT (E18A) YML DLQP EA (T19) H4sH1736 3N 694.8 998.5 529.9 58.2 1019.3 13.3 401.8 775.7 1034, 3 H4sH1736 4N 708.3 997.9 489.9 69.7 1008.4 13.2 384.8 756.2 1075, 7 H4sH1767 0Р 8.1 670.3 374.9 10.7 1062.8 6.2 168.8 614.7 1150, 1

- 52 044060

H4sH1767 5P 19.0 823.8 527.2 6.3 1153.1 5.8 371.1 808.3 1165.0 H4sH1793 0N2 39.0 972.9 665.7 5.8 1245.9 6.8 531.9 1035.4 1330, 3 H4sH2106 4P 19.0 823.8 527.2 6.3 1153.1 5.8 371.1 808.3 1165.0 Control 14.4 10.9 8.4 9.9 8.1 9.0 8.2 9.1 9.6 isotype Only 9.7 6.9 4.9 6.4 6.3 5.5 4.6 4.5 6.5 secondary Not painted 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Example 9. Reformatting of HLA-A2/HPV16E7 antibodies into ScFv for use in chimeric antigen receptors.

Six antibodies against HLA-A2/HPV16E7:11-19 (17363N, 17364N, 17670P, 17675P, 17930N2 and 21064P) were reformatted into single chain VL-VH variable fragments (ScFv) and placed into a chimeric antigen receptor (CAR) construct that used CD8a hinge and transmembrane domain, 4-1BB co-stimulatory domain and CD3Z stimulatory domain (SEQ ID NO: 540-545). Specific HLA-A2/HPV16E7:11-19 CARs were cloned into a lentiviral expression vector (Lenti-X™ with bicistronic expression system (Neo), Clontech Cat #632181), and lentiviral particles were produced using the Lenti-X Packaging Single packaging system -Shot (VSV-G) (Clontech Cat #631276) according to manufacturer's protocols. Jurkat cells engineered to express an NFAT-luciferase reporter (Jurkat/NFATLuc cl.3C7) were then transduced with 6 different CAR constructs using RetroNectin® precoated dishes (Clontech, Cat # T110a) according to the manufacturer's protocols. After selection for at least 2 weeks using 500 μg/ml G418 (Gibco, Cat # 11811-098), CAR-T cell lines were generated.

The activity of CAR-T lines was assessed in a CAR-T/Antigen-presenting cell (APC) bioassay.

To perform the bioassay, 50,000 Jurkat/NFATLuc cl cells. 3C7 CAR-T was added to 96-well white Thermo-Nunc plates (Thermo Scientific, Cat # 136101) in 50 µl of assay medium (RPMI medium with 10% FBS and 1% P/S/G) followed by 3× serial dilution of APC (150,000 cells per 200 cells) in 50 μl of analytical medium. The following APCs were used: CASKI (HLA-A2+/HPV16+), CASKI cells overexpressing a single-chain variant of HLA-A2 presenting peptide 11-19 or 82-90, HEK293 (HLA-A2+/HPV16-) or C33a (HLA-A2+/ HPV16-). The cell mixture was incubated in a humidified incubator at 37°C, 5% CO ₂ for 5 hours. NFAT-luciferase activity was measured using a Promega One-Glo (Cat # E6130) and a Perkin Elmer Envision plate reader. Relative luciferase units (RLU) were generated and plotted in Graphpad Prism using a four-parameter logistic equation over an 8-point response curve to calculate EC ₅₀ values. The zero APC condition for each dose-response curve is also included in the analysis as a continuation of the three-fold serial dilution and is represented as the lowest dose. The maximum activation fold was determined by ratioing the largest RLU value in the curve to the smallest. All six HLA-A2/HPV16E7:11-19 CAR-T cell lines were activated by CASKI cells that overexpressed the HPV16E7:1119 peptide with a maximum activation fold of 2.5-32.3-fold. No CAR-T cell lines were activated by APCs that overexpressed the HPV16E7:82-90 peptide, or by HEK293 and C33a cells. Interestingly, one CAR-T cell line that used ScFv from antibody 17675P was activated by native CASKI cells with a fold activation of 4.1 and EC ₅₀ by 68654 cells (Table 15).

- 53 044060

Table 15

Activation of CAR-T HPV16E7 (11-19) in a CAR-T/APC Jurkat/NFATLuc chimeric antigen receptor construct bioassay

17363N 17364N 17670R 17675R 17930N2 21064Р ARS EC5 0 (cells) Multiplicity of VAC act EC5 0 (cells) Activation frequency EC5 0 (cells) Activation rate EC5 0 (cells) AI activation frequency EC5 0 (cells) How often does it need to be activated? EC5 0 (cells) How often does it need to be activated? CAS KI ND 0.9 ND 1 ND 0.8 6865 4 4.1 ND 0.9 ND 1.2 CAS KI11- 19 510 8 2.5 6632 10.4 414 5 8.5 9703 32.3 788 5 16.8 722 0 20.7 CAS KI 82- 90 ND 0.9 ND 1 ND 0.9 ND 1.5 ND 0.7 ND 0.9 HEK2 93 ND 0.8 ND 0.9 ND 0.8 ND 0.7 ND 0.7 ND 0.8 SZZa ND 0.7 ND 1.2 ND 0.8 ND 0.6 ND 0.6 ND 0.4

ND=EC ₅₀ He defined when the max fold binding was less than or equal to 2-fold.

Increasing the amount of HPV16E7:11-19 presented HLA-A2 peptide should result in increased HLA-HPV16E7:11-19 CAR activation. It has been reported that interferon gamma can increase antigen presentation by MHC class 1 molecules despite activation of the proteasome (Fruh K. and Yang Y. (1999) Curr Opin Immunol. 11(1): 76-81). Based on this observation, it was determined whether wild-type CASKI cells or HEK293 cells pretreated with interferon gamma could lead to increased activation of CAR-T cell lines. CASKI cells and HEK293 cells were pretreated with recombinant human IFN-γ (Peprotech Cat # 300-02) at 500 units/ml for 48 hours and then used in the CAR-T/APC bioassay as described above (Table 16) . CASKI cells pretreated with IFN-γ activated all 6 CAR-T cell lines HPV16E7:11-19 with fold activation in the range of 2.4-10.6.

Table 16

Activation of HPV16E7(11-19) CAR-T in the presence of IFN-γ

CASKI CASKI+IFN-g HEK293 HEK293+IFN-g Jurkat/NFA T Luc CART EC50 (cells) Max magnification EC50 (cell) Max multiplicity EC50 (cells) Max multiplicity EC50 (cell) Max multiple 17363N ND ι,ο 51837 2.4 ND ι,ο ND 0.81 17364N ND ι,ο 6440 5.6 ND 0.86 ND 0.75 17670R ND ι,ο 51360 2.7 ND 0.77 ND 0.78 17675R 13844 1.77 64903 10.6 ND 0.81 ND 0.71 17930N2 ND 0.97 57186 8.1 ND 0.75 ND 0.71 21064Р ND 1.0 55863 8.97 ND 0.8 ND 0.7

ND=EC ₅₀ He defined when the max fold binding was less than or equal to 2-fold.

To further evaluate the specificity of the HPV16E7:11-19 CAR-T lines in the luciferase assay, we used T2 cells as APCs and stimulated with predicted off-target peptides (Table 17). Briefly, T2 cells were stimulated with three-fold serial dilutions of the indicated peptides (1.7 to 100 ng/ml). After stimulation, 50,000 CAR-T cells were added to 96-well white Thermo-Nunc plates (Thermo Scientific, Cat #136101) in 50 μL assay media. 50,000 stimulated T2 cells were then added to the plates in 50 μl of assay medium. The cell mixture was incubated in a humidified incubator at 37°C, 5% CO ₂ for 5 hours. NFAT-luciferase activity was determined using a Promega One-Glo (Cat # E6130) and a Perkin Elmer Envision plate reader. RLU was plotted in Graphpad Prism using a four-parameter logistic equation over a 12-point response curve to calculate EC ₅₀ values. The unstimulated condition for each dose-response curve is also included in the analysis as a continuation of the three-fold serial dilution and is presented as the lowest dose. Maximum multiplicity of activation

- 54 044060 was divided as described previously. All CAR-T cell lines were activated by T2 cells stimulated with HPV16E7:11-19 peptide. The Jurkat/NFATLuc CART line using ScFv from antibody 17364N was nonspecifically activated by T2 cells stimulated with endophilin-B 1 (SH3GLB1: 244-252), chondroitin sulfate synthase 2 (CHPF: 463: 471) and E3 ubiquitin protein ligase CBL (CBL : 83-91). All other CAR-T cell lines had no significant activation with any non-target peptide.

Table 17

Max. fold activation of HPV16E7 (11-19) CAR-T against stimulated T2 cells

Jurkat/NFATLuc chimeric antigen receptor design Peptide 17363N 17364N 17670R 17675R 17930N2 21064Р Max activation factor HPV16E7:11-19 6.5 10.9 1.9 10.6 7.8 12.9 HPV16E7:82-90 0.9 0.9 0.9 0.9 0.9 0.8 SH3GLB1:244252 1.3 6.1 0.9 1.1 1.4 3.4 SAMKK1:388- 396 0.9 1.0 0.9 0.9 0.9 0.8 USP47:691-699 1.0 0.9 0.9 1.0 0.9 1.1 CHPF:463:471 1.1 5.2 0.9 1.0 0.9 1.1 PKD1:2694- 2702 0.8 0.9 1.0 1.0 0.9 0.9 NBRl:357-365 0.9 0.9 0.9 0.9 0.9 0.9 CBL:83-91 1.3 7.2 0.9 1.1 0.9 1.5 PPP4R4:20-28 0.9 0.9 1.0 1.0 0.9 0.9 SBK3:285-293 1.0 0.9 1.0 1.0 0.9 0.9

Example 10. Structural analysis of Fab binding to the HLA-A2+HPV16E7 peptide: 11-19.

In order to better understand the specific interactions between the antibody and the HLA-peptide complex, X-ray crystal structures of the Fab fragment of the antibody associated with HLA-A2/b2m presenting the HPV16E7 peptide were determined: 11-19. One structure contains Fab 17670P and the other structure contains Fab 17363N; together, these two structures cover the sequence space of the six antibodies presented above (eg, Tables 11 and 12). All 9 residues of the HLA-presented peptide FLPV16E7: 11-19 are clearly visible in the electron density maps for both structures 17670P and 17363N. Even at 2.9 Å (structural resolution 17670P), the position and identity of peptide residues are unambiguous, and residue-residue interactions can be accurately determined. The 17363P structure is 2.6A, which improves accuracy.

Fabs 17670P and 17363N bind to the top of the HLA-peptide complex in a manner very similar to the manner in which the TCR binds. Fabs are located and oriented almost identically to each other; both are aligned fairly parallel to the rails flanking the peptide-binding groove, and both are centered on the bound peptide, with the heavy chain CDRs binding to the N-terminal half of the bound peptide and the light chain CDRs binding to the C-terminal half of the peptide. Other published complex antibody structures (eg, PDB codes 1W72 and 4WUU) indicate that the antibody does not have to cover the entire HLA-presented peptide. However, these antibodies with only partial peptide coverage have poor specificity, allowing significant changes in the portion of the peptide not in contact with a small loss of binding affinity.

The structures show that Fab heavy chains 17670P and 17363N bind to residues 11, 14, 15 in the HPV16E7 peptide, whereas Fab light chains bind to residues 15, 17, 18. No Fab contacts occur with side chains of residues 12, 13, 16, or 19 , since they point to the HLA molecule. The bound peptide is numbered according to residue positions in the original FTPVI6 E7 protein as follows:

YMLDLQPET (SEQ ID NO:358)

12 13 14 15 16 17 18 19

Most Fab contacts are made with peptide side chains rather than with the backbone.

Peptide contacts made by 17670P are concentrated almost exclusively in the CDRs of LCDR1 and HCDR3, especially in HCDR3. Specifically, Fab heavy chain residues 100, 101, 102, 105, 109, 110 SEQ ID NO: 34 and light chain residues 30, 31, 32, 50 SEQ ID NO: 42 contact the bound peptide, while Fab heavy chain residues SEQ ID NO: 34 and light chain residues 31, 50, 52, 53, 54, 55, 92 SEQ ID NO: 42 contact HLA. Contact as used herein may include direct or water-mediated hydrogen bonds, charge-charge interactions, or hydrophobic/van der Waals interactions. For 17363N, Fab heavy chain residues 102, 103, 108, 111, 112 SEQ ID NO: 506 and light chain residues 28, 30, 32, 50, 68 SEQ ID NO: 514 bind to the bound

-

Claims (28)

peptide, while Fab heavy chain residues 28, 32, 100, 102, 103, 107, 112 SEQ ID NO: 506 and light chain residues 31, 49, 50, 51, 52, 53, 55, 92 SEQ ID NO: 514 bind to the HLA molecule. Of the six antibodies against HLA-A2:HPV16E7:11-19, 17675P has the highest similarity to 17670P in the peptide binding CDR sequences, with 21064P and 17930N2 also having a high degree of similarity in the CDR peptide binding regions. The key contacts between 17670P and the HLA peptide complex are largely conserved in 17675P, 21064P, and 17930N2, so the mode of binding of these antibodies is likely to be the same as that of 17670P. In contrast, CDR H3 17363N has a very different sequence compared to CDR H3 17670P, and this sequence difference translates into a structural difference in CDR H3, altering contacts with the HLA-peptide complex in this region. For example, Tyr 100 of the heavy chain at 17670P contacts Tyr 11 of the bound peptide. The equivalent residue in 17363N is Tyr 102 (the CDR H3 of this antibody is two residues longer), and this residue does not contact Tyr 11 of the peptide. Instead, Tyr 102 redirected itself to making contact with a nearby HLA molecule. Lead antibody 17364N has a very similar sequence to 17363N and is identical at all residues in contact with the HLA-peptide complex. This antibody should have a binding mechanism very similar to that of 17363N, and thus different from 17670P, 17675P, 17930N2 and 21064P. The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. CLAIM 1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to a conformational epitope of the HLA-A2-presented E7 peptide of human papillomavirus (HPV) 16 (HPV16E7 peptide), wherein the antibody or antigen-binding fragment thereof contains three complementarity determining regions (CDRs) ) heavy chain (HCDR1, HCDR2 and HCDR3); and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain a set of amino acid sequences selected from the group consisting of SEQ ID NO: 4, 6, 8, 12, 14 and 16; 36, 38, 40, 44, 46 and 48; 84, 86, 88, 92, 94 and 96; 196, 198, 200, 204, 206 and 208; 284, 286, 288, 292, 294 and 296 and 508, 510, 512, 516, 518 and 520. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof has a property selected from the group consisting of the following: (a) binds to the monomeric peptide HLA-A2:HPV16E7 11-19 (SEQ ID NO: 538) with a dissociation constant (KD) of less than about 20 nM, as measured in a surface plasmon resonance assay at 25°C; (b) binds to the monomeric peptide HLA-A2:HPV16E7 82-90 (SEQ ID NO: 539) with a dissociation constant (KD) of less than about 25 nM, as measured in a surface plasmon resonance assay at 25°C; (c) binds to cells expressing the HLA-A2:HPV16E7 peptide 11-19 (SEQ ID NO: 538) with an EC ₅₀ of less than about 6 nM and does not bind to cells expressing predicted non-target peptides as determined by luminescence assay ; (d) binds to cells expressing HLA-A2:HPV16E7 peptide 82-90 (SEQ ID NO: 539) with an EC ₅₀ of less than about 1 nM and substantially does not bind to cells expressing predicted non-target peptides as determined by luminescent analysis; (e) binds to cells expressing the HLA-A2:HPV16E7 peptide 11-19 (SEQ ID NO: 538) with an EC ₅₀ of less than about 30 nM, as determined by flow cytometry; and (f) binds to cells expressing the HLA-A2:HPV16E7 peptide 82-90 (SEQ ID NO: 539) with an EC ₅₀ of less than about 75 nM, as determined by flow cytometry. 3. An isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that the HPV16E7 peptide contains the amino acid sequence YMLDLQPET (SEQ ID NO: 538). 4. The isolated antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof is a full-length antibody, Fab, Fab', (Fab') ₂ , Fv, single chain Fv (scFv), T-body construct or CAR. 5. The isolated antibody or its antigen-binding fragment according to claim 4, which is a scFv. 6. An isolated antibody or an antigen-binding fragment thereof according to claim 1, containing a pair of heavy chain variable region (HCVR)/light chain variable region (LCVR) amino acid sequences (HCVR/LCVR), wherein the amino acid sequence is HCVR and the amino acid - 56 044060 the LCVR sequence of each HCVR/LCVR pair, each independently being at least 90% identical to the amino acid sequences of the HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/ 42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418, 426/ 434, 442/450, 458/466, 474/482, 490/498, 506/514 and 522/530. 7. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the HCVR amino acid sequence and the LCVR amino acid sequence of each HCVR/LCVR pair are each independently at least 95% identical to the HCVR/LCVR amino acid sequence of a pair of amino acid sequences selected from the group, consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/ 370, 378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498, 506/514 and 522/530. 8. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the HCVR amino acid sequence and the LCVR amino acid sequence of each HCVR/LCVR pair are each independently at least 98% identical to the amino acid sequences of the HCVR/LCVR amino acid sequence pair selected from the group, consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/ 370, 378/386, 394/402, 410/418, 426/434, 442/450, 458/466, 474/482, 490/498, 506/514 and 522/530. 9. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein the HCVR amino acid sequence and the LCVR amino acid sequence of each HCVR/LCVR pair are selected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/ 58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/226, 234/242, 250/258, 266/274, 282/290, 298/306, 314/322, 330/338, 346/354, 362/370, 378/386, 394/402, 410/418, 426/434, 442/ 450, 458/466, 474/482, 490/498, 506/514 and 522/530. 10. An isolated antibody or an antigen-binding fragment thereof according to claim 9, containing a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10, 34/42, 82/90, 194/202, 282/290 and 506/514. 11. The isolated antibody or its antigen-binding fragment according to claim 1, additionally containing a detectable fragment. 12. A pharmaceutical composition containing an isolated antibody or antigen-binding fragment thereof that binds to HLA-A2:HPV16E7 according to claim 1, and a pharmaceutically acceptable carrier or diluent. 13. A method of treating a patient having a disease or disorder associated with HPV16E7, comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to claim 1 or a pharmaceutical composition according to claim 12, thereby subjecting the patient to treatment. 14. The method of claim 13, wherein the disease or disorder associated with HPV16E7 is an HPV-associated malignancy. 15. The method of claim 14, wherein the HPV-associated malignancy is squamous cell carcinoma. 16. The method of claim 15, wherein the HPV-associated malignancy is cervical cancer, anogenital cancer, head and neck cancer, or oropharyngeal cancer. 17. The method of claim 13, wherein the isolated antibody or antigen-binding fragment thereof is administered to a subject in combination with a second therapeutic agent. 18. The method according to claim 17, characterized in that the second therapeutic agent is selected from the group consisting of a PD-1 inhibitor, a CTLA-4 inhibitor, an antibody to a tumor-specific antigen, an antibody to a virus-infected cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, a bispecific antibody against CD20 and CD3, a dietary supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, surgery, radiation therapy, an NSAID, a corticosteroid, an HPV vaccine, and any other therapy suitable to reduce the intensity of at least one symptom associated with a disease or disorder. 19. The method of claim 13, wherein the isolated antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. 20. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR contains an extracellular binding domain that specifically binds to a conformational epitope of the HLA-A2-presented human papillomavirus (HPV) 16 E7 peptide (HPV16E7 peptide), a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain comprises a human monoclonal antibody against HLA-A2: HPV16E7 or an antigen binding fragment thereof according to claim 1. - 57 044060 21. An expression vector containing an isolated nucleic acid molecule according to claim 20. 22. An isolated immune effector cell containing the vector according to claim 21. 23. The isolated immune effector cell according to claim 22, which is a T body. 24. A method of treating a patient having a disease or disorder associated with HPV, comprising administering to the patient an immune effector cell according to claim 22 or 23. 25. The method of claim 24, wherein the HPV-associated disease or disorder is an HPV-associated malignancy. 26. The method of claim 25, wherein the HPV-associated malignancy is squamous cell carcinoma. 27. The method of claim 25, wherein the HPV-associated malignancy is cervical cancer, anogenital cancer, head and neck cancer, or oropharyngeal cancer. 28. The method of claim 24, wherein the isolated antibody or antigen-binding fragment thereof is administered to a subject in combination with a second therapeutic agent. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2