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WO2025064539A1 - Herv-e antibodies and methods of their use - Google Patents

Herv-e antibodies and methods of their use Download PDF

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Publication number
WO2025064539A1
WO2025064539A1 PCT/US2024/047278 US2024047278W WO2025064539A1 WO 2025064539 A1 WO2025064539 A1 WO 2025064539A1 US 2024047278 W US2024047278 W US 2024047278W WO 2025064539 A1 WO2025064539 A1 WO 2025064539A1
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Prior art keywords
seq
nos
amino acid
antibody
antigen binding
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French (fr)
Inventor
Richard W. Childs
Long Chen
Elena A. CHERKASOVA
Peter Peizhi Luo
Fangyong Du
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Adagene Pte Ltd
US Department of Health and Human Services
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Adagene Pte Ltd
US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This disclosure relates to monoclonal antibodies and antigen binding fragments that specifically bind human endogenous retrovirus type E proteins expressed by renal cell carcinoma, chimeric antigen receptors including the antibodies or antigen binding fragments, and their use for treating renal cell carcinoma.
  • Sequence_Listing_4239-104126-02.xml (129,267 bytes), which was created on September 17, 2024, which is incorporated by reference herein.
  • Renal cell carcinoma is a common solid tumor in the United States.
  • the five-year survival of early stage RCC is 92.6%; however, for metastatic RCC (mRCC) this number drops to 11.7%.
  • mRCC metastatic RCC
  • a transcript derived from human endogenous retrovirus type E (CT-RCC HERV-E) is selectively expressed in clear cell renal cell carcinoma (ccRCC), and encodes an entire envelope gene of this retrovirus.
  • This transcript has one short ORF (CR) and two long ORFs predicted to encode the surface and transmembrane protein of the envelope (SU and TM).
  • mAbs human monoclonal antibodies
  • mAbs chimeric antigen receptors
  • CARs chimeric antigen receptors
  • the antibody or antigen binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l , a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 1 and 2, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 17 and 18, respectively; a heavy chain variable region and a light chain variable region
  • the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 3-8, respectively;
  • the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19-24, respectively;
  • the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 35-40, respectively;
  • the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 51-56 respectively;
  • the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 67- 72, respectively;
  • the antibody comprises VH and VL domains, wherein the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 1 and 2, respectively (for example, comprise SEQ ID NOs: 1 and 2, respectively); the VH and the Vi.
  • the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 33 and 34, respectively (for example, comprise SEQ ID NOs: 33 and 34, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 49 and 50, respectively (for example, comprise SEQ ID NOs: 49 and 50, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 65 and 66, respectively (for example, comprise SEQ ID NOs: 65 and 66, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 81 and 82, respectively (for example, comprise SEQ ID NOs: 81 and 82, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 81 and 82, respectively (for example, comprise SEQ ID
  • the antigen binding fragment is a Fv, Fab, F(ab’)2, scFv, or scFv2 fragment.
  • the antigen binding fragment is a scFv.
  • a bispecific antibody comprising a disclosed antibody or antigen binding fragment is provided.
  • a chimeric antigen receptor (CAR) including a disclosed antibody or antigen binding fragment is provided.
  • the CAR includes an scFv including a VH domain and VL domain of a disclosed antibody.
  • the CAR also includes one or more of a leader sequence, a hinge domain, a transmembrane domain, an intracellular domain, or a combination of two or more thereof.
  • one or more of the leader sequence, hinge domain, and transmembrane domain is from CD8a.
  • the intracellular domain includes a 4- 1BB intracellular domain and a CD3ij intracellular domain.
  • the CAR construct may also include a truncated CD34 domain.
  • the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 130, 137, 139, and 141.
  • the CAR may further include a second antibody or second antigen binding fragment.
  • a conjugate including a disclosed antibody or antigen binding fragment and an effector molecule is provided.
  • an effector molecule such as a detectable marker or a drug
  • a bispecific T cell engager including a disclosed antigen binding fragment (such as a disclosed scFv) linked to a second antigen binding fragment (such as a CD3 scFv) is provided.
  • Nucleic acids encoding the disclosed antibodies or antigen binding fragments are also provided.
  • the antibody or antigen binding fragment is encoded by VH and VL nucleotide sequences comprising SEQ ID NOs: 9 and 10, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 25 and 26, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 41 and 42, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 57 and 58, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 73 and 74, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 89 and 90, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 105 and 106, respectively; or VH and VL nucleotide sequences comprising SEQ ID NOs: 121 and 122, respectively.
  • nucleic acids encoding the disclosed CARs are also provided.
  • the CAR is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 129, 136, 138, and 140.
  • the disclosed nucleic acids are operably linked to a promoter.
  • vectors including the disclosed nucleic acids, and host cells including the vectors.
  • the nucleic acid or vector includes a nucleic acid sequence encoding a CAR and the host cell is an immune cell (such as a T cell or natural killer cell).
  • compositions including the antibody or antigen binding fragment, the chimeric antigen receptor, the conjugate or BiTE, or the nucleic acid or vector; and a pharmaceutically acceptable carrier are provided.
  • the composition includes a host cell including a disclosed nucleic acid and a pharmaceutically acceptable carrier.
  • the host cell is an immune cell (such as a T cell or a natural killer cell) including a nucleic acid encoding a disclosed CAR.
  • methods of treating a subject with renal cell carcinoma are provided.
  • the methods include administering to the subject an effective amount of a disclosed antibody or antigen binding fragment, CAR, conjugate, BiTE, nucleic acids encoding such molecules, or CAR-immune cells (such as CAR-T cells or CAR-NK cells) or a composition thereof.
  • the subject has clear cell renal cell carcinoma.
  • the subject has a renal cell carcinoma expressing a human endogenous retrovirus-E Envelope.
  • FIG. 1 is a heatmap showing binding of antibody candidates screened against RCC and non- RCC cell lines. Cell lines are listed in Table 3.
  • FIGS. 2 A and 2B are heatmaps showing binding of candidate antibodies to lymphocytes (FIG. 2A) or monocytes (FIG. 2B).
  • FIG. 3 is a heatmap showing binding of candidate antibodies to HERV-E+ RCC and normal human tissue.
  • FIG. 4A is a heatmap showing binding of candidate antibodies to CHO cells expressing the indicated CT-RCC HERV-E Env proteins.
  • FIG. 4B is a schematic diagram of the constructs expressed in the CHO cells.
  • FIG. 5 is a schematic diagram of an exemplary TAY058 BBZ CAR construct.
  • FIG. 6 shows expression of TAY058 BBZ CAR in T cells before and after CD34 magnetic sort.
  • FIG. 7 is a graph showing killing of CHO cells expressing HERV-E TM by TAY058 BBZ CAR T cells, but not HERV-E’ Raji cells.
  • FIG. 8 shows high expression of the indicated TAY BBZ CARs in T cells, by measuring CD34.
  • FIG. 9 is a graph showing killing of Raji (CD19 + HERV-E ) cells by the indicated TAY BBZ CAR T cells.
  • FIGS. 10A and 10B show QC of TAY051 BBZ CAR T cells from three donors tested with Raji cells (CD19 + HERV-E’; FIG. 10A) and RCC-U0K171 cells (CD19’ HERV-E’; FIG. 10B).
  • FIGS. 11A-1 ID show specific killing of four different HERV-E+ RCC cell lines by TAY051 BBZ CAR T cells.
  • FIG. 12 is a schematic diagram of an exemplary TAY051 VHVL BBZ CAR construct.
  • FIG. 13 shows high expression of the indicated TAY VHVL BBZ CARs in T cells, by measuring CD34.
  • FIG. 14 shows analysis of live T cells expressing the indicated TAY VHVL BBZ CARs, indicating that TAY052 VHVL BBZ CAR caused fratricide.
  • FIGS. 15A-15H show analysis of killing of HERV-E negative or positive RCC cell lines by TAY BBZ CAR (top) and TAY VHVL BBZ CAR (bottom) T cells.
  • nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable domain of TAY058: EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWGWIRQAPGKGLEWIGEIYHSG NTNYNPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDVYGGYDFDVWG QGTLVTVSS
  • SEQ ID NO: 2 is the amino acid sequence of the light chain variable domain of TAY058: DIQLTQSPS SLS AS VGDRVTITCKS GQSLLHSDGHTYLYWYQQKPGKAPKLLIYD AS NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPSTFGQGTKVEIK
  • SEQ ID NOs: 3-5 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY058.
  • SEQ ID Nos: 6-8 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY058.
  • SEQ ID NO: 9 is a nucleic acid sequence encoding the heavy chain variable domain of TAY058:
  • SEQ ID NO: 10 is a nucleic acid sequence encoding the light chain variable domain of TAY058: GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG GGTGACCATCACCTGCAAGAGCGGCCAGAGCCTGCTGCACAGCGACGGCCACA CCTACCTGTACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCT ACGACGCCAGCAACCTGGAGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGC AGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGAGCTACAGCTGGCCCAGCACCTTCGGCCAGGGCACC AAGGTGGAGATCAAG
  • SEQ ID NOs: 11-13 are nucleic acid sequences encoding HCDR1 , HCDR2, and HCDR3, respectively, of TAY058.
  • SEQ ID NOs: 14-16 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY058.
  • SEQ ID NO: 17 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 18 is the amino acid sequence of the light chain variable domain of TAY051: DIQLTQSPS SLS AS VGDRVTITCQASQDISTYLAWYQQKPGKAPKLLIYAAS SLQS G VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGSTPLTFGQGTKVEIK
  • SEQ ID NOs: 19-21 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY051.
  • SEQ ID Nos: 22-24 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY051.
  • SEQ ID NO: 25 is a nucleic acid sequence encoding the heavy chain variable domain of TAY051:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 26 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 27-29 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY051.
  • SEQ ID NOs: 30-32 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY051.
  • SEQ ID NO: 33 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 34 is the amino acid sequence of the light chain variable domain of TAY052: DIQLTQSPS SLS AS VGDRVTITCGASQD VGTAV AW YQQKPGKAPKLLIYDAS SLES G VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGTFPLTFGQGTKVEIK
  • SEQ ID NOs: 35-37 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY052.
  • SEQ ID Nos: 38-40 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY052.
  • SEQ ID NO: 41 is a nucleic acid sequence encoding the heavy chain variable domain of TAY052:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 42 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 43-45 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY052.
  • SEQ ID NOs: 46-48 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY052.
  • SEQ ID NO: 49 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 50 is the amino acid sequence of the light chain variable domain of TAY053:
  • SEQ ID NOs: 51-53 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY053.
  • SEQ ID Nos: 54-56 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY053.
  • SEQ ID NO: 57 is a nucleic acid sequence encoding the heavy chain variable domain of TAYO53:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 58 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 59-61 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY053.
  • SEQ ID NOs: 62-64 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY053.
  • SEQ ID NO: 65 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 66 is the amino acid sequence of the light chain variable domain of TAY054: DIQLTQSPSSLSASVGDRVTITCRASQDIRKFLAWYQQKPGKAPKLLIYDASNLETG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYHIWTFGQGTKVEIK
  • SEQ ID NOs: 67-69 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY054.
  • SEQ ID Nos: 70-72 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY054.
  • SEQ ID NO: 73 is a nucleic acid sequence encoding the heavy chain variable domain of TAY054:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 74 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 75-77 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY054.
  • SEQ ID NOs: 78-80 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY054.
  • SEQ ID NO: 81 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 82 is the amino acid sequence of the light chain variable domain of TAY059: DIQLTQSPSSLSASVGDRVTITCRASQSISTYLAWYQQKPGKAPKLLIYDASSLESGV PSRFSGSGTDFTLTISSLQPEDFATYYCQQSYHWPPTFGQGTKVEIK
  • SEQ ID NOs: 83-85 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY059.
  • SEQ ID NOs: 86-88 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY059.
  • SEQ ID NO: 89 is a nucleic acid sequence encoding the heavy chain variable domain of TAY059:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 90 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 91-93 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY059.
  • SEQ ID NOs: 94-96 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY059.
  • SEQ ID NO: 97 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 98 is the amino acid sequence of the light chain variable domain of TAY049: DIQLTQSPSSLSASVGDRVTITCRASQDIRSFLGWYQQKPGKAPKLLIYDASNRATGI PSRFSGSGTDFTLTISSLQPEDFATYYCQQAYHLWTFGQGTKVEIK
  • SEQ ID NOs: 99-101 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY049.
  • SEQ ID NOs: 102-104 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY049.
  • SEQ ID NO: 105 is a nucleic acid sequence encoding the heavy chain variable domain of TAY049:
  • CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 106 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 107-109 are nucleic acid sequences encoding HCDR1, HCDR2, and
  • SEQ ID NOs: 110-112 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY049.
  • SEQ ID NO: 113 is the amino acid sequence of the heavy chain variable domain of
  • SEQ ID NO: 114 is the amino acid sequence of the light chain variable domain of
  • SEQ ID NOs: 115-117 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY050.
  • SEQ ID NOs: 118-120 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY050.
  • SEQ ID NO: 121 is a nucleic acid sequence encoding the heavy chain variable domain of
  • SEQ ID NO: 122 is a nucleic acid sequence encoding the light chain variable domain of
  • SEQ ID NOs: 123-125 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY050.
  • SEQ ID NOs: 126-128 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY050.
  • SEQ ID NO: 129 is a nucleic acid sequence encoding an exemplary TAY058 BBZ CAR construct:
  • SEQ ID NO: 130 is the amino acid sequence of an exemplary TAY058 BBZ CAR construct:
  • SEQ ID NO: 131 is a nucleic acid sequence encoding a furin-P2A sequence and truncated CD34:
  • SEQ ID NO: 132 is the amino acid sequence of a furin-P2A and truncated CD34 protein:
  • SEQ ID NOs: 133 and 134 are amino acid sequences of HERV-E TM epitopes.
  • SEQ ID NO: 135 is the amino acid sequence of an exemplary glycine- serine linker:
  • SEQ ID NO: 136 is a nucleic acid sequence encoding an exemplary TAY058 VHVL CAR construct:
  • SEQ ID NO: 138 is a nucleic acid sequence encoding an exemplary TAY051 BBZ CAR construct:
  • SEQ ID NO: 139 is the amino acid sequence of an exemplary TAY051 BBZ CAR construct:
  • SEQ ID NO: 140 is a nucleic acid sequence encoding an exemplary TAY051 VHVL CAR construct: GCCACCATGGCATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGC
  • SEQ ID NO: 141 is the amino acid sequence of an exemplary TAY051 VHVL CAR construct:
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
  • Antibody and Antigen Binding Fragment An immunoglobulin, antigen binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), such as a human endogenous retrovirus E (HERV-E) protein.
  • antibody encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen binding activity.
  • Non- limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen.
  • antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multi-specific antibodies formed from antibody fragments.
  • Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).
  • Antibodies also include genetically engineered forms, such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally- occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
  • immunoglobulin typically has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes.
  • light chain lambda ( ) and kappa (K).
  • heavy chain classes or isotypes which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
  • Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain).
  • the heavy and the light chain variable regions specifically bind the antigen.
  • References to “Vu” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab.
  • References to “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.
  • the VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991).
  • CDRs complementarity-determining regions
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. Sequences of Proteins of Immunological Interest, 5 th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991 ; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol.
  • CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located.
  • VH CDR3 is the CDR3 from the VH of the antibody in which it is found
  • VL CDR1 is the CDR1 from the VL of the antibody in which it is found.
  • Light chain CDRs are sometimes referred to as LCDR1 , LCDR2, and LCDR3.
  • Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.
  • a disclosed antibody includes a heterologous constant domain.
  • the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutation) to increase half-life (see, e.g., Sievers et al., Curr. Opin. HIV AIDS 10: 151-159, 2015).
  • a “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • a “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment.
  • the non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs are substantially identical to corresponding parts of natural human antibody sequences.
  • a “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species.
  • a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and one or more CDRs and/or framework regions from another human antibody.
  • a “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species.
  • a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome.
  • Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).
  • Autologous refers to tissues, cells or nucleic acids taken from an individual’s own tissues. For example, in an autologous transfer or transplantation of immune cells (e.g., T cells or NK cells), the donor and recipient are the same person. Autologous (or “autogeneic” or “autogenous”) is related to self, or originating within an organism itself.
  • Bispecific antibody A recombinant molecule composed of two different antigen binding domains that binds to two different antigenic epitopes.
  • Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains.
  • the antigen binding domains can be linked using a linker.
  • the antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab or scFv), or combinations thereof.
  • a bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.
  • Placement in direct physical association includes both in solid and liquid form, which can take place either in vivo or in vitro.
  • Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody or antigen binding fragment.
  • Contacting can also include contacting a cell, for example by placing an antibody or antigen binding fragment in direct physical association with a cell.
  • a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.
  • Effective amount/therapeutically effective amount The amount of an agent (e.g., a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) that is sufficient to effect at least one beneficial or desired therapeutic result, including clinical results.
  • an agent e.g., a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells
  • An effective amount may vary depending upon one or more of: the subject and disease condition being treated, the sex, weight and age of the subject, the severity of the disease condition, the manner of administration, the ability of the treatment to elicit a desired response in the individual, and the like.
  • the beneficial therapeutic effect can include contributing to diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and/or generally counteracting a disease, symptom, disorder or pathological condition.
  • the term “effective amount” or “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in factors such as age, weight, tumor size, extent of metastasis, and condition of the patient (subject).
  • an “effective amount” (e.g., of a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) may be an amount sufficient to reduce the volume/size of a tumor, the weight of a tumor, the number/extent of metastases, reduce the volume/size of a metastasis, the weight of a metastasis, or combinations thereof, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% (as compared to no administration of the therapeutic agent).
  • an “effective amount” (e.g., of a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) may be an amount sufficient to increase the survival time of a subject, such as a subject with cancer, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, 100%, 200%, 300%, 400%, or 500% (as compared to no administration of the therapeutic agent).
  • Fc region The constant region of an antibody excluding the first heavy chain constant domain.
  • Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM.
  • An Fc region may also include part or all of the flexible hinge N-terminal to these domains.
  • an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain.
  • the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2.
  • the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat.
  • the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.
  • Heterologous Originating from a different genetic source.
  • a nucleic acid molecule that is heterologous to a cell originates from a genetic source other than the cell in which it is expressed.
  • a heterologous nucleic acid molecule encoding a protein, such as an scFv or a CAR is expressed in a cell, such as a mammalian cell.
  • Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.
  • HERV-E Human endogenous retrovirus E
  • HERVs are remnants of ancient exogenous retroviruses integrated into the human genome. HERVs are estimated to comprise 5-8% of the human genome. Most HERVs have accumulated mutations or are transcriptionally silenced and do not produce full-length proteins. However, some HERVs are transcriptionally active in contexts such as tumors.
  • HERV-E is a HERV subtype located on human chromosome 6q. At least three transcripts from HERV-E (e.g., GenBank Accession Nos.
  • EU137846, EU137847, and JQ7339O5) also referred to as HERV-E CT-RCC
  • HERV-E CT-RCC have been identified and are expressed in RCC cells, but not in other tumors or non-tumor cells (Takahashi et al., J. Clin. Oncol. 118:1099-1109, 2008).
  • These transcripts encode for parts of the protease and polymerase as well as the entire envelope genes, respectively.
  • isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids and proteins.
  • An isolated nucleic acid or protein for example an antibody or antigen binding fragment, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
  • Linker A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link domains together (for example, as in a CAR) or to link a detectable marker or drug to an antibody or antigen binding fragment.
  • Non-limiting examples of peptide linkers include glycine- serine linkers.
  • conjugating can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule (such as a detectable marker or drug) to a polypeptide, such as an antibody or antigen binding fragment.
  • the linkage can be either by chemical or recombinant means.
  • “Chemical means” refers to a reaction such that there is a covalent bond formed between the two molecules to form one molecule.
  • NK cells Cells of the immune system that kill target cells in the absence of a specific antigenic stimulus and without restriction according to MHC class.
  • Target cells can be tumor cells or cells harboring viruses.
  • NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells typically comprise approximately 10 to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells without prior immunization or activation. NK cells are thought to provide a “back up” protective mechanism against viruses and tumors that might escape the CTL response by down-regulating MHC class I presentation. In addition to being involved in direct cytotoxic killing, NK cells also serve a role in cytokine production, which can be important to control cancer and infection.
  • a “modified NK cell” is a NK cell transduced or transfected with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins.
  • a heterologous nucleic acid such as one or more of the nucleic acids or vectors disclosed herein
  • the terms “modified NK cell” and “transduced NK cell” are used interchangeably in some examples herein.
  • compositions and formulations suitable for pharmaceutical delivery of the disclosed agents are known to those of ordinary skill in the art. Remington: The Science and Practice of Pharmacy, 22 nd ed. , London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), pH buffering agents, and the like, for example sodium acetate or sorbitan monolaurate.
  • the pharmaceutically acceptable carrier is sterile and suitable for administration to a subject for example, by injection.
  • the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as in a selected quantity in a vial.
  • Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).
  • a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell.
  • the nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.
  • Renal cell carcinoma A tumor originating in the cells of the kidney.
  • RCC is the most common type of kidney cancer in adults.
  • ccRCC cells exhibit clear cytoplasm with acinar or sarcomatoid growth pattern.
  • Additional subtypes include but are not limited to papillary RCC (also originating in cells of the proximal tubule), chromophobic RCC (originating in cells of the cortical collecting duct), oncolytic RCC (a benign neoplasm originating in cells of the cortical collecting duct), and collecting duct RCC (originating in cells of the medullary collecting duct).
  • papillary RCC also originating in cells of the proximal tubule
  • chromophobic RCC originating in cells of the cortical collecting duct
  • oncolytic RCC a benign neoplasm originating in cells of the cortical collecting duct
  • collecting duct RCC originating in cells of the medullary collecting duct.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as non-human primates, pigs, sheep, cows, dogs, cats, rodents, and the like. In one example, a subject is a human.
  • T cell A white blood cell (lymphocyte) that is an important mediator of the immune response.
  • T cells include, but are not limited to, CD4 + T cells and CD8 + T cells.
  • a CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8 + T cells carry the “cluster of differentiation 8” (CD8) marker.
  • a CD8 + T cell is a cytotoxic T lymphocyte (CTL).
  • a CD8 + cell is a suppressor T cell.
  • Activated T cells can be detected by an increase in cell proliferation and/or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNy, or TNFa). Activation of CD8 + T cells can also be detected by an increase in cytolytic activity in response to an antigen.
  • cytokines such as IL-2, IL-4, IL-6, IFNy, or TNFa.
  • transformed and the like encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
  • a nucleic acid molecule such as a DNA or RNA molecule
  • a promoter(s) that is operably linked to the coding sequence of a protein of interest and can express the coding sequence.
  • Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent.
  • a vector is sometimes referred to as a construct.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements.
  • a vector includes a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment or encoding a disclosed chimeric antigen receptor.
  • the vector is a bacterial vector.
  • the vector is a viral vector, such as a nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses.
  • the viral vector is a retroviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the antibodies and antigen binding fragments specifically bind to a HERV-E Envelope.
  • the antibodies and antigen binding fragments specifically bind to the HERV-E transmembrane protein.
  • the antibodies and antigen binding fragments specifically bind to an epitope including or consisting of SEQ ID NO: 133 or SEQ ID NO: 134.
  • the monoclonal antibodies (or antigen binding fragments thereof) provided herein include heavy and/or light chain variable domains comprising a CDR1, CDR2, and/or CDR3 with reference to the IM GT numbering scheme (unless the context indicates otherwise).
  • Various CDR numbering schemes (such as the Kabat, Chothia or IM GT numbering schemes) can be used to determine CDR positions.
  • a monoclonal antibody or antigen binding fragment is provided that comprises the heavy and light chain CDRs of any one of the antibodies described herein.
  • a monoclonal antibody or antigen binding fragment is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein.
  • the antibody or antigen binding fragment includes a VH including the HCDR1 , HCDR2, and HCDR3, and V L including the LCDR1 , LCDR2, and LCDR3 of any one of the TAYO58, TAY051, TAY052, TAY053, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1 , a LCDR2, and a LCDR3 of the V H and V L of SEQ ID NOs: 1 and 2, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 17 and 18, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 33 and 34, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 49 and 50, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 65 and 66, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 81 and 82, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 97 and 98, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 113 and 114, respectively.
  • the CDRs are provided in Table 1.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 1 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 2.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 1 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 2.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 1 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 2 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 17 and a VL including an amino acid sequence at least 90% identical to (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 18.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 17 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 18.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 17 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 18 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 33 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 34.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 33 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 34.
  • the antibody or antigen binding fragment includes a Vu including the amino acid sequence of SEQ ID NO: 33 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 34 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 49 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 50.
  • the antibody or antigen binding fragment includes a Vu with an amino acid sequence including or consisting of SEQ ID NO: 49 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 50.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 49 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 50 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 65 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 66.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 65 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 66. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 65 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 66 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 81 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 82.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 81 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 82.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 81 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 82 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 97 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 98.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 97 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 98.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 97 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 98 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 113 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 114.
  • the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 1 1 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 114.
  • the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 113 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 114 and specifically binds to a HERV-E envelope.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 3-5, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 6-8, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 3-5, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 6-8, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 1 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 2, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 19-21, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 22-24, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 19-21, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 22-24, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 17 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 18, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 35-37, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 38-40, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 35-37, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 38-40, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 33 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 34, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 51-53, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 54-56, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 51-53, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 54-56, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 49 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 50, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 67-69, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 70-72, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 67-69, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 70-72, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 65 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 66, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 83-85, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 86-88, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 83-85, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 86-88, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 81 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 82, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 99-101 , respectively, and a V L including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 102-104, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 99-101, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 102-104, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 97 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 98, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 115-117, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 118-120, respectively.
  • the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 115-117, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 118-120, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 1 13 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 114, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
  • Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a VH and VL and specifically bind a HERV-E envelope. These antibody fragments retain the ability to selectively bind with the antigen and are antigen binding fragments.
  • Non-limiting examples of such fragments include Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; Fv, a genetically engineered fragment containing the VH and VL expressed as two chains; and single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin
  • scFvs with both possible arrangements (VH domain-linker-VL domain or VL domain-linker- VH domain) may be used.
  • a dimer of a single chain antibody scFV q. defined as a dimer of a scFV (also termed a “miniantibody”) is also included in the antigen binding fragments provided herein.
  • antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as a bacterial cell or a mammalian cell) of DNA encoding the fragment.
  • Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies.
  • antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab’ monovalent fragments.
  • a thiol reducing agent such as a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab’ monovalent fragments.
  • Other methods of cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • amino acid sequence variants of the antibodies or antigen binding fragments disclosed herein are provided.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, insertions, and/or substitutions of residues within the amino acid sequences of the antibody or antigen binding fragment. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., specifically binding a HERV-E envelope.
  • variants having one or more amino acid substitutions are provided.
  • Sites of interest for variants include the CDRs and the framework regions.
  • Amino acid substitutions may be introduced and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the variants typically retain amino acid residues necessary for correct folding and stabilization between the VH and the VL regions, and retain the charge characteristics of the residues in order to preserve low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence of any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, and 113.
  • the light chain of the antibody comprises up to 10 (such as up to 1 , up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence of any one of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114.
  • one or more substitutions, insertions, and/or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • only the framework residues are modified so the CDRs are unchanged.
  • the VL and VH segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response.
  • in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the HCDR3 or LCDR3, respectively.
  • the primers are “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions.
  • VH amino acid sequence is one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, and 113.
  • VL amino acid sequence is one of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114.
  • an antibody or antigen binding fragment is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated.
  • Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may he altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the Cl L domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(l):26-32, 1997.
  • the oligosaccharide may include various carbohydrates, e.g.
  • oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.
  • antibody variants have a carbohydrate structure that has a decreased amount of or lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about 3 amino acids upstream or downstream of position 297, e.g., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/01 10282; US 2004/0109865; WO 2003/0851 19; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; WO 2002/031140; Okazaki et al., J. Mol.
  • antibody variants with bisected oligosaccharides are provided, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546.
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function.
  • Such antibody variants are described, e.g., in WO 1997/30087, WO 1998/58964, and WO 1999/22764.
  • the constant region of the antibody comprises one or more amino acid substitutions to optimize in vivo half-life of the antibody.
  • the serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn).
  • the antibody comprises an amino acid substitution that increases binding to the FcRn.
  • substitutions include substitutions at IgG constant regions T250Q and M428L (see, e.g.
  • the disclosed antibodies and antigen binding fragments can be linked to or comprise an Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions.
  • an antibody provided herein may be further modified to contain additional non-proteinaceous moieties.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvin
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.
  • nucleic acids that encode the monoclonal antibodies or antigen binding fragments disclosed herein.
  • Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code.
  • the nucleic acids are codon-optimized, for example for expression in human cells.
  • a nucleic acid encodes a VH including the HCDR1, HCDR2, and HCDR3, and a VL including the LCDR1, LCDR2, and LCDR3 of any one of the TAY058, TAY051, TAY052, TAY053, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein.
  • the nucleic acid includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 9 and 10, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the V H and VL encoded by SEQ ID NOs: 25 and 26, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 41 and 42, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 57 and 58, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the V H and VL encoded by SEQ ID NOs: 73 and 74, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the V H and VL encoded by SEQ ID NOs: 89 and 90, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the V H and VL encoded by SEQ ID NOs: 105 and 106, respectively.
  • the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the V H and VL encoded by SEQ ID NOs: 121 and 122, respectively.
  • the nucleic acids encoding the CDRs are provided in Table 2.
  • an isolated nucleic acid encodes the VH of the TAY058 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 9 or includes or consists of SEQ ID NO: 9.
  • an isolated nucleic acid molecule encodes the VL of the TAY058 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10 or includes or consists of SEQ ID NO: 10.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAYO58 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 9 and 10, respectively.
  • an isolated nucleic acid encodes the VH of the TAY051 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 25 or includes or consists of SEQ ID NO: 25.
  • an isolated nucleic acid molecule encodes the VL of the TAY051 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 26 or includes or consists of SEQ ID NO: 26.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY051 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 25 and 26, respectively.
  • an isolated nucleic acid encodes the VH of the TAY052 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 41 or includes or consists of SEQ ID NO: 41.
  • an isolated nucleic acid molecule encodes the VL of the TAY052 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 42 or includes or consists of SEQ ID NO: 42.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY052 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 41 and 42, respectively.
  • an isolated nucleic acid encodes the VH of the TAY053 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 57 or includes or consists of SEQ ID NO: 57.
  • an isolated nucleic acid molecule encodes the VL of the TAY053 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 58 or includes or consists of SEQ ID NO: 58.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAYO53 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 57 and 58, respectively.
  • an isolated nucleic acid encodes the VH of the TAY054 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 73 or includes or consists of SEQ ID NO: 73.
  • an isolated nucleic acid molecule encodes the VL of the TAY054 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 74 or includes or consists of SEQ ID NO: 74.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY054 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 73 and 74, respectively.
  • an isolated nucleic acid encodes the VH of the TAY059 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 89 or includes or consists of SEQ ID NO: 89.
  • an isolated nucleic acid molecule encodes the VL of the TAY059 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 90 or includes or consists of SEQ ID NO: 90.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY059 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 89 and 90, respectively.
  • an isolated nucleic acid encodes the VH of the TAY049 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 105 or includes or consists of SEQ ID NO: 105.
  • an isolated nucleic acid molecule encodes the VL of the TAY049 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 106 or includes or consists of SEQ ID NO: 106.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY049 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 105 and 106, respectively.
  • an isolated nucleic acid encodes the VH of the TAY050 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 121 or includes or consists of SEQ ID NO: 121.
  • an isolated nucleic acid molecule encodes the VL of the TAY050 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 122 or includes or consists of SEQ ID NO: 122.
  • an isolated nucleic acid molecule encodes the VH and VL of the TAY050 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 121 and 122, respectively.
  • Nucleic acid molecules encoding the antibodies, antigen binding fragments, or other constructs provided herein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods.
  • nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements). Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • the nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect, or mammalian cells.
  • the antibodies, antigen binding fragments, or other constructs can be expressed as individual proteins including the Vn and/or VL, or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011.
  • the Vn- and Vi.-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 135), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker.
  • the single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to a HERV-E envelope and another antigen.
  • the encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains.
  • One or more DNA sequences encoding the antibodies, antigen binding fragments, or other constructs can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as COS, CHO, HeLa, and other cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, may be used.
  • Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.
  • nucleic acids encoding the antibodies, antigen binding fragments, or other constructs described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector.
  • the promoter can be any promoter of interest, including a cytomegalovirus promoter or EF- 1 a promoter.
  • an enhancer such as a cytomegalovirus enhancer, is included in the construct.
  • the vector can be suitable for replication and/or integration in either prokaryotes or eukaryotes. Typical expression vectors contain specific sequences useful for regulation of the expression of the DNA encoding the protein.
  • the vector can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • the vector also encodes a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
  • expression vectors which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • a strong promoter to direct transcription e.g., a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal.
  • control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences).
  • the vector can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the vector can be selected by resistance to antibiotics conferred by genes contained in the vector, such as the amp, gpt, neo and hyg genes.
  • Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
  • the antibodies, antigen binding fragments, or other constructs can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson el al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009).
  • the antibodies, antigen binding fragment, and conjugates need not be 100% pure.
  • the polypeptides should be substantially free of endotoxin.
  • CARs chimeric antigen receptors
  • BiTE bispecific T cell engager constructs
  • antibody-effector conjugates that utilize a disclosed HERV-E-specific antibody or antigen binding fragment thereof.
  • chimeric antigen receptors that include a portion that specifically binds to a HERV-E envelope.
  • the CARs include at least an antigen binding fragment of an antibody provided herein, such as at least one VH domain and at least one VL domain (for example, an scFv).
  • the VH domain and VL domain may be included in the CAR in either order, e.g., in the order VL -linker- VH or VI [-li nker- VL.
  • FIGS. 5 and 12 show exemplary CAR constructs in which the VH domain and VL domain are in the order Vi.-linker- VH (FIG. 5) or in the order Vn-linker-Vi. (FIG. 12).
  • the CAR includes a HERV-E-specific antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular domain, including at least one costimulatory domain. Additional domains may be included in the CAR construct, including, but not limited to a signal peptide (or leader sequence), and/or additional intracellular domain(s).
  • a CAR construct (such as a vector including a CAR) further include a “tag” (such as a truncated CD34 domain), which may be expressed separately from the CAR or cleaved from the CAR after translation.
  • the antigen binding domain is a HERV-E-specific scFv, for example having a VH domain including three HCDRs and a VL domain including three LCDRs as provided in Table 1.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY058 (e.g., SEQ ID NOs: 3-5) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAYO58 (e.g., SEQ ID NOs: 6-8).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 2.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 1 and a Vi. domain with the amino acid sequence of SEQ ID NO: 2.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY051 (e.g., SEQ ID NOs: 19-21) and a VL domain including LCDRI, LCDR2, and LCDR3 from TAY051 (e.g., SEQ ID NOs: 22-24).
  • the CAR includes an scFv including a Vu domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 17 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 18.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 17 and a VL domain with the amino acid sequence of SEQ ID NO: 18.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY052 (e.g., SEQ ID NOs: 35-37) and a V L domain including LCDRI, LCDR2, and LCDR3 from TAY052 (e.g., SEQ ID NOs: 38-40).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 33 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 34.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 33 and a VL domain with the amino acid sequence of SEQ ID NO: 34.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY053 (e.g., SEQ ID NOs: 51-53) and a VL domain including LCDRI, LCDR2, and LCDR3 from TAY053 (e.g., SEQ ID NOs: 54-56).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 49 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 50.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 49 and a VL domain with the amino acid sequence of SEQ ID NO: 50.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY054 (e.g., SEQ ID NOs: 67-69) and a V L domain including LCDR1, LCDR2, and LCDR3 from TAY054 (e.g., SEQ ID NOs: 70-72).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 65 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 66.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 65 and a VL domain with the amino acid sequence of SEQ ID NO: 66.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY059 (e.g., SEQ ID NOs: 83-85) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAY059 (e.g., SEQ ID NOs: 86-88).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 81 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 82.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 81 and a VL domain with the amino acid sequence of SEQ ID NO: 82.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY049 (e.g., SEQ ID NOs: 99-101) and a V L domain including LCDR1, LCDR2, and LCDR3 from TAY049 (e.g., SEQ ID NOs: 102-104).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 97 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 98.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 97 and a VL domain with the amino acid sequence of SEQ ID NO: 98.
  • the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY050 (e.g., SEQ ID NOs: 115-117) and a V L domain including LCDR1, LCDR2, and LCDR3 from TAY050 (e.g., SEQ ID NOs: 118-120).
  • the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1 13 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 114.
  • the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 113 and a VL domain with the amino acid sequence of SEQ ID NO: 114.
  • the hinge domain is a CD8a hinge region.
  • the hinge domain includes the sequence of amino acids 268-312 of SEQ ID NO: 130.
  • Other hinge domains can be used, such as hinge regions from immunoglobulins (for example, IgGl, lgG4, or IgD) or a hinge region from CD28 or CD40.
  • the transmembrane domain is a CD8a transmembrane domain.
  • the transmembrane domain includes the sequence of amino acids 313-336 of SEQ ID NO: 130.
  • the transmembrane domain can also be from other T cell proteins, such as CD28, CD4, CD3 , CD40, OX40L, 41BBL, ICOS, ICOS-L, CD80, CD86, ICAM-1, LFA-1, ICAM-1 , CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2, and CD70.
  • T cell proteins such as CD28, CD4, CD3 , CD40, OX40L, 41BBL, ICOS, ICOS-L, CD80, CD86, ICAM-1, LFA-1, ICAM-1 , CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NK
  • the intracellular domain includes one or more intracellular regions from a co-stimulatory molecule, or a portion thereof.
  • co- stimulatory molecules include CD28, 4-1BB, CD8, CD40, OX-40, ICOS, CD27, and DAP10, OX40-L, 4-1BBL, ICOS-L, CD80, CD86, ICAM-1, LFA-1, CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2, and CD70.
  • the co-stimulatory domain is from 4- IBB.
  • the co-stimulatory domain includes the sequence of amino acids 337-378 of SEQ ID NO: 130.
  • the intracellular domain also includes an intracellular signaling domain from CD3 ⁇ .
  • the intracellular signaling domain includes the sequence of amino acids 379-490 of SEQ ID NO: 130.
  • the intracellular signaling domain is from DAP 10, DAP 12, PDK, or FceRIy.
  • the CAR also includes a signal sequence, which is located N-terminal to the antigen binding domain.
  • the signal sequence is a CD8a signal sequence, an IgG signal sequence, or a GM-CSF signal sequence.
  • the signal sequence is a CD8a signal sequence, such as the sequence of amino acids 1-21 of SEQ ID NO: 130.
  • the CAR does not include the signal sequence and/or an N-terminal methionine; however the signal sequence and/or an N-terminal methionine can be present, for example as a result of the particular expression system used.
  • the CAR construct further includes a domain that can be used to identify and/or isolate cells expressing the CAR.
  • this domain is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide).
  • the selfcleaving peptide is a P2A peptide, such as the sequence of amino acids 1-27 of SEQ ID NO: 132.
  • the domain is a truncated CD34 protein (CD34t), such as a CD34 protein including the extracellular and transmembrane domains, but lacking the intracellular domain.
  • CD34t is expressed on the cell surface, but does not affect activity of cells expressing the truncated protein (Norell et al. , Cancer Immunol. Immunother. 59:851-862, 2010).
  • Cells expressing CD34t can be identified with an anti-CD34 antibody, and can be isolated using flow cytometry or immuno-magnetic methods.
  • the CD34t protein includes or consists of an amino acid sequence having at least 95% (such as at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 28-342 of SEQ ID NO: 132.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 130.
  • the CAR includes or consists of the amino acid sequence of SEQ ID NO: 130.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 130.
  • the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 130.
  • the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 137.
  • the CAR includes or consists of the amino acid sequence of SEQ ID NO: 137.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 137.
  • the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 137.
  • the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 139.
  • the CAR includes or consists of the amino acid sequence of SEQ ID NO: 139.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 139.
  • the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 139.
  • the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 141.
  • the CAR includes or consists of the amino acid sequence of SEQ ID NO: 141.
  • the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 141.
  • the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 141.
  • the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
  • the CAR construct further includes an inducible protein that can be used to eliminate CAR expressing cells (e.g., a “suicide” domain).
  • the inducible protein can be activated in the event of off target side effects (or on target/off tumor effects), such as cytokine release syndrome (“cytokine storm”).
  • expression of the suicide domain is inducible by a small molecule, such as tetracycline or doxycycline (a “TET ON” system) or rapamycin. See, e.g., Gargett et al., Front. Pharmacol. 5:235, 2014; Stavrou et al., Mol. Ther. 6:1266-1276, 2018.
  • the suicide domain is inducible by a Fas domain inducible system.
  • the inducible suicide domain is located N-terminal or C-terminal to the antigen binding domain of the CAR, while in other examples, the inducible suicide domain is located C-terminal to the intracellular signaling domain (for example, the CD3 ⁇ domain) of the CAR.
  • the inducible suicide domain is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide).
  • the inducible suicide domain includes Caspase 9.
  • the CAR construct further includes a domain that increases survival or persistence of a modified immune cell expressing the CAR.
  • the domain is an intracellular domain from a cytokine receptor, for example, an intracellular domain from interleukin (IL) receptor 15, IL- 12 receptor, or IL- 18 receptor.
  • the domain is an intracellular domain from a growth factor receptor, such as an intracellular domain from CD40, NKG2D, NKP40, or NKP46.
  • the domain is located C-terminal to the intracellular signaling domain (for example, the CD3 ⁇ domain) of the CAR (e.g. is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide)).
  • the CAR further includes one or more additional antigen binding domains that specifically bind to an antigen that is co-expressed with HERV-E on tumor cells, or a T cell may express an additional CAR with an additional antigen binding domain.
  • the additional antigen binding domain that specifically binds to a renal cell carcinoma antigen e.g., an antigen binding domain that specifically binds to carbonic anhydrase IX (CAIX), see, e.g., US Pat. No. 10,870,705 and Lo et al., Oncolytics 1 :14003, 2014).
  • CAIX carbonic anhydrase IX
  • a CAIX logic gate CAR may be utilized.
  • Logic Gate CAR T cells are T cells expressing two or more CARs targeting separate tumor antigens. Based on Boolean logic, they can be categorized into “AND,” “OR,” and “NOT” CAR T cells. “AND” CAR T cells are only activated by the presence of both antigens; “OR” CAR T cells may be activated by either antigen; “NOT” CAR T cells are activated only when one antigen is absent. Anti-CAIX CAR T cells cause liver toxicity due to the on-target toxicity by attacking bile duct epithelial cells, as these cells express low-level of CAIX. The “AND”/“OR” Gate CAR T cells simultaneously targeting HERV-E via a disclosed scFV and CAIX may solve this problem, and may also improve the safety profile of the disclosed CARs.
  • Additional antigens that may be targeted by the CAR include, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, CD70, hypoxia-inducible protein 2 (HIG2), von Hippel- Lindau protein (VHL) 5T4 protein, mucin 1 (MUC1), Wilms’ tumor 1 (WT1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), AXL, alpha-fetoprotein, melanoma-associated antigen 4 (MAGEA4), epidermal growth factor receptor (EGFR), mesothelin, programmed cell death 1 ligand 1 (PD-L1), CD56, CD10, CEA cell adhesion molecule 5, epithelial cell adhesion molecule (EPCAM), Erb-B2 receptor tyrosine kinase 3 (ERBB3), ERBB5, CD138, CD133, CD86, CD123, c-Met, TNF superfamily member 13b (TACI), TNF
  • the CAR includes a nucleic acid encoding a VH including the HCDR1, HCDR2, and HCDR3, and a VL including the LCDR1, LCDR2, and LCDR3 of any one of the TAY058, TAY051, TAY052, TAYO53, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAYO58 antibody includes the nucleic acid sequences of SEQ ID NOs: 9 and 10, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY051 antibody and includes the nucleic acid sequences of SEQ ID NOs: 25 and 26, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY052 antibody and includes the nucleic acid sequences of SEQ ID NOs: 41 and 42, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY053 antibody and includes the nucleic acid sequences of SEQ ID NOs: 57 and 58, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY054 antibody and includes the nucleic acid sequences of SEQ ID NOs: 73 and 74, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY059 antibody and includes the nucleic acid sequences of SEQ ID NOs: 89 and 90, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY049 antibody and includes the nucleic acid sequences of SEQ ID NOs: 105 and 106, respectively.
  • the CAR includes a nucleic acid encoding the VH and VL of the TAY050 antibody and includes the nucleic acid sequences of SEQ ID NOs: 121 and 122, respectively.
  • a CAR is encoded by the nucleic acid sequence of any one of SEQ ID NO: 129, 136, 138, and 140.
  • the functional variant can be at least about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR or domain. Substitutions can be made, for example, in one or more of the extracellular targeting domain, hinge domain, transmembrane domain, and intracellular domains.
  • the functional variant includes the amino acid sequence of the parent CAR or domain with at least one conservative amino acid substitution (such as up to 10 conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions).
  • the functional variant includes the amino acid sequence of the parent CAR or domain with at least one non-conservative amino acid substitution (such as up to 10 non-conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non- conservative substitutions).
  • the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved or increased as compared to the parent CAR or domain.
  • the CARs or one or more domains thereof can in some examples, include one or more synthetic amino acids in place of one or more naturally-occurring amino acids.
  • Such synthetic or non-naturally occurring amino acids include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, P-phenylserine P-hydroxyphenylalanine, phenylglycine, a -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4- tetrahydroisoquinoline- 3-carboxylic acid, aminomalonic acid
  • the CARs may also include one or more D-amino acids.
  • the CARs may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N- acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • a nucleic acid molecule encoding a disclosed CAR is included in an expression vector (such as a viral vector) for expression in a host cell, such as a T cell or NK cell.
  • the expression vector includes a promoter operably linked to the nucleic acid molecule encoding the CAR.
  • the promoter is a EF-1A promoter. Additional expression control sequences, such as one or more enhancers, transcription and/or translation terminators, and initiation sequences (such as a Kozak sequence) can also be included in the expression vector.
  • a nucleic acid encoding a CAR provided herein is included in a viral vector.
  • virus vectors examples include retrovirus e.g., MoMLV or lentivirus), adenovirus, adeno- associated virus, vaccinia virus, and fowlpox vectors.
  • retrovirus e.g., MoMLV or lentivirus
  • adenovirus e.g., adeno-associated virus
  • vaccinia virus vaccinia virus
  • fowlpox vectors e.g., vaccinia virus
  • the CAR encoding nucleic acid is included in a MoMLV or lentiviral vector.
  • the vector may be a DNA vector (such as a plasmid).
  • the cells include T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages.
  • the cells are T cells, NK cells, or macrophages expressing a disclosed CAR.
  • the immune cells are transduced or transfected with one or more expression vectors including one or more nucleic acids, including nucleic acids encoding a CAR provided herein.
  • the vector or a DNA encoding the construct
  • the CAR-expressing immune cells are generated using Sleeping Beauty transposon system (see, e.g., Monjezi et al., Leukemia 31 : 186- 194, 2017; Hodge et al., Hum. Gene Ther.
  • the transduced or transfected cells are isolated T cells (such as primary T cells or T cells obtained from a subject), isolated NK cells (such as primary NK cells or NK cells obtained from a subject), isolated NKT cells, isolated DNT cells, isolated neutrophils, or isolated macrophages (such as primary macrophages or macrophages obtained from a subject).
  • the T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages are obtained from peripheral blood.
  • T cells, NK cells, NKT cells, or DNT cells are also enriched, purified, and/or expanded from a sample from a subject, for example before and/or after transduction with one or more of the disclosed CARs.
  • the cell is an NK-92 cell.
  • NK-92 cells are a NK cell line derived from a patient with non- Hodgkin’s lymphoma (e.g., ATCC® CRL-2407TM). This cell line has properties of activated NK cells (see, e.g., Gong et al., Leukemia 8:652-658, 1994).
  • the cell is an NK-92MI cell (e.g., ATCC® CRL-2408TM).
  • the NK-92MI cell line is an interleukin-2 (IL-2) independent NK cell line, derived from NK-92, which stably expresses human IL-2 (see, e.g., Tam et al., Hum. Gene Ther. 10:1359-1373, 1999).
  • NK-92 or NK-92MI cells expressing a CAR can be used as an “off the shelf” immunotherapy, since autologous NK cells do not have to be produced for each subject.
  • Other NK cell lines that can be used with the CARs (or other nucleic acids) described herein include NKL, KHYG-1, and YTS cells.
  • immune cells are transduced with a vector encoding a CAR.
  • cells expressing the CAR can be detected and/or enriched, for example, by flow cytometry using a labeled antibody that binds to CD34t (if present in the CAR construct).
  • the transduced cells (such as NK cells or T cells) are expanded, for example, by cell culture for a period of time following transduction. In some examples, some or all of the modified cells are cryopreserved for later use.
  • multi-specific T cell or NK cell engager constructs including one or more antibody or antigen binding fragment thereof provided herein.
  • the construct is a bispecific T cell engager (BiTE) construct including at least one scFv including HCDRs and LCDRs from an antibody provided herein and an scFv that specifically binds to CD3 linked by a peptide linker.
  • the BiTE includes an scFv including VH domain and VE domain from antibody TAYO58 or TAY051; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized..
  • the construct is a bispecific killer cell engager (BiKE) or trispecific killer cell engager (TRiKE) construct (see, e.g., Davis et al., Semin. Immunol. 31 :64-75, 2017).
  • the BiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and an scFv that specifically binds to CD16.
  • the TRiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and scFvs that specifically bind to CD 16 and a third target (such as NKG2A, KIR2DL1, TIGIT, or PD- 1).
  • the TRiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and scFvs that specifically bind to CD 16 and a third molecule (such as IL-15, IL-7, or IL-21).
  • the BiKE or TRiKE includes an scFv including VH domain and VL domain from antibody TAY058 or TAY051 ; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized.
  • the conjugate includes an antibody or antigen binding fragment including HCDRs and LCDRs from an antibody provided herein linked to an effector, such as monomethyl auristatin E (MMAE), MMAF, MED-2460, auristatin phenylalanine phenylenediamine (AFP), maytansinoid, tubulysin, calicheamicin, duocarmycin, benzodiazepines, camptothecin analogues, or doxorubicin.
  • the antibody may be produced in the format of IgGl, IgG2, IgG3, or IgG4 and linked to an effector.
  • the effector could be linked at a ratio of 1, 2, 3, 4, 5, 6, 7, or 8 molecules per antibody.
  • the antibody and effector may be linked by a cleavable (e.g. , protease cleavable) or a non- cleavable linker (see, e.g., Beck et al., Nat. Rev. Drug Disco. 16:315-337, 2017).
  • cleavable e.g. , protease cleavable
  • non- cleavable linker see, e.g., Beck et al., Nat. Rev. Drug Disco. 16:315-337, 2017.
  • exemplary linkers include thioether (e.g. , succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC)), maleimidocaproyl, P-glucuronide, disulfide, acid-sensitive linkers, or peptide linkers.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexan
  • the antibody and effector may be linked by chemical reactions such as thiosuccinimide linkage at natural amino acid residuals such as lysine, or cysteine.
  • the linkage may be also formed at engineered unnatural amino acids such as p- acetylphenylalanine (pAcPhe) either with or without the presence of enzymes such as formylglycine-generating enzyme.
  • pAcPhe p- acetylphenylalanine
  • the linkage may form at the N-glycosylated Asn297 site by glycocojugation through maleimide chemistry, or other chemoenzymatic methods.
  • the conjugate includes an antibody or antigen binding fragment including HCDRs and LCDRs from an antibody provided herein linked (e.g., conjugated) to a cytotoxic molecule (such as an anti-cancer drug).
  • a cytotoxic molecule such as an anti-cancer drug.
  • the antibody-effector conjugate includes an scFv including the VH domain and the VL domain from antibody TAY058 or TAY051 ; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized.
  • the methods include administering to the subject an antibody, antigen binding fragment, or antibody-effector conjugate provided herein (such as an effective amount of an antibody, antigen binding fragment, or antibody-effector conjugate provided herein).
  • the antibody, antigen binding fragment, or antibody-effector conjugate is administered to the subject intravenously or subcutaneously.
  • the methods include administering to the subject a cell (such as a T cell or NK cell) expressing a CAR provided herein (such as effective amount of a CAR-T or CAR-NK cell expressing a CAR provided herein).
  • the cell is administered to the subject intravenously
  • the methods include administering to the subject a BiTE provided herein.
  • the BiTE is administered to the subject intravenously or subcutaneously.
  • the subject has ccRCC, advanced ccRCC, or metastatic ccRCC.
  • the subject has a ccRCC expressing a HERV-E envelope.
  • compositions include one or more of the disclosed antibodies, antigen binding fragments, conjugates, CARs, nucleic acids encoding such molecules, or CAR-expressing immune cells that are disclosed herein in a pharmaceutically acceptable carrier.
  • the compositions are useful, for example, for the inhibition or treatment of a renal cell carcinoma.
  • the compositions can be prepared in unit dosage forms, such as in a kit, for administration to a subject. The amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes.
  • the composition can be formulated for systemic or local administration. In one example, the composition is formulated for intravenous administration or subcutaneous administration.
  • compositions for administration can include a solution of the antibody, antigen binding fragment, CAR, conjugate, nucleic acid encoding such molecules, or CAR-expressing immune cells dissolved or suspended in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a pharmaceutically acceptable carrier such as an aqueous carrier.
  • aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by any suitable technique.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of antibody (or other agent) in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like in accordance with the particular mode of administration selected and the subject’s
  • Antibodies, or an antigen binding fragment thereof or a nucleic acid encoding such molecules can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration.
  • the antibody solution, or an antigen binding fragment or a nucleic acid encoding such antibodies or antigen binding fragments can then be added to an infusion bag containing 0.9% sodium chloride.
  • Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules can be administered by slow infusion, rather than in an intravenous push or bolus.
  • Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the active agent as a central core. In microspheres, the active agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.
  • Polymers can be used for ion-controlled release of the compositions disclosed herein. Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug.
  • Antibodies, antigen binding fragments, and conjugates thereof can be administered by intravenous infusion. Doses of the antibody, antigen binding fragment, or conjugate vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the antibody or antigen binding fragment can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg.
  • the antibody or antigen binding fragment is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or antigen binding fragment is administered weekly, every two weeks, every three weeks or every four weeks.
  • a subject is administered DNA or RNA encoding a disclosed antibody or antigen binding fragment to provide in vivo antibody production, for example using the cellular machinery of the subject.
  • Any suitable method of nucleic acid administration may be used; nonlimiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637.
  • U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism.
  • One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof can be placed under the control of a promoter to increase expression.
  • the methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.
  • a nucleic acid encoding a disclosed antibody, or antigen binding fragment thereof is introduced directly into tissue.
  • the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOSTM Gene Gun.
  • the nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.
  • DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 5 pg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).
  • Single or multiple administrations of a composition including a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules can be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage can be administered once, but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to treat a subject with cancer without producing unacceptable toxicity to the patient.
  • the composition includes about 10 4 to 10 12 of CAR-expressing immune cells (such as CAR-NK cells or CAR-T cells), for example, about 10 4 -10 8 cells, about 10 6 -10 8 cells, or about 10 6 - 10 12 cells.
  • the composition may be prepared such that about 10 4 to IO 10 modified immune cells cells/kg (such as about 10 4 , 10 s , 10 6 , 10 7 , 10 8 , 10 9 , or IO 10 cells/kg) are administered to a subject.
  • the composition includes at least 10 4 , 10 s , 10 6 , or 10 7 CAR-NK cells or CAR-T cells.
  • the population of modified NK cells or modified T cells is typically administered parenterally, for example intravenously; however, injection or infusion to a tumor or close to a tumor (local administration) or administration to the peritoneal cavity can also be used. Appropriate routes of administration can be determined based on factors such as the subject, the condition being treated, and other factors.
  • the CAR-expressing immune cells further express one or more (such as 1, 2, 3, or more) additional CARs.
  • the CAR-expressing immune cells express a second CAR targeting a second antigen expressed by a RCC cell, for example, CAIX.
  • CAR-NK cells or CAR-T cells can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently.
  • a skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.
  • the subject is also administered at least one, at least one, at least two, at least three, or at least four cytokine(s) (such as IL-2, IL-7, IL-15, IL-21, and/or IL-12) to support survival and/or growth of the CAR-expressing cells.
  • the at least one cytokine includes IL-2 and IL-15.
  • the cytokine(s) are administered before, after, or substantially simultaneously with the CAR-expressing cells.
  • at least one cytokine e.g., IL-2
  • Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages of the disclosed compositions for use in humans.
  • the dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the effective dose can be determined from cell culture assays and animal studies.
  • Isolating and validating scFVs from phage library Peptides were synthesized to cover the surface and transmembrane protein of the envelope (SU and TM) and CR derived human endogenous retrovirus type E (CT-RCC HERV-E). Peptides were used as antigens to screen against the human antibody phage library (Dynamic Precision Library Platform). Positive binding scFVs were further validated against yeast expressing SU, TM and CR and the selected scFVs were finally expressed in the format of IgGl.
  • RCC cells and non-RCC adherent cells were detached using 5 mM EDTA in Ca 2+ and Mg 2+ free PBS and 0.5-1 x 10 6 cells were used for each staining.
  • Cells were first stained with fixable aqua dead cell stain kit (Thermo Fisher) in PBS for 30 minutes at room temperature, washed, and resuspended in 100 pL FACs buffer. TAY Abs or anti-GFP control Ab were added and stained for 30 minutes at 4°C.
  • Cells were washed with FACs buffer, stained with PE-Goat anti-Human IgG Fc specific (Jackson) for 20 minutes at 4°C. After finishing staining, cells were finally resuspended in 150 pL FACs buffer and analyzed by LSRFortessaTM cell analyzer (BD).
  • BD LSRFortessaTM cell analyzer
  • Antibodies were identified by screening a human antibody phage library with TM, SU, and CR proteins. The antibodies were screened against RCC and non-RCC cell lines by flow cytometry (FIG. 1). Cell lines utilized are identified in Table 3.
  • TAY057 showed non-specific binding against monocytes (FIG. 2B).
  • TAY052 and TAY055 showed nonspecific binding to normal human tissue (FIG. 3).
  • a summary of the candidate antibodies is shown in Table 4.
  • Antibodies were validated using CHO cells expressing CT-RCC HERV-E Env proteins.
  • pT2/HB transposon plasmids encoding the CARs (150 pg/mL) and pCMV(CAT)T7-SB100 transposase plasmids (50 pg/mL) were added for each electroporation based on optimization experiments.
  • the electroporation was performed using ExPERT ATxTM (MaxCyte). Cells were returned to the incubator 48 hours and resuspended at desired concentration with fresh TexMACSTM complete medium. Medium changes were done every other day depending on cell growth.
  • CD34 + CAR T cells were sorted using anti-human CD34 magnetic beads (Miltenyi). The percentage of CD34 + cells before and after sorting was measured using another anti-human CD34 mAb (Miltenyi, clone AC 136) binding a different epitope than the one recognized by anti-human CD34 magnetic beads.
  • the sorted cells were expanded with ImmunoCultTM Human CD3/CD28/CD2 T cell activator at the concentration of 12.5 pL/mL following the manufacture’s protocol. After expansion, CAR T cells were used for phenotyping and functional assays.
  • % lysis (1- (Calcein AM Count_treated)/(Calcein AM Count_control))xl00%.
  • GFP GFP was used to measure the live cell after coculture.
  • CHO cells were seeded into 96-well black wall microplate 72 hours before toxicity assay at the optimal density from preliminary experiments. Cells were scanned by Celigo image cytometer to calculate the cell numbers.
  • TAYO58 BBZ CAR and the control FMC63 BBZ CAR were stably expressed in T cells post electroporation; CAR T cells could be further enriched to a very high purity based on CD34 magnetic sort, as shown in FIG. 6.
  • the CAR T cells specifically killed CHO cells expressing HERV-E TM. Raji cells were used as a positive control to validate the killing capability of FMC63 BBZ CAR T cells (FIG. 7).
  • TAY VHVL BBZ CARs VH-VL format CARs
  • Each CAR was highly expressed in donor T cells, based on measuring CD34 by flow cytometry (FIG. 13).
  • TAY052 VHVL BBZ CAR caused fratricide, likely due to the nonspecific binding of TAY052 scFv (FIG. 14).
  • the ability of TAY BBZ CAR-T cells and TAY VHVL BBZ CAR-T cells to kill HERV-E+ RCC cell lines was tested (FIGS. 15A-15H).
  • TAY049 CAR-T cells FIG. 15B
  • TAY050 CAR-T cells FIG.
  • This example describes particular methods that can be used to evaluate the disclosed CARs in a clinical trial. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used.
  • Patients with metastatic RCC are recruited for a single-arm Phase I trial. The study is planned based on a Phase I 3+3 dose escalation design.
  • the maximum tolerated dose (MTD) is defined as the highest dose at which 0 or 1 patient in six has experienced a dose limiting toxicity (DLT).
  • DLT dose limiting toxicity
  • Patients with evaluable advanced/metastatic ccRCC are recruited in up to 3 dose levels.
  • T cells are isolated from 15-20 liter leukapheresis of patients and TAY BBZ or TAY VHVL BBZ CAR T cells (such as TAY051 BBZ CAR or TAY051 VHVL BBZ CAR) are manufactured using ExPERT ATXTM (MaxCyte) as following the protocol described above at the Department of Transfusion Medicine of NIH.
  • TAY BBZ or TAY VHVL BBZ CAR T cells (such as TAY0 1 BBZ CAR or TAY051 VHVL BBZ CAR) are infused into patients 5 days after a non- myeloablative immunosuppressive conditioning regimen of cyclophosphamide and fludarabine.
  • Patients are treated in 4 different cohorts: IxlO 6 T cells/kg, 5xl0 6 T cells/kg, IxlO 7 T cells/kg, 5xl0 7 T cells/kg.
  • Patients are discharged from the clinical center following neutrophil recovery and return for weekly visits for 6-8 weeks, where they undergo standard evaluations including physical exams and body weight, and routine clinical labs (hematology and electrolytes). Restaging using PET and CT imaging using RECIST criteria will be performed 30 days following the T cell infusion, then every 3 months for the first year, and then every 6 months thereafter until evidence for tumor progression occurs.

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Abstract

Monoclonal antibodies and antigen binding fragments that specifically bind human endogenous retrovirus type E proteins expressed by renal cell carcinoma are provided. Also provided are chimeric antigen receptors including the antibodies or antigen binding fragments. Methods of treating renal cell carcinoma with the antibodies, antigen binding fragments, or immune cells expressing the chimeric antigen receptors are also provided.

Description

HERV-E ANTIBODIES AND METHODS OF THEIR USE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 63/539,170, filed September 19, 2023, which is incorporated by reference in its entirety.
FIELD
This disclosure relates to monoclonal antibodies and antigen binding fragments that specifically bind human endogenous retrovirus type E proteins expressed by renal cell carcinoma, chimeric antigen receptors including the antibodies or antigen binding fragments, and their use for treating renal cell carcinoma.
PARTIES TO JOINT RESEARCH AGREEMENT
This application describes and claims certain subject matter that was developed under a written collaborative research agreement between The United States of America, as represented by The Secretary, Department of Health and Human Services and Adagene Pte. Ltd.
SEQUENCE LISTING INCORPORATION BY REFERENCE
The Sequence Listing is submitted as an XML file in the form of the file named Sequence_Listing_4239-104126-02.xml (129,267 bytes), which was created on September 17, 2024, which is incorporated by reference herein.
BACKGROUND
Renal cell carcinoma (RCC) is a common solid tumor in the United States. The five-year survival of early stage RCC is 92.6%; however, for metastatic RCC (mRCC) this number drops to 11.7%. For patients with localized tumors, 30~40% will eventually develop metastasis after surgical treatment. Thus there is a need for effective treatment options for patients with RCC.
SUMMARY
A transcript derived from human endogenous retrovirus type E (CT-RCC HERV-E) is selectively expressed in clear cell renal cell carcinoma (ccRCC), and encodes an entire envelope gene of this retrovirus. This transcript has one short ORF (CR) and two long ORFs predicted to encode the surface and transmembrane protein of the envelope (SU and TM). Disclosed herein are human monoclonal antibodies (mAbs) that specifically bind to an HERV-E Envelope. Also disclosed are chimeric antigen receptors (CARs) including these mAbs, or an antigen binding fragment thereof, which can be used in the treatment of RCC (e.g. , ccRCC).
Isolated monoclonal antibodies or antigen binding fragments that specifically bind to a human endogenous retrovirus-E Envelope (such as a transmembrane protein of the human endogenous retrovirus-E Envelope) are provided. In some aspects, the antibody or antigen binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l , a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 1 and 2, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 17 and 18, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 33 and 34, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 49 and 50, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 65 and 66, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 81 and 82, respectively; a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 97 and 98, respectively; or a heavy chain variable region and a light chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 113 and 114, respectively, and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
In some aspects, the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 3-8, respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19-24, respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 35-40, respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 51-56 respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 67- 72, respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 83-88, respectively; the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 99-104, respectively; or the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 115-120, respectively, and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
In further aspects, the antibody comprises VH and VL domains, wherein the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 1 and 2, respectively (for example, comprise SEQ ID NOs: 1 and 2, respectively); the VH and the Vi. comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 17 and 18, respectively (for example, comprise SEQ ID NOs: 17 and 18, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 33 and 34, respectively (for example, comprise SEQ ID NOs: 33 and 34, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 49 and 50, respectively (for example, comprise SEQ ID NOs: 49 and 50, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 65 and 66, respectively (for example, comprise SEQ ID NOs: 65 and 66, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 81 and 82, respectively (for example, comprise SEQ ID NOs: 81 and 82, respectively); the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 97 and 98, respectively (for example, comprise SEQ ID NOs: 97 and 98, respectively); or the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 113 and 114, respectively (for example, comprise SEQ ID NOs: 113 and 114, respectively), and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
In some aspects, the antigen binding fragment is a Fv, Fab, F(ab’)2, scFv, or scFv2 fragment. In one specific example, the antigen binding fragment is a scFv. In other aspects, a bispecific antibody comprising a disclosed antibody or antigen binding fragment is provided.
In additional aspects, a chimeric antigen receptor (CAR) including a disclosed antibody or antigen binding fragment is provided. In some examples, the CAR includes an scFv including a VH domain and VL domain of a disclosed antibody. The CAR also includes one or more of a leader sequence, a hinge domain, a transmembrane domain, an intracellular domain, or a combination of two or more thereof. In some examples, one or more of the leader sequence, hinge domain, and transmembrane domain is from CD8a. In some examples, the intracellular domain includes a 4- 1BB intracellular domain and a CD3ij intracellular domain. The CAR construct may also include a truncated CD34 domain. In non-limiting examples, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 130, 137, 139, and 141. In additional examples, the CAR may further include a second antibody or second antigen binding fragment.
In further aspects, a conjugate including a disclosed antibody or antigen binding fragment and an effector molecule (such as a detectable marker or a drug) is provided. In other aspects, a bispecific T cell engager including a disclosed antigen binding fragment (such as a disclosed scFv) linked to a second antigen binding fragment (such as a CD3 scFv) is provided.
Nucleic acids encoding the disclosed antibodies or antigen binding fragments are also provided. In some non-limiting examples, the antibody or antigen binding fragment is encoded by VH and VL nucleotide sequences comprising SEQ ID NOs: 9 and 10, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 25 and 26, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 41 and 42, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 57 and 58, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 73 and 74, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 89 and 90, respectively; VH and VL nucleotide sequences comprising SEQ ID NOs: 105 and 106, respectively; or VH and VL nucleotide sequences comprising SEQ ID NOs: 121 and 122, respectively.
In some aspects, nucleic acids encoding the disclosed CARs are also provided. In nonlimiting examples, the CAR is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 129, 136, 138, and 140.
The disclosed nucleic acids, in some examples, are operably linked to a promoter. Also provided are vectors including the disclosed nucleic acids, and host cells including the vectors. In some examples, the nucleic acid or vector includes a nucleic acid sequence encoding a CAR and the host cell is an immune cell (such as a T cell or natural killer cell).
In some examples, compositions including the antibody or antigen binding fragment, the chimeric antigen receptor, the conjugate or BiTE, or the nucleic acid or vector; and a pharmaceutically acceptable carrier are provided. In other examples, the composition includes a host cell including a disclosed nucleic acid and a pharmaceutically acceptable carrier. In particular examples, the host cell is an immune cell (such as a T cell or a natural killer cell) including a nucleic acid encoding a disclosed CAR. In some aspects, methods of treating a subject with renal cell carcinoma are provided. The methods include administering to the subject an effective amount of a disclosed antibody or antigen binding fragment, CAR, conjugate, BiTE, nucleic acids encoding such molecules, or CAR-immune cells (such as CAR-T cells or CAR-NK cells) or a composition thereof. In some examples, the subject has clear cell renal cell carcinoma. In other examples, the subject has a renal cell carcinoma expressing a human endogenous retrovirus-E Envelope.
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a heatmap showing binding of antibody candidates screened against RCC and non- RCC cell lines. Cell lines are listed in Table 3.
FIGS. 2 A and 2B are heatmaps showing binding of candidate antibodies to lymphocytes (FIG. 2A) or monocytes (FIG. 2B).
FIG. 3 is a heatmap showing binding of candidate antibodies to HERV-E+ RCC and normal human tissue.
FIG. 4A is a heatmap showing binding of candidate antibodies to CHO cells expressing the indicated CT-RCC HERV-E Env proteins. FIG. 4B is a schematic diagram of the constructs expressed in the CHO cells.
FIG. 5 is a schematic diagram of an exemplary TAY058 BBZ CAR construct.
FIG. 6 shows expression of TAY058 BBZ CAR in T cells before and after CD34 magnetic sort.
FIG. 7 is a graph showing killing of CHO cells expressing HERV-E TM by TAY058 BBZ CAR T cells, but not HERV-E’ Raji cells.
FIG. 8 shows high expression of the indicated TAY BBZ CARs in T cells, by measuring CD34.
FIG. 9 is a graph showing killing of Raji (CD19+ HERV-E ) cells by the indicated TAY BBZ CAR T cells.
FIGS. 10A and 10B show QC of TAY051 BBZ CAR T cells from three donors tested with Raji cells (CD19+ HERV-E’; FIG. 10A) and RCC-U0K171 cells (CD19’ HERV-E’; FIG. 10B).
FIGS. 11A-1 ID show specific killing of four different HERV-E+ RCC cell lines by TAY051 BBZ CAR T cells.
FIG. 12 is a schematic diagram of an exemplary TAY051 VHVL BBZ CAR construct. FIG. 13 shows high expression of the indicated TAY VHVL BBZ CARs in T cells, by measuring CD34.
FIG. 14 shows analysis of live T cells expressing the indicated TAY VHVL BBZ CARs, indicating that TAY052 VHVL BBZ CAR caused fratricide.
FIGS. 15A-15H show analysis of killing of HERV-E negative or positive RCC cell lines by TAY BBZ CAR (top) and TAY VHVL BBZ CAR (bottom) T cells.
SEQUENCE LISTING
The nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable domain of TAY058: EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWGWIRQAPGKGLEWIGEIYHSG NTNYNPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDVYGGYDFDVWG QGTLVTVSS
SEQ ID NO: 2 is the amino acid sequence of the light chain variable domain of TAY058: DIQLTQSPS SLS AS VGDRVTITCKS GQSLLHSDGHTYLYWYQQKPGKAPKLLIYD AS NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPSTFGQGTKVEIK SEQ ID NOs: 3-5 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY058.
SEQ ID NOs: 6-8 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY058.
SEQ ID NO: 9 is a nucleic acid sequence encoding the heavy chain variable domain of TAY058:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT GAGGCTGAGCTGCGCCGCCAGCGGCTACAGCATCAGCAGCGGCCACTACTGGG GCTGGATCAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCTAC CACAGCGGCAACACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAG CAGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCG AGGACACCGCCGTGTACTACTGCGCCAGGGACGTGTACGGCGGCTACGACTTC GACGTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC
SEQ ID NO: 10 is a nucleic acid sequence encoding the light chain variable domain of TAY058: GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG GGTGACCATCACCTGCAAGAGCGGCCAGAGCCTGCTGCACAGCGACGGCCACA CCTACCTGTACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCT ACGACGCCAGCAACCTGGAGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGC AGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGAGCTACAGCTGGCCCAGCACCTTCGGCCAGGGCACC AAGGTGGAGATCAAG
SEQ ID NOs: 11-13 are nucleic acid sequences encoding HCDR1 , HCDR2, and HCDR3, respectively, of TAY058.
SEQ ID NOs: 14-16 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY058.
SEQ ID NO: 17 is the amino acid sequence of the heavy chain variable domain of
TAY051:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWIGIISPSGGG TNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGIGFAYWGQGTLV TVSS
SEQ ID NO: 18 is the amino acid sequence of the light chain variable domain of TAY051: DIQLTQSPS SLS AS VGDRVTITCQASQDISTYLAWYQQKPGKAPKLLIYAAS SLQS G VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGSTPLTFGQGTKVEIK
SEQ ID NOs: 19-21 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY051.
SEQ ID NOs: 22-24 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY051.
SEQ ID NO: 25 is a nucleic acid sequence encoding the heavy chain variable domain of TAY051:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCATCGGCTTCGCCTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 26 is a nucleic acid sequence encoding the light chain variable domain of
TAY051:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCCAGGCCAGCCAGGACATCAGCACCTACCTGGCCTGGTA
CCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCC
TGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTC
ACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAG
CAGGGCGGCAGCACCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAA G
SEQ ID NOs: 27-29 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY051.
SEQ ID NOs: 30-32 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY051.
SEQ ID NO: 33 is the amino acid sequence of the heavy chain variable domain of
TAY052:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYW1HWVRQAPGKGLEWIG11SPSGG GTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGLGFVYWGQGT LVTVSS
SEQ ID NO: 34 is the amino acid sequence of the light chain variable domain of TAY052: DIQLTQSPS SLS AS VGDRVTITCGASQD VGTAV AW YQQKPGKAPKLLIYDAS SLES G VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGTFPLTFGQGTKVEIK
SEQ ID NOs: 35-37 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY052.
SEQ ID NOs: 38-40 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY052.
SEQ ID NO: 41 is a nucleic acid sequence encoding the heavy chain variable domain of TAY052:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGGCACCAAGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCCTGGGCTTCGTGTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 42 is a nucleic acid sequence encoding the light chain variable domain of
TAY052:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCGGCGCCAGCCAGGACGTGGGCACCGCCGTGGCCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGC
CTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGGGCGGCACCTTCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCA
AG
SEQ ID NOs: 43-45 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY052.
SEQ ID NOs: 46-48 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY052.
SEQ ID NO: 49 is the amino acid sequence of the heavy chain variable domain of
TAYO53:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYW1HWVRQAPGKGLEWIG11SPSGG GTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGIGFAYWGQGTL VTVSS
SEQ ID NO: 50 is the amino acid sequence of the light chain variable domain of TAY053:
DIQLTQSPSSLSASVGDRVTITCRASQDIRSVLAWYQQKPGKAPKLLIYDASSLESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGSFPLTFGQGTKVEIK
SEQ ID NOs: 51-53 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY053.
SEQ ID NOs: 54-56 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY053.
SEQ ID NO: 57 is a nucleic acid sequence encoding the heavy chain variable domain of TAYO53:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC
AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA
GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCATCGGCTTCGCCTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 58 is a nucleic acid sequence encoding the light chain variable domain of
TAYO53:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCAGGGCCAGCCAGGACATCAGGAGCGTGCTGGCCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGC
CTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGGGCGGCAGCTTCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCA
AG
SEQ ID NOs: 59-61 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY053.
SEQ ID NOs: 62-64 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY053.
SEQ ID NO: 65 is the amino acid sequence of the heavy chain variable domain of
TAY054:
EVQLVESGGGLVQPGGSLRLSCAASGYTFSNYW1HWVRQAPGKGLEW1G1ISPSGG
DTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGLGFTYWGQGTL VTVSS
SEQ ID NO: 66 is the amino acid sequence of the light chain variable domain of TAY054: DIQLTQSPSSLSASVGDRVTITCRASQDIRKFLAWYQQKPGKAPKLLIYDASNLETG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYHIWTFGQGTKVEIK
SEQ ID NOs: 67-69 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY054.
SEQ ID NOs: 70-72 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY054.
SEQ ID NO: 73 is a nucleic acid sequence encoding the heavy chain variable domain of TAY054:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTACACCTTCAGCAACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGACACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC
AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA
GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCCTGGGCTTCACCTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 74 is a nucleic acid sequence encoding the light chain variable domain of
TAY054:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCAGGGCCAGCCAGGACATCAGGAAGTTCCTGGCCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAAC
CTGGAGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGGGCTACCACATCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG
SEQ ID NOs: 75-77 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY054.
SEQ ID NOs: 78-80 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY054.
SEQ ID NO: 81 is the amino acid sequence of the heavy chain variable domain of
TAY059:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWIHWVRQAPGKGLEWIGIISPSGGS
TNYAQKFQGRVT1SRDNSKNTLYLQLNSLRAEDTAVYYCARGYLGFAYWGQGTL VTVSS
SEQ ID NO: 82 is the amino acid sequence of the light chain variable domain of TAY059: DIQLTQSPSSLSASVGDRVTITCRASQSISTYLAWYQQKPGKAPKLLIYDASSLESGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYHWPPTFGQGTKVEIK
SEQ ID NOs: 83-85 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY059.
SEQ ID NOs: 86-88 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY059.
SEQ ID NO: 89 is a nucleic acid sequence encoding the heavy chain variable domain of TAY059:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCAGCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC
AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA
GGACACCGCCGTGTACTACTGCGCCAGGGGCTACCTGGGCTTCGCCTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 90 is a nucleic acid sequence encoding the light chain variable domain of
TAY059:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCACCTACCTGGCCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGC
CTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGAGCTACCACTGGCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAGATCA
AG
SEQ ID NOs: 91-93 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY059.
SEQ ID NOs: 94-96 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY059.
SEQ ID NO: 97 is the amino acid sequence of the heavy chain variable domain of
TAY049:
EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYW1HWVRQAPGKGLEW1G1ISPSGG DTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGLGFAYWGQGT LVTVSS
SEQ ID NO: 98 is the amino acid sequence of the light chain variable domain of TAY049: DIQLTQSPSSLSASVGDRVTITCRASQDIRSFLGWYQQKPGKAPKLLIYDASNRATGI PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYHLWTFGQGTKVEIK
SEQ ID NOs: 99-101 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY049.
SEQ ID NOs: 102-104 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY049.
SEQ ID NO: 105 is a nucleic acid sequence encoding the heavy chain variable domain of TAY049:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTACACCTTCAGCGACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGACACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC
AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA
GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCCTGGGCTTCGCCTACTGGGG
CCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 106 is a nucleic acid sequence encoding the light chain variable domain of
TAY049:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCAGGGCCAGCCAGGACATCAGGAGCTTCCTGGGCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAAC
AGGGCCACCGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA GCAGGCCTACCACCTGTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG SEQ ID NOs: 107-109 are nucleic acid sequences encoding HCDR1, HCDR2, and
HCDR3, respectively, of TAY049.
SEQ ID NOs: 110-112 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY049.
SEQ ID NO: 113 is the amino acid sequence of the heavy chain variable domain of
TAY050:
EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYWIHWVRQAPGKGLEWIGIISPSGG
GTNYAQKFQGRVT1SRDNSKNTLYLQLNSLRAEDTAVYYCARGGVGFAYWGQGT LVTVSS
SEQ ID NO: 114 is the amino acid sequence of the light chain variable domain of
TAY050:
DIQLTQSPSSLSASVGDRVTITCRASQSVGSYLAWYQQKPGKAPKLLIYDASSLESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGSFPLTFGQGTKVEIK
SEQ ID NOs: 115-117 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, of TAY050.
SEQ ID NOs: 118-120 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, of TAY050.
SEQ ID NO: 121 is a nucleic acid sequence encoding the heavy chain variable domain of
TAY050:
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGGCTGAGCTGCGCCGCCAGCGGCTACACCTTCAGCGACTACTGGATCCACTG
GGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCATCATCAGCCCCA
GCGGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCAGC
AGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGGCGGCGTGGGCTTCGCCTACTGGG GCCAGGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 122 is a nucleic acid sequence encoding the light chain variable domain of
TAY050:
GACATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAG
GGTGACCATCACCTGCAGGGCCAGCCAGAGCGTGGGCAGCTACCTGGCCTGGT
ACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGC
CTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCA
GCAGGGCGGCAGCTTCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCA AG
SEQ ID NOs: 123-125 are nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3, respectively, of TAY050.
SEQ ID NOs: 126-128 are nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3, respectively, of TAY050.
SEQ ID NO: 129 is a nucleic acid sequence encoding an exemplary TAY058 BBZ CAR construct:
GCCACCATGGCATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGC
ATGCTGCCAGACCTGACATCCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCT
CTGTGGGCGACAGAGTGACCATCACATGCAAGAGCGGCCAGAGCCTGCTGCAC
TCTGATGGCCACACATACCTGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCCT
AAGCTGCTGATCTACGATGCCAGCAACCTGGAAACCGGCGTGCCCAGCAGATTT
TCTGGCAGCGGAAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCT
GAGGACTTCGCCACCTACTACTGCCAGCAGTCCTACAGCTGGCCTAGCACATTT
GGCCAGGGCACCAAGGTGGAAATCAAAGGCGGCGGAGGATCTGGCGGAGGTG
GAAGTGGCGGAGGCGGATCTGAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTG
GTTCAACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTACTCTATC
AGCTCTGGCCATTACTGGGGCTGGATCAGACAGGCCCCTGGCAAAGGACTGGA
ATGGATCGGCGAGATCTACCACAGCGGCAACACCAACTACAACCCCAGCCTGA
AGTCCCGCGTGACCATCTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAG
CTGAACAGCCTGAGAGCCGAGGATACCGCCGTGTACTACTGCGCCAGAGATGT
GTACGGCGGCTACGACTTTGATGTGTGGGGCCAGGGAACCCTGGTCACCGTTTC
TTCTACCACAACCCCAGCACCTCGGCCCCCTACACCAGCACCCACCATCGCCTC
TCAGCCTCTGAGCCTGCGGCCAGAGGCCTGTAGACCAGCAGCAGGAGGAGCAG
TGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTACATCTGGGCACCTCTGG
CAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGC GGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTTATGAGGCCTGTGC
AGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCAGAAGAGGAGGAG
GGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGCAGCGCCGATGCACCTGCATAC
CAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGA
GTATGACGTGCTGGATAAGAGGCGGGGCCGGGACCCCGAGATGGGAGGCAAGC
CTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAA
GATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCA
AGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACATAT
GATGCCCTGCACATGCAGGCCCTGCCACCCAGA
SEQ ID NO: 130 is the amino acid sequence of an exemplary TAY058 BBZ CAR construct:
MALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCKSGQSLLHSDGHT
YLYWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
QQSYSWPSTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS
CAASGYSISSGHYWGWIRQAPGKGLEWIGEIYHSGNTNYNPSLKSRVTISRDNSKN
TLYLQLNSLRAEDTAVYYCARDVYGGYDFDVWGQGTLVTVSSTTTPAPRPPTPAP
TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK
RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ
GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 131 is a nucleic acid sequence encoding a furin-P2A sequence and truncated CD34:
AGGCGCAAGAGGTCCGGATCTGGAGCAACCAACTTTAGCCTGCTGAAGCAGGC
AGGCGACGTGGAGGAGAATCCTGGACCTCCAAGGGGATGGACAGCCCTGTGCC
TGCTGTCCCTGCTGCCATCTGGCTTCATGAGCCTGGATAACAATGGCACAGCCA
CCCCTGAGCTGCCAACACAGGGCACCTTTTCCAACGTGTCTACCAATGTGAGCT
ACCAGGAGACAACCACACCATCCACACTGGGAAGCACCTCCCTGCACCCCGTG
AGCCAGCACGGAAACGAGGCAACCACAAATATCACCGAGACAACAGTGAAGTT
CACATCTACCAGCGTGATCACATCCGTGTATGGCAACACCAATAGCTCCGTGCA
GAGCCAGACATCCGTGATCTCTACCGTGTTTACCACACCCGCCAACGTGAGCAC
CCCTGAGACAACCCTGAAGCCATCCCTGTCTCCCGGCAATGTGAGCGACCTGTC
CACCACAAGCACATCCCTGGCCACATCCCCCACCAAGCCTTACACCTCTAGCTC
CCCTATCCTGTCTGATATCAAGGCCGAGATCAAGTGCAGCGGCATCCGGGAGGT
GAAGCTGACACAGGGCATCTGCCTGGAGCAGAACAAGACCTCTAGCTGTGCCG AGTTCAAGAAGGACAGGGGAGAGGGCCTGGCACGGGTGCTGTGCGGCGAGGA
GCAGGCCGACGCCGATGCCGGCGCCCAGGTGTGCTCCCTGCTGCTGGCACAGTC
TGAGGTGCGGCCACAGTGTCTGCTGCTGGTGCTGGCCAATAGAACCGAGATCTC
CTCTAAGCTGCAGCTGATGAAGAAGCACCAGAGCGATCTGAAGAAGCTGGGCA
TCCTGGACTTTACCGAGCAGGATGTGGCCAGCCACCAGTCTTACAGCCAGAAGA
CACTGATCGCCCTGGTGACCTCCGGCGCCCTGCTGGCCGTGCTGGGCATCACCG
GCTATTTTCTGATGAATCGGAGGTCTTGGAGCCCTACCGGGGAACGACTGGAAC TGGAACCCTGA
SEQ ID NO: 132 is the amino acid sequence of a furin-P2A and truncated CD34 protein:
RRKRSGSGATNFSLLKQAGDVEENPGPPRGWTALCLLSLLPSGFMSLDNNGTATPE
LPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITS
VYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKP
YTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEE
QADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILD
FTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLELEP
SEQ ID NOs: 133 and 134 are amino acid sequences of HERV-E TM epitopes.
SEQ ID NO: 135 is the amino acid sequence of an exemplary glycine- serine linker:
GGGGSGGGGSGGGGS
SEQ ID NO: 136 is a nucleic acid sequence encoding an exemplary TAY058 VHVL CAR construct:
GCCACCATGGCATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGC
ATGCTGCTAGACCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGACTTGTTCAGC
CTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTACAGCATCAGCTCTG
GACATTACTGGGGCTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGATC
GGCGAGATCTACCACAGCGGCAACACCAACTACAACCCCAGCCTGAAGTCCAG
AGTGACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACA
GCCTGAGAGCCGAGGATACCGCCGTGTACTACTGCGCCAGAGATGTGTACGGC
GGCTACGACTTTGATGTGTGGGGCCAGGGAACCCTGGTCACAGTTTCTAGCGGA
GGCGGAGGATCTGGTGGTGGTGGCTCTGGTGGCGGCGGATCTGATATTCAGCTG
ACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACAATTAC
CTGCAAGAGCGGCCAGAGCCTGCTGCACAGTGATGGCCACACATACCTGTACT
GGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGATGCCAGC
AACCTGGAAACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGAC
TTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGC CAGCAGTCCTACAGCTGGCCTAGCACATTTGGCCAGGGCACCAAGGTGGAAAT
CAAGACCACAACCCCAGCACCTCGGCCCCCTACACCAGCACCCACCATCGCCTC
TCAGCCTCTGAGCCTGCGGCCAGAGGCCTGTAGACCAGCAGCAGGAGGAGCAG
TGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTACATCTGGGCACCTCTGG
CAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGC
GGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTTATGAGGCCTGTGC
AGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCAGAAGAGGAGGAG
GGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGCAGCGCCGATGCACCTGCATAC
CAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGA
GTATGACGTGCTGGATAAGAGGCGGGGCCGGGACCCCGAGATGGGAGGCAAGC
CTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAA
GATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCA
AGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACATAT
GATGCCCTGCACATGCAGGCCCTGCCACCCAGA
SEQ ID NO: 137 is the amino acid sequence of an exemplary TAY058 VHVL CAR construct:
MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYW
GWIRQAPGKGLEWIGEIYHSGNTNYNPSLKSRVTISRDNSKNTLYLQLNSLRAEDT
AVYYCARDVYGGYDFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLS
ASVGDRVTITCKSGQSLLHSDGHTYLYWYQQKPGKAPKLLIYDASNLETGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQSYSWPSTFGQGTKVEIKTTTPAPRPPTPAPTI
ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 138 is a nucleic acid sequence encoding an exemplary TAY051 BBZ CAR construct:
GCCACCATGGCATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGC
ATGCTGCCAGACCTGACATCCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCT
CTGTGGGCGACAGAGTGACCATTACCTGTCAGGCCAGCCAGGACATCAGCACC
TACCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTAT
GCCGCTAGCTCTCTGCAGTCTGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCT
GGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACC
TACTACTGTCAGCAAGGCGGCAGCACCCCTCTGACATTTGGCCAGGGAACAAA GGTGGAAATCAAAGGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGC
GGATCTGAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTGGTTCAACCTGGCGG
ATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACTGGAT
CCACTGGGTCCGACAGGCCCCTGGCAAAGGACTGGAATGGATCGGCATCATCA
GCCCTAGTGGCGGCGGAACAAACTACGCCCAGAAATTCCAGGGCCGCGTGACC
ATCAGCCGGGACAACTCTAAGAACACCCTGTACCTGCAGCTGAACAGCCTGAG
AGCCGAGGATACCGCCGTGTACTATTGTGCCAGAGGCGGCATCGGCTTTGCCTA
TTGGGGACAGGGAACCCTGGTCACCGTTAGCTCTACCACAACCCCAGCACCTCG
GCCCCCTACACCAGCACCCACCATCGCCTCTCAGCCTCTGAGCCTGCGGCCAGA
GGCCTGTAGACCAGCAGCAGGAGGAGCAGTGCACACCAGAGGCCTGGACTTCG
CCTGCGATATCTACATCTGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGC
TGAGCCTGGTCATCACCCTGTACTGCAAGCGGGGCAGAAAGAAGCTGCTGTAT
ATCTTCAAGCAGCCCTTTATGAGGCCTGTGCAGACAACCCAGGAGGAGGACGG
CTGCTCCTGTAGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGA
AGTTTTCTCGCAGCGCCGATGCACCTGCATACCAGCAGGGACAGAATCAGCTGT
ATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTATGACGTGCTGGATAAGAGG
CGGGGCCGGGACCCCGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGG
AGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAG
ATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACC
AGGGCCTGTCCACAGCCACCAAGGACACATATGATGCCCTGCACATGCAGGCC
CTGCCACCCAGA
SEQ ID NO: 139 is the amino acid sequence of an exemplary TAY051 BBZ CAR construct:
MALPVTALLLPLALLLHAARPDIQLTQSPSSLSASVGDRVTITCQASQDISTYLAWY
QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGST
PLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFT
FSSYWIHWVRQAPGKGLEWIGIISPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNS
LRAEDTAVYYCARGGIGFAYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEA
CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG
RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 140 is a nucleic acid sequence encoding an exemplary TAY051 VHVL CAR construct: GCCACCATGGCATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGC
ATGCTGCTAGACCTGAGGTGCAGCTGGTGGAATCTGGCGGAGGACTTGTTCAGC
CTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCT
ATTGGATCCACTGGGTCCGACAGGCCCCTGGCAAAGGACTGGAATGGATCGGC
ATCATCAGCCCTAGCGGCGGAGGCACAAATTACGCCCAGAAATTCCAGGGCAG
AGTGACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGCTGAACA
GCCTGAGAGCCGAGGATACCGCCGTGTACTATTGTGCCAGAGGCGGCATCGGC
TTTGCCTATTGGGGACAGGGAACCCTGGTCACCGTTTCTAGCGGAGGCGGAGGA
TCTGGTGGTGGTGGCTCTGGTGGCGGCGGATCTGATATTCAGCTGACACAGAGC
CCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACAATTACCTGCCAGGCC
AGCCAGGACATCAGCACATACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGC
CCCTAAGCTGCTGATCTACGCTGCTAGCTCTCTGCAGTCTGGCGTGCCCAGCAG
ATTTTCTGGCAGCGGCAGCGGAACCGACTTCACCCTGACCATATCTAGCCTGCA
GCCTGAGGACTTCGCCACCTACTACTGTCAGCAAGGCGGCAGCACCCCTCTGAC
ATTTGGCCAGGGAACAAAGGTGGAAATCAAGACCACAACCCCAGCACCTCGGC
CCCCTACACCAGCACCCACCATCGCCTCTCAGCCTCTGAGCCTGCGGCCAGAGG
CCTGTAGACCAGCAGCAGGAGGAGCAGTGCACACCAGAGGCCTGGACTTCGCC
TGCGATATCTACATCTGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTG
AGCCTGGTCATCACCCTGTACTGCAAGCGGGGCAGAAAGAAGCTGCTGTATATC
TTCAAGCAGCCCTTTATGAGGCCTGTGCAGACAACCCAGGAGGAGGACGGCTG
CTCCTGTAGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGT
TTTCTCGCAGCGCCGATGCACCTGCATACCAGCAGGGACAGAATCAGCTGTATA
ACGAGCTGAATCTGGGCCGGAGAGAGGAGTATGACGTGCTGGATAAGAGGCGG
GGCCGGGACCCCGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGG
GCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATC
GGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGG
GCCTGTCCACAGCCACCAAGGACACATATGATGCCCTGCACATGCAGGCCCTGC
CACCCAGA
SEQ ID NO: 141 is the amino acid sequence of an exemplary TAY051 VHVL CAR construct:
MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIH
WVRQAPGKGLEWIGIISPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDT
AVYYCARGGIGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASV
GDRVTITCQASQDISTYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQGGSTPLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
DETAILED DESCRIPTION
I. Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
Antibody and Antigen Binding Fragment: An immunoglobulin, antigen binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), such as a human endogenous retrovirus E (HERV-E) protein. The term “antibody” encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen binding activity.
Non- limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multi-specific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer-Verlag, 2010). Antibodies also include genetically engineered forms, such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).
An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally- occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda ( ) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen. References to “Vu” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.
The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, e.g., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991 ; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; also referred to as the “Chothia” numbering scheme), and Lefranc el al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27(l):55-77, 2003; referred to as the “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1 , LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.
In some aspects, a disclosed antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutation) to increase half-life (see, e.g., Sievers et al., Curr. Opin. HIV AIDS 10: 151-159, 2015).
A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. See, for example, Greenfield (Ed.), Antibodies: A Laboratory’ Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.
A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one aspect, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, in some examples, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.
A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and one or more CDRs and/or framework regions from another human antibody.
A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some aspects, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: Cold Spring Harbor Laboratory Press, 2004; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).
Autologous: Refers to tissues, cells or nucleic acids taken from an individual’s own tissues. For example, in an autologous transfer or transplantation of immune cells (e.g., T cells or NK cells), the donor and recipient are the same person. Autologous (or “autogeneic” or “autogenous”) is related to self, or originating within an organism itself.
Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab or scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.
CD34: A cell surface glycoprotein that functions as a cell-cell adhesion molecule. CD34 is a single-pass transmembrane protein with a highly glycosylated extracellular domain, a transmembrane domain, and an intracellular signaling domain. CD34 is expressed on hematopoietic cells and plays a role in cell migration. Exemplary human CD34 sequences include GenBank Accession Nos. NM_001025109 and NM_001773 (nucleic acid sequences) and NP_001020280 and NP_001764 (amino acid sequences). In some examples, CD34 is a truncated CD34 protein (CD34t) lacking the intracellular signaling domain. The CD34t protein includes the extracellular and transmembrane regions of CD34, and as a result, it is expressed on the cell surface, but does not affect activity of cells expressing the truncated protein (Norell et al., Cancer Immunol. Immunother. 59:851-862, 2010).
Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as one or more single domain antibodies or scFvs) and an intracellular domain, such as an intracellular domain from a T cell receptor (e.g. CD3 . Typically, CARs include an antigenbinding portion(s), a transmembrane domain, and an intracellular domain. CARs also may include a hinge domain and/or a signal peptide (or leader sequence). The intracellular domain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (IT AM), such as CD3^ or FceRIy. In some instances, the intracellular domain also includes the intracellular portion of at least one additional co- stimulatory domain, such as CD28, 4- IBB (CD 137), ICOS, 0X40 (CD134), CD27, and/or DAP10.
Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one aspect, an antibody or antigen binding fragment is linked to an effector molecule; for example, an antibody or antigen binding fragment that specifically binds to a HERV- E protein may be covalently linked to an effector molecule, such as a detectable label or a drug. The linkage can be by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody or antigen binding fragment and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (e.g., a short peptide sequence) can optionally be included between the antibody or antigen binding fragment and the effector molecule.
Conservative variants: “Conservative’' amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, an antibody or antigen binding fragment disclosed herein can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody or antigen binding fragment sequence and retain specific binding activity for a HERV-E protein, such as a HERV-E Envelope. The term conservative variant also includes the use of a substituted amino acid in place of an unsubstituted amino acid. Thus, a conservative substitution does not alter the basic function of a protein of interest.
Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are also considered to be conservative variants, where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
The following are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody or antigen binding fragment. Contacting can also include contacting a cell, for example by placing an antibody or antigen binding fragment in direct physical association with a cell.
Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.
Effective amount/therapeutically effective amount: The amount of an agent (e.g., a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) that is sufficient to effect at least one beneficial or desired therapeutic result, including clinical results.
An effective amount may vary depending upon one or more of: the subject and disease condition being treated, the sex, weight and age of the subject, the severity of the disease condition, the manner of administration, the ability of the treatment to elicit a desired response in the individual, and the like. The beneficial therapeutic effect can include contributing to diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and/or generally counteracting a disease, symptom, disorder or pathological condition. The term “effective amount” or “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in factors such as age, weight, tumor size, extent of metastasis, and condition of the patient (subject).
In some aspects, an “effective amount” (e.g., of a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) may be an amount sufficient to reduce the volume/size of a tumor, the weight of a tumor, the number/extent of metastases, reduce the volume/size of a metastasis, the weight of a metastasis, or combinations thereof, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% (as compared to no administration of the therapeutic agent). In other aspects, an “effective amount” (e.g., of a disclosed antibody, antigen binding fragment, chimeric antigen receptor, or CAR expressing immune cells) may be an amount sufficient to increase the survival time of a subject, such as a subject with cancer, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, 100%, 200%, 300%, 400%, or 500% (as compared to no administration of the therapeutic agent).
Fc region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.
Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originates from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as an scFv or a CAR, is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.
Human endogenous retrovirus E (HERV-E): HERVs are remnants of ancient exogenous retroviruses integrated into the human genome. HERVs are estimated to comprise 5-8% of the human genome. Most HERVs have accumulated mutations or are transcriptionally silenced and do not produce full-length proteins. However, some HERVs are transcriptionally active in contexts such as tumors. HERV-E is a HERV subtype located on human chromosome 6q. At least three transcripts from HERV-E (e.g., GenBank Accession Nos. EU137846, EU137847, and JQ7339O5), also referred to as HERV-E CT-RCC, have been identified and are expressed in RCC cells, but not in other tumors or non-tumor cells (Takahashi et al., J. Clin. Oncol. 118:1099-1109, 2008). These transcripts encode for parts of the protease and polymerase as well as the entire envelope genes, respectively.
Isolated: A biological component (such as a nucleic acid or protein, for example an antibody or antigen binding fragment) that has been substantially separated, produced apart from, or purified away from other biological components, for example other nucleic acids and/or proteins. Thus, isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids and proteins. An isolated nucleic acid or protein, for example an antibody or antigen binding fragment, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link domains together (for example, as in a CAR) or to link a detectable marker or drug to an antibody or antigen binding fragment. Non-limiting examples of peptide linkers include glycine- serine linkers.
The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule (such as a detectable marker or drug) to a polypeptide, such as an antibody or antigen binding fragment. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction such that there is a covalent bond formed between the two molecules to form one molecule.
Natural Killer (NK) cells: Cells of the immune system that kill target cells in the absence of a specific antigenic stimulus and without restriction according to MHC class. Target cells can be tumor cells or cells harboring viruses. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells typically comprise approximately 10 to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells without prior immunization or activation. NK cells are thought to provide a “back up” protective mechanism against viruses and tumors that might escape the CTL response by down-regulating MHC class I presentation. In addition to being involved in direct cytotoxic killing, NK cells also serve a role in cytokine production, which can be important to control cancer and infection.
Tn some examples, a “modified NK cell” is a NK cell transduced or transfected with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins. The terms “modified NK cell” and “transduced NK cell” are used interchangeably in some examples herein.
Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers of use are known to those of ordinary skill in the art. Remington: The Science and Practice of Pharmacy, 22nd ed. , London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), pH buffering agents, and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody or antigen binding fragment) is more enriched than the peptide or protein is in its original environment, such as within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.
Renal cell carcinoma (RCC): A tumor originating in the cells of the kidney. RCC is the most common type of kidney cancer in adults. There are multiple histological subtypes of RCC, including clear cell renal cell carcinoma (ccRCC), which accounts for 60-70% of RCC and originates in the cells of the proximal tubule. ccRCC cells exhibit clear cytoplasm with acinar or sarcomatoid growth pattern. Additional subtypes include but are not limited to papillary RCC (also originating in cells of the proximal tubule), chromophobic RCC (originating in cells of the cortical collecting duct), oncolytic RCC (a benign neoplasm originating in cells of the cortical collecting duct), and collecting duct RCC (originating in cells of the medullary collecting duct).
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as non-human primates, pigs, sheep, cows, dogs, cats, rodents, and the like. In one example, a subject is a human.
T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker. In one aspect, a CD8+ T cell is a cytotoxic T lymphocyte (CTL). In another aspect, a CD8+ cell is a suppressor T cell.
Activated T cells can be detected by an increase in cell proliferation and/or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNy, or TNFa). Activation of CD8+ T cells can also be detected by an increase in cytolytic activity in response to an antigen.
In some examples, a “modified T cell” is a T cell transduced with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins. The terms “modified T cell’’ and “transduced T cell” are used interchangeably in some examples herein. Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
Vector: A nucleic acid molecule (such as a DNA or RNA molecule) including a promoter(s) that is operably linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. In some aspects, a vector includes a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment or encoding a disclosed chimeric antigen receptor. In some examples, the vector is a bacterial vector. In other examples, the vector is a viral vector, such as a nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses. In some aspects, the viral vector is a retroviral vector or an adeno-associated virus (AAV) vector.
II. Monoclonal Antibodies
Disclosed herein are monoclonal antibodies and antigen binding fragments that specifically bind to a HERV-E Envelope. In particular aspects, the antibodies and antigen binding fragments specifically bind to the HERV-E transmembrane protein. In some aspects, the antibodies and antigen binding fragments specifically bind to an epitope including or consisting of SEQ ID NO: 133 or SEQ ID NO: 134.
The monoclonal antibodies (or antigen binding fragments thereof) provided herein include heavy and/or light chain variable domains comprising a CDR1, CDR2, and/or CDR3 with reference to the IM GT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (such as the Kabat, Chothia or IM GT numbering schemes) can be used to determine CDR positions. In some aspects, a monoclonal antibody or antigen binding fragment is provided that comprises the heavy and light chain CDRs of any one of the antibodies described herein. In other aspects, a monoclonal antibody or antigen binding fragment is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein.
In some aspects, the antibody or antigen binding fragment includes a VH including the HCDR1 , HCDR2, and HCDR3, and VL including the LCDR1 , LCDR2, and LCDR3 of any one of the TAYO58, TAY051, TAY052, TAY053, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein. In some examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1 , a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 1 and 2, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 17 and 18, respectively. In further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 33 and 34, respectively. In other examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 49 and 50, respectively. In further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 65 and 66, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 81 and 82, respectively. In still further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 97 and 98, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 113 and 114, respectively. The CDRs are provided in Table 1.
Table 1. TAY monoclonal antibody CDR amino acid sequences (defined by IM GT system)
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
In some aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 1 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 2. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 1 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 2. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 1 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 2 and specifically binds to a HERV-E envelope.
In additional aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 17 and a VL including an amino acid sequence at least 90% identical to (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 18. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 17 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 18. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 17 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 18 and specifically binds to a HERV-E envelope.
In other aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 33 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 34. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 33 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 34. In other aspects, the antibody or antigen binding fragment includes a Vu including the amino acid sequence of SEQ ID NO: 33 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 34 and specifically binds to a HERV-E envelope.
In further aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 49 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 50. In some examples, the antibody or antigen binding fragment includes a Vu with an amino acid sequence including or consisting of SEQ ID NO: 49 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 50. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 49 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 50 and specifically binds to a HERV-E envelope.
In some aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 65 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 66. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 65 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 66. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 65 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 66 and specifically binds to a HERV-E envelope.
In other aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 81 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 82. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 81 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 82. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 81 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 82 and specifically binds to a HERV-E envelope.
In further aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 97 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 98. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 97 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 98. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 97 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 98 and specifically binds to a HERV-E envelope. In additional aspects, the antibody or antigen binding fragment includes a VH including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 113 and a VL including an amino acid sequence at least 90% identical to (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) the amino acid sequence of SEQ ID NO: 114. In some examples, the antibody or antigen binding fragment includes a VH with an amino acid sequence including or consisting of SEQ ID NO: 1 1 and a VL with an amino acid sequence including or consisting of SEQ ID NO: 114. In other aspects, the antibody or antigen binding fragment includes a VH including the amino acid sequence of SEQ ID NO: 113 and specifically binds to a HERV-E envelope. In other aspects, the antibody or antigen binding fragment includes a VL including the amino acid sequence of SEQ ID NO: 114 and specifically binds to a HERV-E envelope.
In some aspects, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 3-5, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 6-8, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 3-5, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 6-8, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 1 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 2, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In other aspects, the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 19-21, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 22-24, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 19-21, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 22-24, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 17 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 18, and the antibody or antigen binding fragment specifically binds a HERV-E Env. In some aspects, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 35-37, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 38-40, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 35-37, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 38-40, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 33 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 34, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In additional aspects, the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 51-53, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 54-56, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 51-53, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 54-56, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 49 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 50, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In other aspects, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 67-69, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 70-72, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 67-69, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 70-72, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 65 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 66, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In additional aspects, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 83-85, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 86-88, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 83-85, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 86-88, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 81 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 82, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In still further aspects, the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 99-101 , respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 102-104, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 99-101, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 102-104, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 97 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 98, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
In additional aspects, the antibody or antigen binding fragment includes a VH including the HCDR1 , the HCDR2, and the HCDR3 of SEQ ID NOs: 115-117, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 118-120, respectively. In some examples, the antibody or antigen binding fragment includes a VH including the HCDR1, the HCDR2, and the HCDR3 of SEQ ID NOs: 115-117, respectively, and a VL including the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 118-120, respectively, wherein the VH includes an amino acid sequence at least 90% identical to (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 1 13 and the VL includes an amino acid sequence at least 90% identical to (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) SEQ ID NO: 114, and the antibody or antigen binding fragment specifically binds a HERV-E Env.
Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a VH and VL and specifically bind a HERV-E envelope. These antibody fragments retain the ability to selectively bind with the antigen and are antigen binding fragments. Non-limiting examples of such fragments include Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; Fv, a genetically engineered fragment containing the VH and VL expressed as two chains; and single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1 155/2012/980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). The intramolecular orientation of the VH domain and the VL domain in a scFv, is not decisive for the provided antibodies. Thus, scFvs with both possible arrangements (VH domain-linker-VL domain or VL domain-linker- VH domain) may be used. A dimer of a single chain antibody (scFV q. defined as a dimer of a scFV (also termed a “miniantibody”) is also included in the antigen binding fragments provided herein.
Any suitable method of producing the above-discussed antigen binding fragments may be used. Non- limiting examples are provided in Harlow and Lane, Antibodies: A Laboratory’ Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 2013. In some examples, antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as a bacterial cell or a mammalian cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab’ monovalent fragments. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
In some aspects, amino acid sequence variants of the antibodies or antigen binding fragments disclosed herein are provided. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, insertions, and/or substitutions of residues within the amino acid sequences of the antibody or antigen binding fragment. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., specifically binding a HERV-E envelope.
In some aspects, variants having one or more amino acid substitutions are provided. Sites of interest for variants (such as substitutions) include the CDRs and the framework regions. Amino acid substitutions may be introduced and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
The variants typically retain amino acid residues necessary for correct folding and stabilization between the VH and the VL regions, and retain the charge characteristics of the residues in order to preserve low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield.
In some aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence of any one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, and 113. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1 , up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence of any one of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114.
In some aspects, one or more substitutions, insertions, and/or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In some aspects of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions. In other aspects of the variant VH and VL sequences provided above, only the framework residues are modified so the CDRs are unchanged.
To increase binding affinity of the antibody, the VL and VH segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the HCDR3 or LCDR3, respectively. In this process, the primers are “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be tested to determine the binding affinity for a HERV-E envelope (such as the TM protein). In particular examples, the VH amino acid sequence is one of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, and 113. In other examples, the VL amino acid sequence is one of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 114.
In some aspects, an antibody or antigen binding fragment is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody includes an Fc region, the carbohydrate attached thereto may he altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the Cl L domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(l):26-32, 1997. The oligosaccharide may include various carbohydrates, e.g. , mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.
In one aspect, antibody variants have a carbohydrate structure that has a decreased amount of or lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about 3 amino acids upstream or downstream of position 297, e.g., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/01 10282; US 2004/0109865; WO 2003/0851 19; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; WO 2002/031140; Okazaki et al., J. Mol. Biol., 336(5):1239- 1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5 ) :614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. , Arch. Biochem. Biophys. 249(2):533-545, 1986; US Pat. Appl. No. US 2003/0157108 and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng. , 87(5): 614-622, 2004; Kanda etal., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).
In other examples, antibody variants with bisected oligosaccharides are provided, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087, WO 1998/58964, and WO 1999/22764.
In several aspects, the constant region of the antibody comprises one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several aspects, the antibody comprises an amino acid substitution that increases binding to the FcRn. Non-limiting examples of such substitutions include substitutions at IgG constant regions T250Q and M428L (see, e.g. , Hinton et al., J Immunol., 176(l):346-356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen binding fragments can be linked to or comprise an Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions.
In some aspects, an antibody provided herein may be further modified to contain additional non-proteinaceous moieties. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non- limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.
Also provided are nucleic acids that encode the monoclonal antibodies or antigen binding fragments disclosed herein. Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In some aspects, the nucleic acids are codon-optimized, for example for expression in human cells.
In some aspects, a nucleic acid encodes a VH including the HCDR1, HCDR2, and HCDR3, and a VL including the LCDR1, LCDR2, and LCDR3 of any one of the TAY058, TAY051, TAY052, TAY053, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein. In some examples, the nucleic acid includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 9 and 10, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 25 and 26, respectively. In further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 41 and 42, respectively. In other examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 57 and 58, respectively. In further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 73 and 74, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 89 and 90, respectively. In still further examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 105 and 106, respectively. In additional examples, the antibody or antigen binding fragment includes a heavy chain variable region and a light chain variable region including a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2, and a LCDR3 of the VH and VL encoded by SEQ ID NOs: 121 and 122, respectively. The nucleic acids encoding the CDRs are provided in Table 2.
Table 2. TAY monoclonal antibody CDR nucleic acid sequences (defined by IMGT system)
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
In some aspects an isolated nucleic acid encodes the VH of the TAY058 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 9 or includes or consists of SEQ ID NO: 9. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY058 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 10 or includes or consists of SEQ ID NO: 10. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAYO58 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 9 and 10, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY051 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 25 or includes or consists of SEQ ID NO: 25. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY051 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 26 or includes or consists of SEQ ID NO: 26. In a non- limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY051 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 25 and 26, respectively. In some aspects an isolated nucleic acid encodes the VH of the TAY052 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 41 or includes or consists of SEQ ID NO: 41. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY052 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 42 or includes or consists of SEQ ID NO: 42. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY052 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 41 and 42, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY053 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 57 or includes or consists of SEQ ID NO: 57. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY053 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 58 or includes or consists of SEQ ID NO: 58. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAYO53 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 57 and 58, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY054 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 73 or includes or consists of SEQ ID NO: 73. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY054 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 74 or includes or consists of SEQ ID NO: 74. In a non- limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY054 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 73 and 74, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY059 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 89 or includes or consists of SEQ ID NO: 89. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY059 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 90 or includes or consists of SEQ ID NO: 90. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY059 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 89 and 90, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY049 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 105 or includes or consists of SEQ ID NO: 105. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY049 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 106 or includes or consists of SEQ ID NO: 106. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY049 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 105 and 106, respectively.
In some aspects an isolated nucleic acid encodes the VH of the TAY050 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 121 or includes or consists of SEQ ID NO: 121. In another aspect, an isolated nucleic acid molecule encodes the VL of the TAY050 antibody or antigen binding fragment and includes a nucleic acid sequence with at least 90% sequence identity (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 122 or includes or consists of SEQ ID NO: 122. In a non-limiting example, an isolated nucleic acid molecule encodes the VH and VL of the TAY050 antibody or antigen binding fragment and includes the nucleic acid sequences of SEQ ID NOs: 121 and 122, respectively.
Nucleic acid molecules encoding the antibodies, antigen binding fragments, or other constructs provided herein (such as bispecific antibodies, CARs, or antibody-effector molecule conjugates) that specifically bind to a HERV-E envelope can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods.
Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements). Nucleic acids can also be prepared by amplification methods.
Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).
The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect, or mammalian cells. The antibodies, antigen binding fragments, or other constructs can be expressed as individual proteins including the Vn and/or VL, or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011.
In some examples, to create a scFv the Vn- and Vi.-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 135), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to a HERV-E envelope and another antigen. The encoded VH and VLoptionally can include a furin cleavage site between the VH and VL domains.
One or more DNA sequences encoding the antibodies, antigen binding fragments, or other constructs can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as COS, CHO, HeLa, and other cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, may be used. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.
The expression of nucleic acids encoding the antibodies, antigen binding fragments, or other constructs described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The promoter can be any promoter of interest, including a cytomegalovirus promoter or EF- 1 a promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The vector can be suitable for replication and/or integration in either prokaryotes or eukaryotes. Typical expression vectors contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the vector can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. In some examples, the vector also encodes a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The vector can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the vector can be selected by resistance to antibiotics conferred by genes contained in the vector, such as the amp, gpt, neo and hyg genes.
Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
Once expressed, the antibodies, antigen binding fragments, or other constructs can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson el al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylactically, the polypeptides should be substantially free of endotoxin.
Methods for expression of antibodies, antigen binding fragments, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 2014; Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009; and Ward et al., Nature 341(6242): 544- 546, 1989.
III. Chimeric Antigen Receptors and Modified Antibodies
Provided herein are chimeric antigen receptors (CARs), bispecific T cell engager (BiTE) constructs, and antibody-effector conjugates that utilize a disclosed HERV-E-specific antibody or antigen binding fragment thereof.
A. Chimeric Antigen Receptors
Provided herein are chimeric antigen receptors (CARs) that include a portion that specifically binds to a HERV-E envelope. In some aspects, the CARs include at least an antigen binding fragment of an antibody provided herein, such as at least one VH domain and at least one VL domain (for example, an scFv). The VH domain and VL domain may be included in the CAR in either order, e.g., in the order VL -linker- VH or VI [-li nker- VL. FIGS. 5 and 12 show exemplary CAR constructs in which the VH domain and VL domain are in the order Vi.-linker- VH (FIG. 5) or in the order Vn-linker-Vi. (FIG. 12).
In some aspects, the CAR includes a HERV-E-specific antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular domain, including at least one costimulatory domain. Additional domains may be included in the CAR construct, including, but not limited to a signal peptide (or leader sequence), and/or additional intracellular domain(s). In some examples, a CAR construct (such as a vector including a CAR) further include a “tag” (such as a truncated CD34 domain), which may be expressed separately from the CAR or cleaved from the CAR after translation.
In some aspects, the antigen binding domain is a HERV-E-specific scFv, for example having a VH domain including three HCDRs and a VL domain including three LCDRs as provided in Table 1. In one non-limiting example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY058 (e.g., SEQ ID NOs: 3-5) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAYO58 (e.g., SEQ ID NOs: 6-8). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 2. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 1 and a Vi. domain with the amino acid sequence of SEQ ID NO: 2.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY051 (e.g., SEQ ID NOs: 19-21) and a VL domain including LCDRI, LCDR2, and LCDR3 from TAY051 (e.g., SEQ ID NOs: 22-24). In another example, the CAR includes an scFv including a Vu domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 17 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 18. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 17 and a VL domain with the amino acid sequence of SEQ ID NO: 18.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY052 (e.g., SEQ ID NOs: 35-37) and a VL domain including LCDRI, LCDR2, and LCDR3 from TAY052 (e.g., SEQ ID NOs: 38-40). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 33 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 34. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 33 and a VL domain with the amino acid sequence of SEQ ID NO: 34.
In another non-limiting example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY053 (e.g., SEQ ID NOs: 51-53) and a VL domain including LCDRI, LCDR2, and LCDR3 from TAY053 (e.g., SEQ ID NOs: 54-56). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 49 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 50. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 49 and a VL domain with the amino acid sequence of SEQ ID NO: 50.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY054 (e.g., SEQ ID NOs: 67-69) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAY054 (e.g., SEQ ID NOs: 70-72). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 65 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 66. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 65 and a VL domain with the amino acid sequence of SEQ ID NO: 66.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY059 (e.g., SEQ ID NOs: 83-85) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAY059 (e.g., SEQ ID NOs: 86-88). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 81 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 82. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 81 and a VL domain with the amino acid sequence of SEQ ID NO: 82.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY049 (e.g., SEQ ID NOs: 99-101) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAY049 (e.g., SEQ ID NOs: 102-104). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 97 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 98. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 97 and a VL domain with the amino acid sequence of SEQ ID NO: 98.
In another example, the CAR includes an scFv with a VH domain including HCDR1, HCDR2, and HCDR3 from TAY050 (e.g., SEQ ID NOs: 115-117) and a VL domain including LCDR1, LCDR2, and LCDR3 from TAY050 (e.g., SEQ ID NOs: 118-120). In another example, the CAR includes an scFv including a VH domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1 13 and a VL domain with an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 114. In one non-limiting example, the CAR includes an scFv including a VH domain with the amino acid sequence of SEQ ID NO: 113 and a VL domain with the amino acid sequence of SEQ ID NO: 114.
In some aspects, the hinge domain is a CD8a hinge region. In one example, the hinge domain includes the sequence of amino acids 268-312 of SEQ ID NO: 130. Other hinge domains can be used, such as hinge regions from immunoglobulins (for example, IgGl, lgG4, or IgD) or a hinge region from CD28 or CD40.
In additional aspects, the transmembrane domain is a CD8a transmembrane domain. In one example, the transmembrane domain includes the sequence of amino acids 313-336 of SEQ ID NO: 130. The transmembrane domain can also be from other T cell proteins, such as CD28, CD4, CD3 , CD40, OX40L, 41BBL, ICOS, ICOS-L, CD80, CD86, ICAM-1, LFA-1, ICAM-1 , CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2, and CD70.
In further aspects, the intracellular domain includes one or more intracellular regions from a co-stimulatory molecule, or a portion thereof. Exemplary co- stimulatory molecules include CD28, 4-1BB, CD8, CD40, OX-40, ICOS, CD27, and DAP10, OX40-L, 4-1BBL, ICOS-L, CD80, CD86, ICAM-1, LFA-1, CD56, CTLA-4, PD-1, TIM-3, NKP30, NKP44, NKP40, NKP46, B7-H3, PD-L1, PD-2, and CD70. In particular examples, the co-stimulatory domain is from 4- IBB. In one example, the co-stimulatory domain includes the sequence of amino acids 337-378 of SEQ ID NO: 130. In particular aspects, the intracellular domain also includes an intracellular signaling domain from CD3^. In one example, the intracellular signaling domain includes the sequence of amino acids 379-490 of SEQ ID NO: 130. In other examples, the intracellular signaling domain is from DAP 10, DAP 12, PDK, or FceRIy.
In some aspects, the CAR also includes a signal sequence, which is located N-terminal to the antigen binding domain. In some examples, the signal sequence is a CD8a signal sequence, an IgG signal sequence, or a GM-CSF signal sequence. In one example, the signal sequence is a CD8a signal sequence, such as the sequence of amino acids 1-21 of SEQ ID NO: 130. In some examples, the CAR does not include the signal sequence and/or an N-terminal methionine; however the signal sequence and/or an N-terminal methionine can be present, for example as a result of the particular expression system used.
In some aspects, the CAR construct further includes a domain that can be used to identify and/or isolate cells expressing the CAR. In some examples, this domain is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide). In one example, the selfcleaving peptide is a P2A peptide, such as the sequence of amino acids 1-27 of SEQ ID NO: 132. In one example, the domain is a truncated CD34 protein (CD34t), such as a CD34 protein including the extracellular and transmembrane domains, but lacking the intracellular domain. As a result, CD34t is expressed on the cell surface, but does not affect activity of cells expressing the truncated protein (Norell et al. , Cancer Immunol. Immunother. 59:851-862, 2010). Cells expressing CD34t can be identified with an anti-CD34 antibody, and can be isolated using flow cytometry or immuno-magnetic methods. In one example, the CD34t protein includes or consists of an amino acid sequence having at least 95% (such as at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 28-342 of SEQ ID NO: 132.
In some non-limiting aspects, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 130. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of SEQ ID NO: 130. In other examples, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 130. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 130. In additional examples, the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
In other non-limiting aspects, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 137. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of SEQ ID NO: 137. In other examples, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 137. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 137. In additional examples, the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
In additional non-limiting aspects, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 139. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of SEQ ID NO: 139. In other examples, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 139. In other non- limiting examples, the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 139. In additional examples, the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
In further non-limiting aspects, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 141. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of SEQ ID NO: 141. In other examples, the CAR has an amino acid sequence with at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 22-490 of SEQ ID NO: 141. In other non-limiting examples, the CAR includes or consists of the amino acid sequence of amino acids 22-490 of SEQ ID NO: 141. In additional examples, the CAR construct further includes the amino acid sequence of SEQ ID NO: 132.
In additional aspects, the CAR construct further includes an inducible protein that can be used to eliminate CAR expressing cells (e.g., a “suicide” domain). The inducible protein can be activated in the event of off target side effects (or on target/off tumor effects), such as cytokine release syndrome (“cytokine storm”). In some examples, expression of the suicide domain is inducible by a small molecule, such as tetracycline or doxycycline (a “TET ON” system) or rapamycin. See, e.g., Gargett et al., Front. Pharmacol. 5:235, 2014; Stavrou et al., Mol. Ther. 6:1266-1276, 2018. In other examples, the suicide domain is inducible by a Fas domain inducible system. In some examples, the inducible suicide domain is located N-terminal or C-terminal to the antigen binding domain of the CAR, while in other examples, the inducible suicide domain is located C-terminal to the intracellular signaling domain (for example, the CD3^ domain) of the CAR. The inducible suicide domain is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide). In some aspects, the inducible suicide domain includes Caspase 9.
In other aspects, the CAR construct further includes a domain that increases survival or persistence of a modified immune cell expressing the CAR. In some examples, the domain is an intracellular domain from a cytokine receptor, for example, an intracellular domain from interleukin (IL) receptor 15, IL- 12 receptor, or IL- 18 receptor. In other examples, the domain is an intracellular domain from a growth factor receptor, such as an intracellular domain from CD40, NKG2D, NKP40, or NKP46. In some examples, the domain is located C-terminal to the intracellular signaling domain (for example, the CD3^ domain) of the CAR (e.g. is separated from the CAR by a self-cleaving peptide (such as a P2A peptide or T2A peptide)).
In some examples, the CAR further includes one or more additional antigen binding domains that specifically bind to an antigen that is co-expressed with HERV-E on tumor cells, or a T cell may express an additional CAR with an additional antigen binding domain. In some nonlimiting examples, the additional antigen binding domain that specifically binds to a renal cell carcinoma antigen (e.g., an antigen binding domain that specifically binds to carbonic anhydrase IX (CAIX), see, e.g., US Pat. No. 10,870,705 and Lo et al., Oncolytics 1 :14003, 2014). In some examples, a CAIX logic gate CAR may be utilized. Logic Gate CAR T cells are T cells expressing two or more CARs targeting separate tumor antigens. Based on Boolean logic, they can be categorized into “AND,” “OR,” and “NOT” CAR T cells. “AND” CAR T cells are only activated by the presence of both antigens; “OR” CAR T cells may be activated by either antigen; “NOT” CAR T cells are activated only when one antigen is absent. Anti-CAIX CAR T cells cause liver toxicity due to the on-target toxicity by attacking bile duct epithelial cells, as these cells express low-level of CAIX. The “AND”/“OR” Gate CAR T cells simultaneously targeting HERV-E via a disclosed scFV and CAIX may solve this problem, and may also improve the safety profile of the disclosed CARs.
Additional antigens that may be targeted by the CAR include, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, CD70, hypoxia-inducible protein 2 (HIG2), von Hippel- Lindau protein (VHL) 5T4 protein, mucin 1 (MUC1), Wilms’ tumor 1 (WT1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), AXL, alpha-fetoprotein, melanoma-associated antigen 4 (MAGEA4), epidermal growth factor receptor (EGFR), mesothelin, programmed cell death 1 ligand 1 (PD-L1), CD56, CD10, CEA cell adhesion molecule 5, epithelial cell adhesion molecule (EPCAM), Erb-B2 receptor tyrosine kinase 3 (ERBB3), ERBB5, CD138, CD133, CD86, CD123, c-Met, TNF superfamily member 13b (TACI), TNF superfamily member 10b (DR5), ectonucleotide pyrophosphatase/phosphodi esterase 3, and hepatitis A virus cellular receptor 1. Also provided are nucleic acids encoding the CARs disclosed herein. In some aspects, the CAR includes a nucleic acid encoding a VH including the HCDR1, HCDR2, and HCDR3, and a VL including the LCDR1, LCDR2, and LCDR3 of any one of the TAY058, TAY051, TAY052, TAYO53, TAY054, TAY059, TAY049, or TAY050 antibodies provided herein. In some aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAYO58 antibody includes the nucleic acid sequences of SEQ ID NOs: 9 and 10, respectively. In other aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY051 antibody and includes the nucleic acid sequences of SEQ ID NOs: 25 and 26, respectively. In additional aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY052 antibody and includes the nucleic acid sequences of SEQ ID NOs: 41 and 42, respectively. In further aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY053 antibody and includes the nucleic acid sequences of SEQ ID NOs: 57 and 58, respectively. In other aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY054 antibody and includes the nucleic acid sequences of SEQ ID NOs: 73 and 74, respectively. In additional aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY059 antibody and includes the nucleic acid sequences of SEQ ID NOs: 89 and 90, respectively. In some aspects the CAR includes a nucleic acid encoding the VH and VL of the TAY049 antibody and includes the nucleic acid sequences of SEQ ID NOs: 105 and 106, respectively. In other aspects, the CAR includes a nucleic acid encoding the VH and VL of the TAY050 antibody and includes the nucleic acid sequences of SEQ ID NOs: 121 and 122, respectively. In non-limiting examples, a CAR is encoded by the nucleic acid sequence of any one of SEQ ID NO: 129, 136, 138, and 140.
Also provided are functional variants of the CARs or one or more domains thereof described herein, which retain the biological activity of the CAR of which it is a variant or retains the biological activity of the particular domain. The functional variant can be at least about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR or domain. Substitutions can be made, for example, in one or more of the extracellular targeting domain, hinge domain, transmembrane domain, and intracellular domains.
In some examples, the functional variant includes the amino acid sequence of the parent CAR or domain with at least one conservative amino acid substitution (such as up to 10 conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions). In other examples, the functional variant includes the amino acid sequence of the parent CAR or domain with at least one non-conservative amino acid substitution (such as up to 10 non-conservative amino acid substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non- conservative substitutions). In this case, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved or increased as compared to the parent CAR or domain.
The CARs or one or more domains thereof can in some examples, include one or more synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic or non-naturally occurring amino acids include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, P-phenylserine P-hydroxyphenylalanine, phenylglycine, a -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4- tetrahydroisoquinoline- 3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a- aminocyclohexane carboxylic acid, oc- aminocycloheptane carboxylic acid, -(2-amino-2- norbomanej-carboxylic acid, y-diaminobutyric acid, a,P-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine. The CARs may also include one or more D-amino acids. The CARs may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N- acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
In some aspects, a nucleic acid molecule encoding a disclosed CAR is included in an expression vector (such as a viral vector) for expression in a host cell, such as a T cell or NK cell. In some examples, the expression vector includes a promoter operably linked to the nucleic acid molecule encoding the CAR. In one non- limiting example, the promoter is a EF-1A promoter. Additional expression control sequences, such as one or more enhancers, transcription and/or translation terminators, and initiation sequences (such as a Kozak sequence) can also be included in the expression vector. In some aspects, a nucleic acid encoding a CAR provided herein is included in a viral vector. Examples of suitable virus vectors include retrovirus e.g., MoMLV or lentivirus), adenovirus, adeno- associated virus, vaccinia virus, and fowlpox vectors. In specific examples, the CAR encoding nucleic acid is included in a MoMLV or lentiviral vector. In other examples, the vector may be a DNA vector (such as a plasmid).
Also provided herein are cells (for example, immune cells) that express the disclosed CARs. In particular aspects, the cells include T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages. In some aspects, the cells are T cells, NK cells, or macrophages expressing a disclosed CAR.
In some examples, the immune cells are transduced or transfected with one or more expression vectors including one or more nucleic acids, including nucleic acids encoding a CAR provided herein. In other examples, the vector (or a DNA encoding the construct) may be introduced by contacting the cells with a nanoparticle including the vector or DNA. In additional examples, the CAR-expressing immune cells are generated using Sleeping Beauty transposon system (see, e.g., Monjezi et al., Leukemia 31 : 186- 194, 2017; Hodge et al., Hum. Gene Ther. 28:842-855, 2017) or a CRISPR-Cas9/AAV system (see, e.g., Eyquem et al., Nature 543:113-117, 2017; Knott et al., Science 361:866-869, 2018).
In some examples, the transduced or transfected cells are isolated T cells (such as primary T cells or T cells obtained from a subject), isolated NK cells (such as primary NK cells or NK cells obtained from a subject), isolated NKT cells, isolated DNT cells, isolated neutrophils, or isolated macrophages (such as primary macrophages or macrophages obtained from a subject). In some examples, the T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages are obtained from peripheral blood. In some examples, T cells, NK cells, NKT cells, or DNT cells are also enriched, purified, and/or expanded from a sample from a subject, for example before and/or after transduction with one or more of the disclosed CARs.
In one non-limiting aspect, the cell is an NK-92 cell. NK-92 cells are a NK cell line derived from a patient with non- Hodgkin’s lymphoma (e.g., ATCC® CRL-2407™). This cell line has properties of activated NK cells (see, e.g., Gong et al., Leukemia 8:652-658, 1994). In another aspect, the cell is an NK-92MI cell (e.g., ATCC® CRL-2408™). The NK-92MI cell line is an interleukin-2 (IL-2) independent NK cell line, derived from NK-92, which stably expresses human IL-2 (see, e.g., Tam et al., Hum. Gene Ther. 10:1359-1373, 1999). NK-92 or NK-92MI cells expressing a CAR can be used as an “off the shelf” immunotherapy, since autologous NK cells do not have to be produced for each subject. Other NK cell lines that can be used with the CARs (or other nucleic acids) described herein include NKL, KHYG-1, and YTS cells.
In some non-limiting aspects, immune cells are transduced with a vector encoding a CAR. Following transduction, cells expressing the CAR can be detected and/or enriched, for example, by flow cytometry using a labeled antibody that binds to CD34t (if present in the CAR construct). In some examples, the transduced cells (such as NK cells or T cells) are expanded, for example, by cell culture for a period of time following transduction. In some examples, some or all of the modified cells are cryopreserved for later use. B. Multispecific T cell or NK Cell Engagers
Also provided are multi- specific T cell or NK cell engager constructs including one or more antibody or antigen binding fragment thereof provided herein. In some examples, the construct is a bispecific T cell engager (BiTE) construct including at least one scFv including HCDRs and LCDRs from an antibody provided herein and an scFv that specifically binds to CD3 linked by a peptide linker. In one non- limiting example the BiTE includes an scFv including VH domain and VE domain from antibody TAYO58 or TAY051; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized..
In other examples, the construct is a bispecific killer cell engager (BiKE) or trispecific killer cell engager (TRiKE) construct (see, e.g., Davis et al., Semin. Immunol. 31 :64-75, 2017). In one example, the BiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and an scFv that specifically binds to CD16. In other examples, the TRiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and scFvs that specifically bind to CD 16 and a third target (such as NKG2A, KIR2DL1, TIGIT, or PD- 1). In other examples, the TRiKE includes at least one scFv including HCDRs and LCDRs from an antibody provided herein and scFvs that specifically bind to CD 16 and a third molecule (such as IL-15, IL-7, or IL-21). In some non-limiting examples the BiKE or TRiKE includes an scFv including VH domain and VL domain from antibody TAY058 or TAY051 ; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized.
C. Antibody-Effector Conjugates
Also provided are antibody-effector conjugates utilizing the antibodies provided herein, or an antigen binding fragment thereof. In particular examples, the conjugate includes an antibody or antigen binding fragment including HCDRs and LCDRs from an antibody provided herein linked to an effector, such as monomethyl auristatin E (MMAE), MMAF, MED-2460, auristatin phenylalanine phenylenediamine (AFP), maytansinoid, tubulysin, calicheamicin, duocarmycin, benzodiazepines, camptothecin analogues, or doxorubicin.. The antibody may be produced in the format of IgGl, IgG2, IgG3, or IgG4 and linked to an effector. The effector could be linked at a ratio of 1, 2, 3, 4, 5, 6, 7, or 8 molecules per antibody.
The antibody and effector may be linked by a cleavable (e.g. , protease cleavable) or a non- cleavable linker (see, e.g., Beck et al., Nat. Rev. Drug Disco. 16:315-337, 2017). Exemplary linkers include thioether (e.g. , succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC)), maleimidocaproyl, P-glucuronide, disulfide, acid-sensitive linkers, or peptide linkers. In some aspects, the antibody and effector may be linked by chemical reactions such as thiosuccinimide linkage at natural amino acid residuals such as lysine, or cysteine. The linkage may be also formed at engineered unnatural amino acids such as p- acetylphenylalanine (pAcPhe) either with or without the presence of enzymes such as formylglycine-generating enzyme. In some aspects, the linkage may form at the N-glycosylated Asn297 site by glycocojugation through maleimide chemistry, or other chemoenzymatic methods.
In some aspects, the conjugate includes an antibody or antigen binding fragment including HCDRs and LCDRs from an antibody provided herein linked (e.g., conjugated) to a cytotoxic molecule (such as an anti-cancer drug). In one non-limiting example the antibody-effector conjugate includes an scFv including the VH domain and the VL domain from antibody TAY058 or TAY051 ; however, an scFv (or other antigen binding domain) from any of the disclosed antibodies could be utilized.
IV. Methods of Treating Renal Cell Carcinoma
Disclosed herein are methods of treating or inhibiting RCC in a subject. In some aspects, the methods include administering to the subject an antibody, antigen binding fragment, or antibody-effector conjugate provided herein (such as an effective amount of an antibody, antigen binding fragment, or antibody-effector conjugate provided herein). In some aspects, the antibody, antigen binding fragment, or antibody-effector conjugate is administered to the subject intravenously or subcutaneously. In other aspects, the methods include administering to the subject a cell (such as a T cell or NK cell) expressing a CAR provided herein (such as effective amount of a CAR-T or CAR-NK cell expressing a CAR provided herein). In some aspects, the cell is administered to the subject intravenously In further examples, the methods include administering to the subject a BiTE provided herein. In some aspects, the BiTE is administered to the subject intravenously or subcutaneously. In some examples, the subject has ccRCC, advanced ccRCC, or metastatic ccRCC. In particular examples, the subject has a ccRCC expressing a HERV-E envelope.
Compositions are provided that include one or more of the disclosed antibodies, antigen binding fragments, conjugates, CARs, nucleic acids encoding such molecules, or CAR-expressing immune cells that are disclosed herein in a pharmaceutically acceptable carrier. The compositions are useful, for example, for the inhibition or treatment of a renal cell carcinoma. The compositions can be prepared in unit dosage forms, such as in a kit, for administration to a subject. The amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes. The composition can be formulated for systemic or local administration. In one example, the composition is formulated for intravenous administration or subcutaneous administration.
The compositions for administration can include a solution of the antibody, antigen binding fragment, CAR, conjugate, nucleic acid encoding such molecules, or CAR-expressing immune cells dissolved or suspended in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by any suitable technique. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody (or other agent) in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like in accordance with the particular mode of administration selected and the subject’s needs.
Antibodies, or an antigen binding fragment thereof or a nucleic acid encoding such molecules, can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution, or an antigen binding fragment or a nucleic acid encoding such antibodies or antigen binding fragments, can then be added to an infusion bag containing 0.9% sodium chloride. Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules, can be administered by slow infusion, rather than in an intravenous push or bolus.
Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the active agent as a central core. In microspheres, the active agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.
Polymers can be used for ion-controlled release of the compositions disclosed herein. Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug.
Antibodies, antigen binding fragments, and conjugates thereof can be administered by intravenous infusion. Doses of the antibody, antigen binding fragment, or conjugate vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the antibody or antigen binding fragment can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg. The antibody or antigen binding fragment is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or antigen binding fragment is administered weekly, every two weeks, every three weeks or every four weeks.
In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody or antigen binding fragment to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; nonlimiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.
In one aspect, a nucleic acid encoding a disclosed antibody, or antigen binding fragment thereof, is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 5 pg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466). Single or multiple administrations of a composition including a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once, but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to treat a subject with cancer without producing unacceptable toxicity to the patient.
In other aspects, the composition includes about 104 to 1012 of CAR-expressing immune cells (such as CAR-NK cells or CAR-T cells), for example, about 104-108 cells, about 106-108 cells, or about 106- 1012 cells. For example, the composition may be prepared such that about 104 to IO10 modified immune cells cells/kg (such as about 104, 10 s, 106, 107, 108, 109, or IO10 cells/kg) are administered to a subject. In specific examples, the composition includes at least 104, 10s, 106, or 107 CAR-NK cells or CAR-T cells. The population of modified NK cells or modified T cells is typically administered parenterally, for example intravenously; however, injection or infusion to a tumor or close to a tumor (local administration) or administration to the peritoneal cavity can also be used. Appropriate routes of administration can be determined based on factors such as the subject, the condition being treated, and other factors. In some aspects, the CAR-expressing immune cells further express one or more (such as 1, 2, 3, or more) additional CARs. In one nonlimiting example, the CAR-expressing immune cells express a second CAR targeting a second antigen expressed by a RCC cell, for example, CAIX.
Multiple doses of the population of the CAR-expressing immune cells can be administered to a subject. For example, CAR-NK cells or CAR-T cells can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. A skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.
In additional examples, the subject is also administered at least one, at least one, at least two, at least three, or at least four cytokine(s) (such as IL-2, IL-7, IL-15, IL-21, and/or IL-12) to support survival and/or growth of the CAR-expressing cells. In specific, non-limiting examples, the at least one cytokine includes IL-2 and IL-15. The cytokine(s) are administered before, after, or substantially simultaneously with the CAR-expressing cells. In specific examples, at least one cytokine (e.g., IL-2) is administered simultaneously, for example, with CAR-T cells or CAR-NK cells.
Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages of the disclosed compositions for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The effective dose can be determined from cell culture assays and animal studies.
EXAMPLES
The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.
Example 1
Monoclonal Antibody Isolation and Characterization
Isolating and validating scFVs from phage library: Peptides were synthesized to cover the surface and transmembrane protein of the envelope (SU and TM) and CR derived human endogenous retrovirus type E (CT-RCC HERV-E). Peptides were used as antigens to screen against the human antibody phage library (Dynamic Precision Library Platform). Positive binding scFVs were further validated against yeast expressing SU, TM and CR and the selected scFVs were finally expressed in the format of IgGl.
Flow cytometry: RCC cells and non-RCC adherent cells (including CHO cells expressing SU, TM, CR) were detached using 5 mM EDTA in Ca2+ and Mg2+ free PBS and 0.5-1 x 106 cells were used for each staining. Cells were first stained with fixable aqua dead cell stain kit (Thermo Fisher) in PBS for 30 minutes at room temperature, washed, and resuspended in 100 pL FACs buffer. TAY Abs or anti-GFP control Ab were added and stained for 30 minutes at 4°C. Cells were washed with FACs buffer, stained with PE-Goat anti-Human IgG Fc specific (Jackson) for 20 minutes at 4°C. After finishing staining, cells were finally resuspended in 150 pL FACs buffer and analyzed by LSRFortessa™ cell analyzer (BD).
Antibodies were identified by screening a human antibody phage library with TM, SU, and CR proteins. The antibodies were screened against RCC and non-RCC cell lines by flow cytometry (FIG. 1). Cell lines utilized are identified in Table 3.
Table 3. Cell lines utilized for antibody screening
Figure imgf000068_0001
Figure imgf000069_0001
The antibodies were also screened for binding to monocytes and normal human tissue. TAY057 showed non-specific binding against monocytes (FIG. 2B). TAY052 and TAY055 showed nonspecific binding to normal human tissue (FIG. 3). A summary of the candidate antibodies is shown in Table 4.
Table 4. Candidate antibody summary
Figure imgf000069_0002
Figure imgf000070_0001
Antibodies were validated using CHO cells expressing CT-RCC HERV-E Env proteins.
TAYO58 showed specific binding to CHO cells expressing TM (FIG. 4). TAY052 and TAY055 showed non-specific binding to all CHO cells (FIG. 4). Table 5 provides a summary of epitope mapping and target validation for the antibodies.
Table 5. Additional candidate antibody characterization
Figure imgf000070_0002
Figure imgf000071_0001
Example 2 Preparation and Characterization of TAY058 CAR
Sleeping Beauty vector construction: The backbone transposon plasmid pT2/HB was Addgene plasmid # 26557. The transposase plasmid pCMV(CAT)T7-SB 100 was Addgene plasmid # 34879. CAR sequences (FIG. 5) were synthesized from Eurofins Genomics and cloned into pT2/HB vector through In-Fusion® HD Cloning Plus kit (Takara Bio) following manufacturer’s instructions. Anti-CD19 clone FMC63 sequence was retrieved from IMGT database and cloned into pT2/HB backbone using the same method as a negative control.
CAR-T cells electroporation, sort and expansion: T cells were resuspended in TexMACS™ medium (Miltenyi) with 3% heat inactivated human AB serum (Sigma), penicillinstreptomycin IX (Thermo Fisher), 10 ng/mL rhIL-7 (PeproTech) and 5 ng/mL rhIL-15 (PeproTech), designated as TexMACS™ complete medium. ImmunoCult™ Human CD3/CD28/CD2 T cell activator (StemCell Technologies) was added to the medium to activate T cells at the concentration of 25 pL/mL for 72 hours. After activation, cells were harvested, counted and resuspended in Opti-MEM® medium at the concentration of 2xl08 /mL. pT2/HB transposon plasmids encoding the CARs (150 pg/mL) and pCMV(CAT)T7-SB100 transposase plasmids (50 pg/mL) were added for each electroporation based on optimization experiments. The electroporation was performed using ExPERT ATx™ (MaxCyte). Cells were returned to the incubator 48 hours and resuspended at desired concentration with fresh TexMACS™ complete medium. Medium changes were done every other day depending on cell growth. On day 7 post electroporation, CD34+ CAR T cells were sorted using anti-human CD34 magnetic beads (Miltenyi). The percentage of CD34+ cells before and after sorting was measured using another anti-human CD34 mAb (Miltenyi, clone AC 136) binding a different epitope than the one recognized by anti-human CD34 magnetic beads. The sorted cells were expanded with ImmunoCult™ Human CD3/CD28/CD2 T cell activator at the concentration of 12.5 pL/mL following the manufacture’s protocol. After expansion, CAR T cells were used for phenotyping and functional assays.
In vitro toxicity assay against CHO and Raji cells: Cytotoxicity of CAR T cells was measured using the Celigo™ image cytometer. Raji cells were harvested and resuspended in PBS at the concentration of IxlO6 /m . Calecin AM (BD) was added into the cell suspension for 30 minutes in the incubator for labelling the cells according to manufacturer’s protocol. Cells were washed and 10,000 cells were aliquoted into a 96- well clear V-bottom plate (Corning) in 100 pL TexMACS™ complete medium. Effector CAR T cells were added at different effectortarget ratio (E:T ratio) in 100 pL TexMACS™ complete medium and co-cultured for 4 hours in the incubator. After co-culture, 100 pL of the mixture was transferred to 96- well black wall microplate and scanned by Celigo™ image cytometer. The percent lysis was calculated as: % lysis = (1- (Calcein AM Count_treated)/(Calcein AM Count_control))xl00%. For CHO cells expressing HERV-E SU, TM, TM-native, CR and SU-TM-CR (Comb), GFP was used to measure the live cell after coculture. In brief, CHO cells were seeded into 96-well black wall microplate 72 hours before toxicity assay at the optimal density from preliminary experiments. Cells were scanned by Celigo image cytometer to calculate the cell numbers. Effector CAR T cells were added at 10:1 effector: target ratio in 100 pL TexMACS™ complete medium and were incubated for 4 hours and scanned by Celigo image cytometer. The percent lysis was calculated as: % lysis = (1- (GFP Count_treated)/(GFP Count_control))xl00%.
TAYO58 BBZ CAR and the control FMC63 BBZ CAR were stably expressed in T cells post electroporation; CAR T cells could be further enriched to a very high purity based on CD34 magnetic sort, as shown in FIG. 6. In addition, the CAR T cells specifically killed CHO cells expressing HERV-E TM. Raji cells were used as a positive control to validate the killing capability of FMC63 BBZ CAR T cells (FIG. 7).
Example 3 Preparation and Characterization of additional TAY CARs
CAR sequences for TAY049, TAY050, TAY051, TAY052, TAY053, TAY054, and TAY059 using the format shown in FIG. 5 (“TAY BBZ” CARs) were synthesized and expressed in T cells as described in Example 2. High expression of CD34 was observed in each CAR-T cell preparation (FIG. 8). Toxicity of the CAR-T cells against Raji cells was carried out as described in Example 2 (FIG. 9). TAY051 BBZ CAR-T cells specifically killed HERV-E+ cells but not the control Raji cells (FIGS. 10-11). Example 4
Preparation and Characterization of VH-VL Format TAY CARs
All TAY candidates were converted into VH-VL format CARs (FIG. 12; “TAY VHVL BBZ” CARs). Each CAR was highly expressed in donor T cells, based on measuring CD34 by flow cytometry (FIG. 13). TAY052 VHVL BBZ CAR caused fratricide, likely due to the nonspecific binding of TAY052 scFv (FIG. 14). The ability of TAY BBZ CAR-T cells and TAY VHVL BBZ CAR-T cells to kill HERV-E+ RCC cell lines was tested (FIGS. 15A-15H). TAY049 CAR-T cells (FIG. 15B), TAY050 CAR-T cells (FIG. 15C), TAY053 CAR-T cells (FIG. 15E), TAY054 CAR-T cells (FIG. 15F), and TAY059 CAR-T cells (FIG. 15H) (both BBZ and VHVL BBZ CARs) did not kill HERV-E+ RCC cell lines. TAYO58 BBZ CAR-T cells killed CHO cells expressing TM, but did not kill HERV-E+ RCC cells (FIG. 15G). TAY052 BBZ CAR-T cells did not kill HERV-E+ RCC cell lines. However, TAY052 VHVL BBZ CAR-T cells killed HERV-E+ RCC cell lines, likely in a non-specific way (FIG. 15D). Both TAY051 BBZ CAR- and TAY051 VHVL BBZ CAR-T cells killed several HERV-E+ RCC cell lines (FIG. 15 A).
Example 5 Efficacy of CARs in Mouse Xenograft Model
This example describes particular methods that can be used to evaluate efficacy of the disclosed CARs in a mouse model of cancer. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used.
NSG mice at 6-12 week are s.c. injected with 2 x 106 RCC cells in the flank region 2 weeks prior to treatment. Mice are randomized into 3 groups receiving: TAY BBZ or TAY VHVL BBZ CAR T cells (such as TAY051 BBZ CAR or TAY051 VHVL BBZ CAR), FMC63 BBZ CAR T cells, or no treatment. Methods to produce the CAR T cells are described in Examples 2 and 4. CAR T cells are administered via tail vein injection in 100 pL PBS at the concentration of 5xl07 cells/mL. Tumor size is monitored, for example, using bioluminescent imaging (PerkinElmer), such as twice a week.
Example 6
Evaluation of CARs in Human Trial
This example describes particular methods that can be used to evaluate the disclosed CARs in a clinical trial. However, one skilled in the art will appreciate that methods that deviate from these specific methods can also be used. Patients with metastatic RCC are recruited for a single-arm Phase I trial. The study is planned based on a Phase I 3+3 dose escalation design. The maximum tolerated dose (MTD) is defined as the highest dose at which 0 or 1 patient in six has experienced a dose limiting toxicity (DLT). Patients with evaluable advanced/metastatic ccRCC are recruited in up to 3 dose levels.
T cells are isolated from 15-20 liter leukapheresis of patients and TAY BBZ or TAY VHVL BBZ CAR T cells (such as TAY051 BBZ CAR or TAY051 VHVL BBZ CAR) are manufactured using ExPERT ATX™ (MaxCyte) as following the protocol described above at the Department of Transfusion Medicine of NIH. TAY BBZ or TAY VHVL BBZ CAR T cells (such as TAY0 1 BBZ CAR or TAY051 VHVL BBZ CAR) are infused into patients 5 days after a non- myeloablative immunosuppressive conditioning regimen of cyclophosphamide and fludarabine. Patients are treated in 4 different cohorts: IxlO6 T cells/kg, 5xl06 T cells/kg, IxlO7 T cells/kg, 5xl07 T cells/kg. Patients are discharged from the clinical center following neutrophil recovery and return for weekly visits for 6-8 weeks, where they undergo standard evaluations including physical exams and body weight, and routine clinical labs (hematology and electrolytes). Restaging using PET and CT imaging using RECIST criteria will be performed 30 days following the T cell infusion, then every 3 months for the first year, and then every 6 months thereafter until evidence for tumor progression occurs.
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:
1. An isolated monoclonal antibody or antigen binding fragment thereof, comprising: a) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 17 and 18, respectively; b) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 1 and 2, respectively; c) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 33 and 34, respectively; d) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 49 and 50, respectively; e) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 65 and 66, respectively; f) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 81 and 82, respectively; g) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 97 and 98, respectively; or h) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL of SEQ ID NOs: 113 and 114, respectively; and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
2. The antibody or antigen binding fragment of claim 1 , wherein a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 19-24, respectively; b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 3-8, respectively; c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 35-40, respectively; d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 51-56 respectively; e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 67-72, respectively; f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 83-88, respectively; g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 99-104, respectively; or h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences of SEQ ID NOs: 115-120, respectively, and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
3. The antibody or antigen binding fragment of claim 1 or claim 2, wherein a) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 17 and 18, respectively; b) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 1 and 2, respectively; c) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 33 and 34, respectively; d) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 49 and 50, respectively; e) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 65 and 66, respectively; f) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 81 and 82, respectively; g) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 97 and 98, respectively; or h) the VH and the VL comprise amino acid sequences at least 90% identical to the amino acid sequences of SEQ ID NOs: 113 and 1 14, respectively, and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
4. The antibody or antigen binding fragment of any one of claims 1 to 3, wherein: a) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 17 and 18, respectively; b) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 1 and 2, respectively; c) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 33 and 34, respectively; d) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 49 and 50, respectively; e) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 65 and 66, respectively; f) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 81 and 82, respectively; g) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 97 and 98, respectively; or h) the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 113 and 114, respectively, and wherein the antibody or antigen binding fragment specifically binds to a human endogenous retrovirus-E Envelope.
5. The antibody or antigen binding fragment of any one of claims 1 to 4, wherein the antibody or antigen binding fragment specifically binds to a transmembrane protein of the human endogenous retrovirus-E Envelope.
6. The antigen binding fragment of any one of claims 1 to 5, wherein the antigen binding fragment is a Fv, Fab, F(ab’)2, scFV, or scFv2 fragment.
7. A bispecific antibody comprising the antibody or antigen binding fragment of any one of claims 1 to 6.
8. A chimeric antigen receptor comprising the antibody or antigen binding fragment of any one of claims 1 to 6.
9. The chimeric antigen receptor of claim 8, wherein the chimeric antigen receptor further comprises a leader sequence, a hinge domain, a transmembrane domain, and an intracellular domain.
10. The chimeric antigen receptor of claim 9, wherein one or more of the leader sequence, hinge domain, and transmembrane domain is from CD8a.
11. The chimeric antigen receptor of claim 9 or claim 10, wherein the intracellular domain comprises a 4- IBB intracellular domain and a CD3^ intracellular domain.
12. The chimeric antigen receptor of any one of claims 8 to 11, further comprising a truncated CD34 domain.
13. The chimeric antigen receptor of any one of claims 8 to 12, wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 141, 139, 130, and 137.
14. The chimeric antigen receptor of any one of claims 8 to 13, further comprising a second antibody or antigen binding fragment.
15. The antibody or antigen binding fragment of any one of claims 1 to 6, conjugated to a detectable marker or a drug.
16. An isolated nucleic acid encoding the antibody or antigen binding fragment of any one of claims 1 to 6 or the chimeric antigen receptor of any one of claims 8 to 14.
17. The isolated nucleic acid of claim 16, wherein: a) the VH and VL nucleotide sequences comprise SEQ ID NOs: 25 and 26, respectively; b) the VH and VL nucleotide sequences comprise SEQ ID NOs: 9 and 10, respectively; c) the VH and VL nucleotide sequences comprise SEQ ID NOs: 41 and 42, respectively; d) the VH and VL nucleotide sequences comprise SEQ ID NOs: 57 and 58, respectively; e) the VH and VL nucleotide sequences comprise SEQ ID NOs: 73 and 74, respectively; f) the VH and VL nucleotide sequences comprise SEQ ID NOs: 89 and 90, respectively; g) the VH and VL nucleotide sequences comprise SEQ ID NOs: 105 and 106, respectively; or h) the VH and VL nucleotide sequences comprise SEQ ID NOs: 121 and 122, respectively.
18. The isolated nucleic acid of claim 16 or claim 17, wherein the nucleic acid encodes a chimeric antigen receptor.
19. The isolated nucleic acid of claim 18, comprising the nucleic acid sequence of any one of SEQ ID NOs: 138, 140, 129, and 136.
20. The isolated nucleic acid of any one of claims 16 to 19, operably linked to a promoter.
21. A vector comprising the isolated nucleic acid of any one of claims 16 to 20.
22. A host cell comprising the isolated nucleic acid of any one of claims 16 to 20 or the vector of claim 21.
23. The host cell of claim 22, wherein the cell is a T cell or natural killer cell.
24. A composition comprising the antibody or antigen binding fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 14, the conjugate of claim 15, the nucleic acid of any one of claims 16 to 20, the vector of claim 21, or the host cell of claim 22 or 23 and a pharmaceutically acceptable carrier.
25. A method of treating renal cell carcinoma, comprising administering to a subject with renal cell carcinoma the composition of claim 24.
26. The method of claim 25, wherein the subject with renal cell carcinoma has clear cell renal cell carcinoma.
27. The method of claim 25 or claim 26, wherein the subject has a renal cell carcinoma expressing a human endogenous retrovirus-E Envelope.
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