CD70 anti-idiotype antibody

Abstract

Provided herein are anti-idiotypic antibodies that specifically recognize anti-CD70 antibody portions, particularly anti-CD70 antibody portions present in recombinant receptors, including chimeric antigen receptors (CARs). The present disclosure further relates to the use of anti-idiotypic antibodies to specifically identify and / or select cells expressing such recombinant receptors, such as anti-CD70 CAR T cells. The present disclosure further relates to the use of anti-idiotypic antibodies to specifically activate such cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 387,334, filed December 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy, created on November 20, 2023, is named AT-056_02WO_SL.xml and is 41,688 bytes in size.

[0003] The present disclosure, in some aspects, relates to anti-idiotypic (anti-id) antibodies that specifically recognize anti-CD70 antibody portions, particularly anti-CD70 antibody portions present in recombinant receptors, including chimeric antigen receptors (CARs). The present disclosure further relates to the use of anti-idiotypic antibodies to specifically identify, quantitate, or select cells expressing such recombinant receptors, such as, for example, anti-CD70 CAR T cells. The present disclosure further relates to the use of anti-idiotypic antibodies to specifically activate such cells. BACKGROUND OF THE INVENTION

[0004] Anti-idiotypic antibodies are a subset of antibodies raised against immunizing antibodies. These anti-idiotypic antibodies exhibited specific binding to the idiotope (a unique antigenic determinant on the surface of an antibody) of the immunizing antibody. Anti-idiotypic antibodies can generally be classified into three distinct groups: (1) antibodies that recognize idiotopes distinct from the antigen-binding site (ABS) on the immunizing antibody, (2) antibodies that recognize epitopes within the ABS and mimic the structure of the nominal antigen, and (3) antibodies that recognize epitopes within the ABS without mimicking the structure of the nominal antigen (see, e.g., Pan et al., (1995) FASEB J 9:43-49).

[0005] Traditional methods for detecting CAR expression, such as anti-CD70 CAR expression (e.g., using soluble human CD70-Fc fusion proteins), can underestimate the true number of CAR-expressing cells due to lack of specificity, batch-to-batch variability, low signal intensity in analytical methods (e.g., flow cytometry), and difficulty in producing sufficient quantities.

[0006] Thus, there is a need for robust reagents that accurately detect anti-CD70 CAR expression in engineered T cells or in vitro assays. Provided herein are methods and compositions that address this and other needs. Summary of the Invention

[0007] Provided herein are agents that specifically bind to antibodies, including antibody fragments, such as single-chain variable fragments (scFv), and chimeric molecules containing antibody fragments, such as chimeric antigen receptors. Also provided herein, among other embodiments, are uses and methods of using such agents, including the detection, use, manipulation, and / or stimulation of cells or therapies containing or suspected of containing the antibody or chimeric molecule, such as the detection, stimulation, or use of CAR-expressing cells.

[0008] In some embodiments, the antibody is, or comprises, 4F11, an anti-idiotypic (anti-id) antibody or antigen-binding fragment thereof that specifically binds to a target antibody that is, or contains, the variable region(s) of the antibody designated 4F11, and / or specifically binds to a chimeric molecule containing an antibody fragment such as a CAR having a binding domain containing an antibody variable region or portion thereof derived from 4F11, such as in the form of an scFv.

[0009] In some embodiments, the anti-idiotype antibody is a non-blocking anti-idiotype antibody, e.g., binding of the anti-idiotype antibody to a target antibody, e.g., an anti-CD70 antibody, does not block binding of the target antibody to a target antigen, e.g., CD70.

[0010] In some embodiments, the anti-idiotype antibody is a blocking anti-idiotype antibody, e.g., an anti-idiotype antibody binding to a target antibody, e.g., an anti-CD70 antibody, that blocks or reduces binding of the target antibody to a target antigen, e.g., CD70.

[0011] In some embodiments, an anti-CD70 scFv derived from 4F11 comprises an amino acid sequence of SEQ ID NO: 35 that has at least 80%, at least 90%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the isolated antibody does not bind to a framework region of 4F11. In some embodiments, the isolated antibody has a dissociation constant K of 10 nM or less, 5 nM or less, 1 nM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, or 20 pM or less as determined by a Biacore assay at 25°C. D and binds to an anti-CD70 scFv derived from 4F11. In various embodiments, the antigen-binding molecule is selected from the group consisting of an antibody, scFv, Fab, Fab', Fv, F(ab'), dAb, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, IgE antibody, IgD antibody, IgM antibody, IgG1 antibody, IgG1 antibody with at least one mutation in the hinge region, IgG2 antibody, IgG2 antibody with at least one mutation in the hinge region, IgG3 antibody, IgG3 antibody with at least one mutation in the hinge region, IgG4 antibody, IgG4 antibody with at least one mutation in the hinge region, an antibody comprising at least one non-naturally occurring amino acid, and any combination thereof.

[0012] In further embodiments, the isolated anti-idiotype antibody comprises a heavy chain (HC), and in particular embodiments, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 2. Further particular embodiments of the antibodies provided herein comprise a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 6, 7, and 8. Additional particular embodiments of the antibodies provided herein comprise a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 9 and 10. Still other embodiments of the antibodies provided herein comprise a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 11. In still further embodiments, the heavy chain of the antibodies provided herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and each CDR comprises the amino acid sequence set forth in Table 1c.

[0013] In some embodiments, the anti-idiotype antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VH of an antigen-binding molecule provided herein.

[0014] In some embodiments, the isolated anti-idiotype antibodies provided herein comprise a light chain (LC), and in various embodiments, the LC comprises the light chain variable region (VL) sequence of SEQ ID NO: 4. In some embodiments, the light chain variable region (VL) of the antibodies provided herein comprises one or more of (a) CDR1, (b) CDR2, and (c) CDR3. In more specific embodiments, the light chain CDR1 of the antibodies provided herein may comprise SEQ ID NO: 12. In other embodiments, the light chain CDR2 of the antibodies provided herein may comprise SEQ ID NO: 13. In yet further embodiments, the light chain CDR3 of the antibodies provided herein may comprise SEQ ID NO: 14. In still further embodiments, the light chain of the antibodies provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and each CDR comprises the amino acid sequence of Table 1d.

[0015] In some embodiments, provided herein are VL amino acid sequences that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VL of an antigen-binding molecule.

[0016] In some embodiments, the anti-idiotype antibodies provided herein comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 2, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, the antibodies provided herein comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 7, or 8, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 9 or 10, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0017] In further embodiments, the isolated anti-idiotype antibody comprises a heavy chain (HC), and in particular embodiments, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 21. Further particular embodiments of the antibodies provided herein comprise a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 25, 26, and 27. Additional particular embodiments of the antibodies provided herein comprise a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 28 and 29. Still other embodiments of the antibodies provided herein comprise a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 30. In still further embodiments, the heavy chain of the antibodies provided herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and each CDR comprises the amino acid sequence set forth in Table 1c.

[0018] In some embodiments, the anti-idiotype antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VH of an antigen-binding molecule provided herein.

[0019] In some embodiments, the isolated anti-idiotype antibodies provided herein comprise a light chain (LC), and in various embodiments, the LC comprises the light chain variable region (VL) sequence of SEQ ID NO: 23. In some embodiments, the light chain variable region (VL) of the antibodies provided herein comprises one or more of (a) CDR1, (b) CDR2, and (c) CDR3. In more specific embodiments, the light chain CDR1 of the antibodies provided herein may comprise SEQ ID NO: 31. In other embodiments, the light chain CDR2 of the antibodies provided herein may comprise SEQ ID NO: 32. In yet further embodiments, the light chain CDR3 of the antibodies provided herein may comprise SEQ ID NO: 33. In still further embodiments, the light chain of the antibodies provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and each CDR comprises the amino acid sequence of Table 1d.

[0020] In some embodiments, provided herein are VL amino acid sequences that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VL of an antigen-binding molecule.

[0021] In some embodiments, the anti-idiotype antibodies provided herein comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the antibodies provided herein comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, 26, or 27, (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 28 or 29, (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 31, (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 33.

[0022] In some embodiments, the anti-idiotype antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VH of an antigen-binding molecule provided herein.

[0023] In some embodiments, the VL amino acid sequence is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VL of an antigen-binding molecule provided herein.

[0024] In some embodiments, the anti-idiotype antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the VH of an antigen-binding molecule provided herein.

[0025] In some of any such embodiments, the targeting antibody or antigen-binding fragment is a single-chain fragment. In some aspects, the fragment contains antibody variable regions linked by a flexible linker. In some of any such embodiments, the fragment contains an scFv.

[0026] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope of the target antibody or antigen-binding fragment thereof that is the same as or overlaps with an epitope specifically bound by an anti-id antibody or antigen-binding fragment thereof according to any one of the embodiments described herein.

[0027] In some of any such embodiments, the targeting antibody or antigen-binding fragment is within or contained in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor, and / or the anti-id antibody or antigen-binding fragment specifically binds to the targeting antibody or antigen-binding fragment contained within or contained in the antigen-binding domain of the extracellular portion of the CAR. In some embodiments, the targeting antibody or antigen-binding fragment is an scFv, and the anti-id antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR.

[0028] In some of any such embodiments, the antibody or fragment specifically binds to an scFv derived from antibody 4F11 contained in the extracellular portion of the chimeric antigen receptor, and optionally the scFv derived from antibody 4F11 comprises a heavy chain variable region set forth in SEQ ID NO: 15 and / or a light chain variable region set forth in SEQ ID NO: 16, and / or comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0029] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope within or comprising all or a portion of a complementarity-determining region (CDR) of the target antibody or antigen-binding fragment.

[0030] In some of any such embodiments, the anti-idiotypic antibody or fragment is an antagonist antibody of the CAR containing the targeting antibody or antigen-binding fragment. In some of any such embodiments, the antibody or fragment is an antagonist of the CAR containing the targeting antibody or antigen-binding fragment.

[0031] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is monoclonal.

[0032] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from among a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable fragment (scFv), or a single domain antibody.

[0033] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof contains at least a portion of an immunoglobulin constant region. In some embodiments, the at least a portion of the immunoglobulin constant region contains an Fc region or a portion of an Fc containing a CH2 domain and a CH3 domain. In some aspects, the constant region is derived from human IgG. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is an intact antibody or a full-length antibody.

[0034] Provided herein is a vector containing a nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell containing an anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, or a nucleic acid molecule according to any one of the embodiments described herein.

[0035] Provided herein is a method for producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising expressing a heavy chain and / or a light chain encoded by a nucleic acid molecule according to any one of the embodiments described herein, or a vector according to any one of the embodiments described herein, in a suitable host cell, and recovering or isolating the antibody. In some embodiments, the method for producing an anti-idiotype antibody or antigen-binding fragment comprises culturing a cell according to any one of the embodiments described herein under conditions in which the heavy chain and / or the light chain is expressed, and recovering or isolating the antibody. Also provided herein is an anti-idiotype antibody or antigen-binding fragment thereof produced by a method according to any one of the embodiments described herein.

[0036] Also provided are methods for producing isolated antibodies disclosed herein that specifically bind to a molecule comprising an anti-CD70 scFv comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16 (e.g., an anti-CD70 scFv comprising the amino acid sequence of SEQ ID NO: 35, an anti-CD70 scFv comprising the amino acid sequence of SEQ ID NO: 35, or an anti-CD70 scFv comprising the amino acid sequence of SEQ ID NO: 16), wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 35 or the anti-CD70 chimeric antigen receptor ("CAR") comprises the amino acid sequence of SEQ ID NO: 1 ("4F11 CAR") or 34 ("4F11-RSRQR" or "4F11-QR3," where "R" refers to the rituximab-recognized epitope and "Q" refers to the CD34 epitope recognized by QBEND10), the methods comprising growing or culturing, under suitable conditions, cells comprising a polynucleotide encoding such isolated antibodies.

[0037] Also provided are detection methods using any of the provided agents, such as anti-idiotype antibodies. In some embodiments, methods are provided for detecting a target antibody or antigen-binding fragment thereof, e.g., a CAR containing the same, comprising: (a) contacting a composition containing a target antibody (e.g., one having a variable region derived from antibody 4F11, or an antigen-binding fragment of 4F11) with an anti-idiotype antibody or antigen-binding fragment thereof; and (b) detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment, and / or detecting the presence or absence of the target antibody or agent.

[0038] In some embodiments, provided methods involve detecting a CAR containing a targeting antibody or antigen-binding fragment thereof of any of the embodiments, such as a CAR containing a variable domain derived from 4F11. In some aspects, the method includes (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a targeting antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or antigen-binding fragment thereof 4F11 described in any one of the described embodiments, or a conjugate described in any one of the described embodiments, which specifically binds to the targeting antibody that is antibody 4F11 or an antigen-binding fragment thereof, and (b) detecting cells bound to the anti-idiotype antibody. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection.

[0039] In some embodiments, a method of selecting cells from a cell population is provided, the method comprising: (a) contacting a cell population expressing a chimeric antigen receptor (CAR) containing a targeting antibody or cells bound to the targeting antibody with an anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, or a conjugate of any one of the embodiments described herein, that specifically binds to a targeting antibody, wherein the targeting antibody is antibody 4F11 or an antigen-binding fragment thereof; and (b) selecting cells that bind to the anti-idiotype antibody.

[0040] In some examples, cells bound by the anti-idiotype antibody are selected by affinity-based separation. In some aspects, the affinity-based separation is immunoaffinity-based separation. In some of any such embodiments, the affinity-based separation is by flow cytometry.

[0041] Also provided are methods for stimulating cells using agents, such as stimulating cells containing a molecule such as a CAR that is or contains a targeting antibody recognized by an anti-idiotype antibody. In some aspects, the method comprises incubating an input composition containing cells expressing a chimeric antigen receptor (CAR) containing a targeting antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or antigen-binding fragment thereof 4F11 of any one of the described embodiments, or a conjugate of any one of the described embodiments, that specifically binds to the targeting antibody that is antibody 4F11 or an antigen-binding fragment thereof, thereby generating an output composition containing stimulated cells.

[0042] In some embodiments, the method results in proliferation, activation, stimulation, cytokine release, or other functional outcome, such as upregulation of activation markers, or cytokine release or production, of cells expressing a chimeric receptor, such as a CAR, that is recognized by an anti-id antibody.

[0043] In some aspects, the CAR contains a targeting antibody that specifically binds to CD70. In some embodiments, the targeting antibody is antibody 4F11 or an antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an anti-idiotype antibody or antigen-binding fragment thereof described in any one of the described embodiments that specifically binds to a targeting antibody that is antibody 4F11 or an antigen-binding fragment thereof.

[0044] In some such embodiments, the targeted antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:15 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:16.

[0045] In some embodiments, a method for purifying an antibody or antigen-binding fragment thereof is provided, comprising the steps of: (a) contacting a composition or sample containing a target antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, or a conjugate of any one of the embodiments described herein, which specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof; and (b) isolating the complex containing the anti-idiotype antibody. In some embodiments, the method comprises: (a) contacting a composition or sample containing a target antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, or a conjugate of any one of the embodiments described herein, which specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof; and (b) isolating the complex containing the anti-idiotype antibody.

[0046] In some of any such embodiments, the antigen-binding fragment contains the variable heavy chain region and / or the variable light chain region of the target antibody. In some embodiments, the antigen-binding fragment is a single-chain fragment. In some aspects, the antigen-binding fragment is an scFv. In some of any such embodiments, the antigen-binding fragment is within or contained in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR).

[0047] In some of any such embodiments, the targeting antibody binds to CD70. In some embodiments, the antigen-binding fragment of the targeting antibody is derived from antibody 4F11, and optionally, the antigen-binding fragment of the targeting antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. In some such embodiments, the antigen-binding fragment of the targeting antibody is a single-chain variable fragment (scFv) derived from antibody 4F11, and optionally, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0048] In some embodiments, a method of depleting cells is provided, the method comprising administering to a subject a composition comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, which specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof, or a conjugate of any one of the embodiments described herein, wherein the subject is administered cells expressing a chimeric antigen receptor (CAR) comprising the target antibody that is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the method comprises administering to a subject a composition comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, which specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof, or a conjugate of any one of the embodiments described herein, wherein the subject is administered cells expressing a chimeric antigen receptor (CAR) comprising the target antibody that is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the depletion occurs via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0049] In various embodiments, the antibodies provided herein further comprise a detectable label, which may be selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radiolabel, and a hapten.If fluorescent labeling is desired, the fluorescent label may be Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhocamin B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutant), GFP ( Y66F mutant), EBFP, EBFP2, Azurite, GFPUv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutant), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutant), Greenish cyan, wild-type GFP, GFP (S65C mutant), TurboGFP, TagGFP, GFP (S6 5L mutant), Emerald, GFP (S65T mutant), EGFP, Thistle Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (RFP), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-Phycoerythrin (RPE), B-Phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In certain embodiments, the fluorescent label can be R-phycoerythrin (PE) or allophycocyanin (APC).

[0050] Also provided herein are compositions comprising the antibodies provided herein and, optionally, a pharmaceutically acceptable carrier or vehicle.

[0051] Provided herein is a polynucleotide encoding the heavy chain of an isolated antibody of the antibody provided herein. Additionally, a polynucleotide encoding the light chain of an isolated antibody of the antibody provided herein is also provided. Also provided is a vector comprising a polynucleotide encoding the heavy chain of an isolated antibody of the antibody provided herein and a polynucleotide encoding the light chain of an isolated antibody of the antibody provided herein. Also provided is a cell comprising such a vector, and in various embodiments, the cell comprises a cell selected from the group consisting of a CHO cell, an Sp2 / 0 cell, a rabbit cell, and an Escherichia coli (E. coli) cell.

[0052] Methods of producing the isolated antibodies provided herein are also provided and can include incubating the cells provided herein under appropriate conditions.

[0053] Methods for determining the number of cells expressing a 4F11-derived scFv are provided and may include contacting a sample with an isolated antibody that specifically binds to a 4F11-derived scFv linked to a detectable label, and determining the number of cells expressing the 4F11-derived scFv in the sample. In some embodiments, the isolated antibody that specifically binds to a 4F11-derived scFv is an antibody provided herein or a humanized form thereof.

[0054] Also provided is a method for determining the number of cells that present a polypeptide comprising an anti-CD70 scFv derived from 4F11, the method comprising: (a) providing a sample containing cells known or suspected of presenting a polypeptide comprising an anti-CD70 scFv derived from 4F11; (b) contacting the sample with the isolated antigen-binding molecule provided herein, or a humanized form thereof, under conditions that allow binding of the polypeptide to the antigen-binding molecule; and (c) determining the number of cells that present the polypeptide in the sample.

[0055] Provided herein is a method for determining the presence or absence of a polypeptide comprising a 4F11-derived anti-CD70 scFv. The method comprises: (a) providing a sample known or suspected to contain a polypeptide comprising a 4F11-derived anti-CD70 scFv; (b) contacting the sample with an isolated antigen-binding molecule provided herein or a humanized form thereof under conditions that allow binding of the polypeptide to the antigen-binding molecule; and (c) detecting the presence or absence of a polypeptide:antigen-binding molecule complex. In one embodiment of this method, the sample is a formalin-fixed sample. In another embodiment, the 4F11-derived scFv is a component of a chimeric antigen receptor (CAR). In a further embodiment, the cell expressing the 4F11-derived scFv CAR is an immune cell selected from the group consisting of CD8+ T cells, CD4+ T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. In some embodiments, the isolated antigen-binding molecule is detectably labeled, and the detectable label may be selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radiolabel, and a hapten.When the detectable label is a fluorescent label, the fluorescent label may be Atto dye, Alexafluor dye, quantum dot, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine-Rhocamin B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutant), GFP ( Y66F mutant), EBFP, EBFP2, Azurite, GFPUv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutant), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutant), Greenish cyan, wild-type GFP, GFP (S65C mutant), TurboGFP, TagGFP, GFP (S6 5L mutant), Emerald, GFP (S65T mutant), EGFP, Thistle Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (RFP), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-Phycoerythrin (RPE), B-Phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In certain embodiments, the fluorescent label is R-phycoerythrin (PE) or allophycocyanin (APC).In some embodiments, the cells expressing the 4F11-derived scFv CAR are immune cells, and the immune cells are T cells, which can be located in vitro or in vivo. In some embodiments, the T cells are located in blood, excised tissue, ex vivo cultured tissue, or cell culture medium. In one embodiment, the T cells are autologous T cells. In another embodiment, the T cells are allogeneic T cells.

[0056] In a further aspect, provided herein are methods of making and using anti-idiotype antibodies. In a related aspect, provided herein are methods of quantifying or determining the number of cells that express an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NOs: 15 and 16, the method comprising contacting a cell with an anti-idiotype antibody disclosed herein and determining the number of cells that express the anti-CD70 antibody in the cell.

[0057] In another aspect, the disclosure provides a method for determining the presence or absence of a cell expressing an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NO: 15 and / or 16, the method comprising contacting the cell with an isolated antibody disclosed herein and determining the presence or absence of a cell expressing the anti-CD70 antibody in the cell. In yet another aspect, the disclosure provides a method for purifying an anti-CD70 antibody or antigen-binding fragment thereof, wherein the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 15 and / or 16, the method comprising: (a) contacting a sample comprising the anti-CD70 antibody with an anti-idiotypic antibody disclosed herein to form a complex; and (b) isolating or purifying the complex comprising the anti-CD70 antibody and the isolated antibody from the sample. In some embodiments, the anti-CD70 antibody is an scFv comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the cell expresses an anti-CD70 chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34. In some embodiments, the cell is a CAR T cell.

[0058] Further provided are methods for selecting cells expressing an anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34, the method comprising contacting the cells with an anti-idiotype antibody described herein and selecting cells that bind to the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody further comprises a detectable label. In some embodiments, cells that bind to the anti-idiotype antibody are selected by affinity-based separation.

[0059] Also provided are methods of stimulating anti-CD70 CAR T cells comprising contacting the anti-CD70 CAR T cells with an antibody described herein. In some embodiments, the antibody is conjugated to a bead or solid surface. In certain embodiments, the anti-CD70 CAR T cells are present in a drug substance or drug product. [Brief explanation of the drawings]

[0060] [Figure 1A-1B] Flow cytometry determination of the specificity of anti-idiotypic antibodies clone 37 (Figure 1A, first and second row panels viewed from above) and clone 60 (Figure 1A, third and fourth row panels viewed from above, and Figure 1B). [Figure 2A-2B] Flow cytometric comparison of the anti-idiotypic antibody clone 60 conjugated to PE (phycoerythrin, Fig. 2A) or unconjugated (Fig. 2B), either recombinantly produced or purified from hybridoma cultures. [Figure 3] Flow cytometry demonstration of simultaneous binding of anti-id antibodies to CD70 CAR, or blocking of binding of anti-id antibodies to CD70 CAR, in the presence (top panel) or absence (bottom panel) of target antigen CD70 protein. [Figure 4A-4B] Flow cytometric demonstration of simultaneous binding with target antigen or blocking. NTD, non-transduced. [Figure 5] Surface plasmon resonance determination of affinity. [Figures 6A-6B] Surface plasmon resonance determination of simultaneous binding with the target antigen hCD70. DETAILED DESCRIPTION OF THE INVENTION

[0061] Provided herein are agents, such as anti-idiotypic (anti-id) antibodies and antigen-binding fragments (such as single-chain fragments including Fv, Fab, F(ab')2, single-domain Ab, VHH, or scFv) that specifically recognize anti-CD70 antibody portions (such as anti-CD70 antibody portions present in recombinant receptors, including chimeric antigen receptors). Also provided are uses and methods of use, as well as compositions and articles of manufacture, that include such agents, including those for specifically identifying, quantifying, selecting, isolating, purifying, and / or stimulating and / or activating cells that express or contain the target antibody or fragment, such as anti-CD70 CAR T cells. In some embodiments, the provided antibodies may be used to identify, quantify, and / or select various anti-CD70 CARs, such as CARs bound to or expressed on the cell surface, and may also be used to specifically activate cells that express the target CAR, such as CAR T cells. In some embodiments, antibodies specific to the anti-CD70 antibody designated 4F11, or antibody fragments derived therefrom, are provided, including antibodies containing variable regions derived from said antibody and CARs, and / or antibodies containing idiotopes contained therein.

[0062] In some embodiments, the provided anti-idiotypic antibodies offer advantages over conventional reagents for detecting, identifying, manipulating, and / or influencing and / or manipulating cells expressing a CAR, particularly CARs containing an anti-CD70 antibody scFv extracellular domain, or CARs containing a recognized idiotype. In certain available methods, detecting the presence, absence, or amount of a CAR or CAR-expressing cell in a sample (and / or CAR or CAR-expressing cell stimulation or manipulation) is performed by assessing the presence, absence, or amount of a surrogate molecule, such as one included in a construct encoding the CAR and thus serving as an indirect or surrogate marker for its expression.

[0063] In some embodiments, the provided anti-idiotypic antibodies and antigen-binding fragments overcome the challenges of low binding affinity associated with target antibody ligands and nonspecific binding associated with antibody reagents directed against target antibody constant regions, providing reagents with both high affinity and specificity for their target antibodies or antigen-binding fragments. In some embodiments, the provided antibodies exhibit higher specificity and binding affinity for their target antibodies or antigen-binding fragments, such as the anti-CD70 antibody designated 4F11, compared to CD70-Fc and other reagents currently available for detecting or identifying CARs, including anti-CD70 antibodies.

[0064] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), as well as any other modified configuration of an immunoglobulin molecule containing an antigen recognition site, including, but not limited to, single-chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising antibodies. Antibodies include antibodies of any class (or subclass thereof), such as IgG, IgA, or IgM; an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG1, and IgG2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0065] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single-domain antibody (dAb) fragment (see, e.g., Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0066] An antibody, antibody conjugate, or polypeptide that "specifically binds" to a target is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for longer duration, and / or with higher affinity than with another cell or substance. An antibody "specifically binds" to a target if it binds with higher affinity, avidity, more readily, and / or for longer duration than it binds to other substances. By reading this definition, it is also understood that, for example, an antibody (or moiety or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding.

[0067] Chimeric antigen receptor (CAR) can refer to a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). Upon binding to the target antigen, the CAR expressed on an immune cell can activate the immune cell to attack and destroy cells bearing that antigen (e.g., cancer cells). CARs can also incorporate costimulatory or signaling domains to increase their efficacy. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797; Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0068] CARs can be expressed on the surface membrane of cells. Thus, CARs can contain a transmembrane domain. Suitable transmembrane domains for the CARs disclosed herein have (a) the ability to be expressed on the surface of cells, for example, immune cells, such as, but not limited to, lymphocytes (e.g., T cells) or natural killer (NK) cells, and (b) the ability to interact with a ligand binding domain and an intracellular signaling domain to induce a cellular response of the immune cell against a predetermined target cell. The transmembrane domain can be derived from either natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a domain of a T cell receptor, such as the α, β, γ, or δ polypeptide that constitutes the CD3 complex, an IL-2 receptor, for example, p55 (α chain), p75 (β chain or γ chain), an Fc receptor, particularly a subunit chain of Fcγ receptor III, or a CD protein. Alternatively, the transmembrane domain can be synthetic and primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1). The transmembrane domain may further contain a stalk domain between the extracellular ligand-binding domain and the transmembrane domain. The stalk domain can comprise up to 300 amino acids, e.g., 10 to 100 amino acids or 25 to 50 amino acids. The stalk region may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, the stalk domain may be a synthetic sequence corresponding to a natural stalk sequence, or may be an entirely synthetic stalk sequence. In some embodiments, the stalk domain is a portion of the human CD8 α chain (e.g., NP_001139345 and its isoforms). In another specific embodiment, the transmembrane domain comprises a portion of the human CD8 α chain. In some embodiments, the CAR can be introduced into immune cells as a transgene via a vector (e.g., a plasmid vector).In some embodiments, vectors, eg, plasmid vectors, can also contain a selectable marker, eg, that provides for identification and / or selection of cells that have received the vector.

[0069] The intracellular (cytoplasmic) domain of a CAR of the present disclosure can provide for activation of at least one normal effector function of an immune cell involving the CAR, e.g., signal 1 / activation, and / or signal 2 / costimulation. Effector function of a T cell can refer to, for example, cytolytic activity or helper activity, including cytokine secretion.

[0070] In some embodiments, activating intracellular signaling domains for use in CARs may be, for example, but not limited to, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same function.

[0071] Suitable (e.g., activation) intracellular domains include, but are not limited to, CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokines, and the like. Kine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domains comprise a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0072] In addition to the activation domain described above, the intracellular domain of the CAR of the present disclosure can incorporate a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule or costimulatory domain) to increase its efficacy. The costimulatory domain can provide a signal in addition to the primary signal provided by the activation molecule described herein.

[0073] As used herein, a "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In certain embodiments, an anti-idiotype antibody is multispecific. Multispecific binding molecules include, for example, multispecific antibodies, including bispecific antibodies. Multispecific binding partners, such as antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for the anti-CD70 antibody moiety, and the other is for a different antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of the anti-CD70 antibody moiety. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing the anti-CD70 antibody moiety on their surface, such as anti-CD70CAR T cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Bispecific antibodies include multispecific single-chain antibodies, such as diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs.

[0074] Furthermore, in certain embodiments, anti-idiotype antibodies and antigen-binding fragments may be selected as agonists or antagonists of chimeric receptors comprising their target antibodies or antigen-binding fragments, allowing for selective detection, isolation, ablation, and / or depletion (e.g., killing via antibody-dependent cell-mediated cytotoxicity, ADCC), and / or stimulation or activation of cells bearing such chimeric receptors bound to or expressed on the surface of cells. In some embodiments, anti-idiotype antibodies may be humanized or fully human. Provided herein are anti-idiotype antibody agonists that exhibit activity to stimulate (e.g., activate) CARs comprising the extracellular binding domain derived from the anti-CD70 antibody designated 4F11. In some aspects, such antibodies may be used in methods for stimulating and expanding specific CAR-expressing cells, including processes for generating and preparing CAR-expressing cells. In some embodiments, the stimulation and expansion methods may be in vitro methods. In some embodiments, the stimulation and expansion methods may be integrated into a CAR T manufacturing process. In some embodiments, the stimulation and expansion methods may be in vivo methods. In some embodiments, the anti-idiotype antibody may be a humanized or fully human antibody.

[0075] Also provided are nucleic acids encoding the anti-idiotype antibodies and fragments provided herein, and cells, such as recombinant cells, for expressing and producing these anti-idiotype antibodies and fragments. Also provided are methods of making and using the anti-idiotype antibodies and fragments, and cells that express or contain the anti-idiotype antibodies and fragments.

[0076] The scFv portion of some chimeric antigen receptors (CARs) is derived from the fully human antibody clone 4F11, which has high affinity for CD70. The present disclosure provides reagents for detecting anti-CD70 CARs containing scFv portions derived from antibody clone 4F11. Disclosed herein are anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11. The anti-id antibodies against the 4F11-derived molecules disclosed herein exhibit specific, high-affinity binding to chimeric antigen receptors (CARs) containing 4F11-derived scFvs. A non-limiting example of a 4F11-derived scFv comprises the amino acid sequence of SEQ ID NO: 35.

[0077] When conjugated to a bright fluorescent dye, the anti-id antibodies disclosed herein stain cells expressing chimeric antigen receptors (CARs) containing 4F11-derived scFvs with high MFI and low background. The antibodies described herein can be used in methods for detecting anti-CD70 CAR expression. These antibodies can be used for identification by both flow cytometry and immunohistochemistry. These antibodies can also be used in the production of immune cells containing 4F11-derived scFvs in connection with non-clinical studies, as clinical flow-based pharmacokinetic reagents, and in clinical immunogenicity studies.

[0078] 4F11 is a CD70 monoclonal antibody that recognizes human CD70. Single-chain variable fragments (scFvs) formed from 4F11 contain several chimeric antigen receptor (CAR) targeting components. In some embodiments, scFvs derived from CD70 monoclonal antibody 4F11 comprise a portion of the CD70 monoclonal antibody 4F11 immunoglobulin gamma 1 heavy chain (SEQ ID NO: 15) and a portion of the anti-CD70 monoclonal antibody 4F11 immunoglobulin kappa light chain (SEQ ID NO: 16), linked together by a flexible linker. In some embodiments, the scFv comprises a variable fragment of the anti-CD70 monoclonal antibody 4F11 immunoglobulin gamma 1 heavy chain and a variable fragment of the anti-CD70 monoclonal antibody 4F11 immunoglobulin kappa light chain, linked together by a flexible linker.

[0079] The anti-CD70 monoclonal antibody 4F11 immunoglobulin gamma 1 heavy chain variable domain comprises the amino acid sequence ("4F11 VH"), with bolded residues indicating CDR sequences according to the Kabat naming system, and underlined residues indicating CDR sequences according to the Chothia naming system. QVTLKESGPVLVKPTETLTLTCTVSGFSLS NARMGVT WIRQPPGKALEWLA HIFSNDEKSYSTSLKS RLTISKDTSKTQVVLTMTTNMDPVDTATYYCAR IRDYYDISSYYDY WGQGTLVSVSS (SEQ ID NO: 15)

[0080] The anti-CD70 monoclonal antibody 4F11 immunoglobulin kappa light chain variable domain comprises the amino acid sequence (4F11 VL), with bolded residues indicating CDR sequences according to the Kabat naming system, and underlined residues indicating CDR sequences according to the Chothia naming system. DIQMTQSPSAMSASVGDRVTITC RASQDISNYLA WFQQKPGKVPKRLIY AASSLQS GVPSRFSGSGSGSGTEFTLTISSLLPEDFATYC LQLNSFPFT FGGGTKVEIN (SEQ ID NO: 16)

[0081] An exemplary 4F11-derived chimeric antigen receptor ("CAR") comprises, or alternatively consists of, the following amino acid sequence (underlined is the exemplary signal peptide MALPVTALLLPLALLLHAARP SEQ ID NO:39): MALPVTALLLPLALLLHAARP QVTLKESGPVLVKPTETLTLTCTVS GFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASV GDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1) or the mature protein without the signal peptide. QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLV SVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGG GTKVEINTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 37).

[0082] A second exemplary 4F11-derived chimeric antigen receptor ("CAR") comprises, or alternatively consists of, the following amino acid sequence (underlined is an exemplary signal peptide, e.g., from CD8a): MALPVTALLLPLALLLHAARP (SEQ ID NO: 34) or the mature protein without the signal peptide. (SEQ ID NO: 38).

[0083] In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16, with or without a signal sequence. In some embodiments, the scFv has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16 and / or SEQ ID NO: 35.

[0084] An exemplary 4F11-derived scFv comprises, or alternatively consists of, the following amino acid sequence: QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEIN (SEQ ID NO: 35)

[0085] An exemplary 4F11-derived scFv with a His tag comprises the following amino acid sequence: QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINGGSGHHHHHHHHHH (SEQ ID NO: 36)

[0086] In some embodiments, the scFv has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 34. Disclosed herein are antigen-binding molecules comprising antibodies that specifically bind to anti-CD70 scFv derived from 4F11, as well as molecules comprising these sequences, and cells that display such molecules. Humanized forms of the antigen-binding molecules are also embodiments of the present disclosure. Applications and uses of these antigen-binding molecules are also disclosed.

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0088] I. Antigen-binding molecules and polynucleotides encoding antigen-binding molecules As used herein, "antigen-binding domain" refers to any polypeptide that binds to a specific target antigen; for example, the specific target antigen may be the CD70 protein or a fragment thereof (interchangeably referred to herein as a "CD70 antigen," "CD70 target antigen," or "CD70 target"). In the context of the anti-idiotype antibodies of the present disclosure, the target antigen is an antigen-binding molecule that specifically binds to CD70 (e.g., antibody clone 4F11, and antigen-binding molecules derived from or related to 4F11, including scFvs).

[0089] In some embodiments, the antigen binding domain binds to the CD70 antigen on a tumor cell. In some embodiments, the antigen binding domain binds to the CD70 antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen.

[0090] Antigen-binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen-binding domain refers to a species of antigen-binding domain that contains a portion of an antibody (regardless of how the portion is obtained or synthesized) that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to a target antigen. Such fragments are biologically active in that they can bind to a target antigen and compete with other antigen-binding domains, including intact antibodies, for binding to a given epitope. In some embodiments, the fragment is a neutralizing fragment. In some embodiments, the fragment can block or reduce the activity of an anti-CD70 CAR (e.g., a blocking effect). In some embodiments, the fragment can antagonize the activity of an anti-CD70 CAR.

[0091] In certain embodiments, the anti-id antibody of the present disclosure is the antibody identified herein as clone 60, or clone 37, each of which comprises the heavy and light chain amino acids, variable regions, CDR sequences, and nucleotide sequences encoding such sequences, as presented and labeled herein.

[0092] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (such as Fab', F(ab')2), one or more complementarity-determining regions ("CDRs"), diabodies (a light chain variable domain and a heavy chain variable domain on the same polypeptide linked via a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen-binding domains (comprising two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments may be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. As will be appreciated by those skilled in the art, antigen-binding domains may comprise non-proteinaceous components.

[0093] Variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (CDRs). The CDRs of the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the N-terminus to the C-terminus, both light chain variable regions and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are typically referred to as LC CDR1, CDR2, and CDR3.

[0094] In some embodiments, an antigen-binding domain comprises one or more complementary binding regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments, a single heavy and / or light chain or portion thereof. These fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the antigen-binding domain comprising an intact antibody.

[0095] In some embodiments, the antigen-binding domain is an antibody fragment thereof, comprising one or more of its complementarity-determining regions (CDRs), hi some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3.

[0096] The assignment of amino acids to each of the framework, CDR, and variable domains typically follows Kabat numbering (see, e.g., Kabat et al., in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda, Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), contact numbering, or the AbM scheme (Antibody Modeling program, Oxford Molecular).

[0097] Thus, in some embodiments, the CDRs of the anti-idiotype antibodies presented herein are numbered according to the Kabat numbering scheme. In other embodiments, the CDRs of the anti-idiotype antibodies presented herein are numbered according to the Chothia numbering scheme. In other embodiments, the CDRs of the anti-idiotype antibodies presented herein are numbered according to the contact numbering scheme. In other embodiments, the CDRs of the anti-idiotype antibodies presented herein are numbered according to the AbM numbering scheme.

[0098] Humanized antibodies are described herein and can be prepared by known techniques. In some embodiments, a humanized monoclonal antibody comprises the variable domain of an anti-id antibody (or all or part of its antigen-binding site) and a constant domain derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen-binding site of a mouse or rabbit monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Techniques for producing engineered monoclonal antibodies include those described, for example, in Riechmann et al. (1988) Nature 332:323, Liu et al. (1987) Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al. (1989) Bio / Technology 7:934, and Winter et al. (1993) TIPS 14:139. In some embodiments, a chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are described, for example, in U.S. Patent Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, 6,881,557, Padlan et al. (1995) FASEB J. 9:133-39, Tamura et al. (2000) J. Immunol. 164:1432-41; Zhang et al. (2005) Mol. Immunol. 42(12):1445-1451; Hwang et al., Methods. (2005) 36(1):35-42, Dall'Acqua et al. (2005) Methods 36(1):43-60, and Clark, (2000) Immunology Today 21(8):397-402.

[0099] Anti-idiotype antibody variants are also within the scope of the present disclosure, for example, variants comprising variable light chain and / or variable heavy chain domains or regions each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% homology to the amino acid sequence of the antigen-binding domain sequence described herein. In some embodiments, the anti-idiotype antibody comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% homologous to a heavy chain variable region sequence provided in Table 1a and / or a light chain variable region sequence provided in Table 1b, and optionally further comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% homologous to a heavy chain constant region sequence provided in Table 1e and / or a light chain constant region sequence provided in Table 1e.

[0100] In some instances, such molecules contain at least one heavy chain and one light chain, while in other instances, variant forms contain two variable light chains and two variable heavy chains (or portions thereof). Those skilled in the art can use well-known techniques to determine suitable variants of the anti-idiotype antibodies described herein. In certain embodiments, those skilled in the art can identify appropriate regions of the molecule that can be altered without destroying activity by targeting regions not believed to be important for activity.

[0101] The anti-id antibodies of the present disclosure may also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any of a number of techniques familiar to those of skill in the art, including, but not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of splenocytes from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other techniques known in the art and based on the disclosure herein.

[0102] Anti-id antibodies 4F11 that specifically bind to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11 are said to be "selective" if they bind to one target more strongly than they bind to a second target.

[0103] An anti-id antibody that specifically binds to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11 is said to "specifically bind" its target antigen (e.g., murine 4F11 and molecules derived from 4F11) when the dissociation constant (Kd) is approximately 1 nM. An antigen-binding domain specifically binds to an antigen with "high affinity" when the Kd is between 1 and 5 nM, and with "ultra-high affinity" when the Kd is between 0.1 and 0.5 nM. In one embodiment, the antigen-binding domain has a Kd of approximately 1 nM. In one embodiment, the off-rate is <1 x 10 -5 In other embodiments, the antigen-binding domain is about 1 x 10 -7 M~1×10 -12 M, and in yet another embodiment, the antigen-binding domain binds with a Kd of about 1 x 10 -5 ~1×10 -12 It binds with a Kd of

[0104] As provided herein, anti-id antibodies of the present disclosure specifically bind to 4F11 and 4F11-derived molecules (e.g., 4F11-derived CARs). In certain embodiments, anti-id antibodies of the present disclosure specifically bind to 4F11 and 4F11-derived molecules at 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M or 1 x 10 -9 In certain embodiments, the anti-id antibody binds 4F11 and 4F11-derived molecules with a KD of less than 1×10 -7 In another embodiment, the anti-id antibody binds 4F11 and 4F11-derived molecules with a KD of less than 1×10 -8 In some embodiments, the anti-id antibody binds 4F11 and 4F11-derived molecules with a KD of less than about 1 x 10 -7 M, approx. 2 x 10 -7 M, about 3 x 10 -7 M, approx. 4 x 10 -7 M, about 5 x 10 -7 M, about 6 x 10 -7 M, about 7 x 10 -7 M, about 8 x 10 -7 M, about 9 x 10 -7 M, about 1 x 10 -8 M, approx. 2 x 10 -8 M, about 3 x 10 -8 M, approx. 4 x 10 -8 M, about 5 x 10 -8 M, about 6 x 10 -8 M, about 7 x 10 -8 M, about 8 x 10 -8 M, about 9 x 10 -8 M, about 1 x 10 -9 M, approx. 2 x 10 -9 M, about 3 x 10 -9 M, approx. 4 x 10 -9 M, about 5 x 10 -9 M, about 6 x 10 -9 M, about 7 x 10 -9 M, about 8 x 10 -9 M, about 9 x 10 -9 M, about 1 x 10 -10 M, or approximately 5 x 10 -10 In certain embodiments, the Kd is off / K onIt is calculated as the quotient of K on and K. off is determined using a monovalent antibody, such as a Fab fragment, as measured, for example, by BIAcore® surface plasmon resonance technology. In other embodiments, Kd is off / K on It is calculated as the quotient of K on and K. off is determined using a bivalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology.

[0105] In some embodiments, the anti-id antibody is administered to 4F11 and 4F11-derived molecules at a concentration of 1×10 -4 M -1 s -1 Less than 2 x 10 -4 M -1 s -1 Less than 3 x 10 -4 M -1 s -1 Less than 4 x 10 -4 M -1 s -1 Less than 5 x 10 -4 M -1 s -1 Less than 7 x 10 -4 M -1 s -1 Less than 8 x 10 -4 M -1 s -1 Less than 9 x 10 -4 M -1 s -1 Less than 1×10 -5 M -1 s -1 Less than 2 x 10 -5 M -1 s -1 Less than 3 x 10 -5 M -1 s -1 Less than 4 x 10 -5 M -1 s -1 Less than 5 x 10 -5 M -1 s -1 Less than 6 x 10 -5 M -1 s -1 Less than 7 x 10-5 M -1 s -1 Less than 8 x 10 -5 M -1 s -1 Less than 9 x 10 -5 M -1 s -1 Less than 1×10 -6 M -1 s -1 Less than 2 x 10 -6 M -1 s -1 Less than 3 x 10 -6 M -1 s -1 Less than 4 x 10 -6 M -1 s -1 Less than 5 x 10 -6 M -1 s -1 Less than 6 x 10 -6 M -1 s -1 Less than 7 x 10 -6 M -1 s -1 Less than 8 x 10 -6 M -1 s -1 Less than 9 x 10 -6 M -1 s -1 Less than or equal to 1 x 10 -7 M -1 s -1 Association rate (k on In certain embodiments, k on is determined using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology. on is determined, for example, using bivalent antibodies measured by BIAcore® surface plasmon resonance technology.

[0106] In some embodiments, the anti-id antibody is administered to 4F11 and 4F11-derived molecules at a concentration of 1×10 -2 s -1 Less than 2 x 10 -2 s -1 Less than 3 x 10 -2 s -1 Less than 4 x 10 -2s -1 Less than 5 x 10 -2 s -1 Less than 6 x 10 -2 s -1 Less than 7 x 10 -2 s -1 Less than 8 x 10 -2 s -1 Less than 9 x 10 -2 s -1 Less than 1×10 -3 s -1 Less than 2 x 10 -3 s -1 Less than 3 x 10 -3 s -1 Less than 4 x 10 -3 s -1 Less than 5 x 10 -3 s -1 Less than 6 x 10 -3 s -1 Less than 7 x 10 -3 s -1 Less than 8 x 10 -3 s -1 Less than 9 x 10 -3 s -1 Less than 1×10 -4 s -1 Less than 2 x 10 -4 s -1 Less than 3 x 10 -4 s -1 Less than 4 x 10 -4 s -1 Less than 5 x 10 -4 s -1 Less than 6 x 10 -4 s -1 Less than 7 x 10 -4 s -1 Less than 8 x 10 -4 s -1 Less than 9 x 10 -4 s -1 Less than 1×10 -5 s -1 Less than or 5 x 10 -5 s -1 Dissociation constant (k off In certain embodiments, k off is determined using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology.off is determined, for example, using bivalent antibodies measured by BIAcore® surface plasmon resonance technology.

[0107] Provided herein are anti-idiotype antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11, comprising a variable heavy chain (VH), wherein the amino acid sequence or polynucleotide sequence of the VH is selected from the VH sequences presented in Table 1a (CDRs under the Kabat naming system or according to the Kabat naming system are in bold, and CDRs under the Chothia naming system or according to the Chothia naming system are underlined).

[0108] [Table 1a]

[0109] Provided herein are anti-idiotype antibodies that specifically bind to anti-CD70 clone 4F11, comprising a variable light chain (VL), wherein the amino acid sequence or polynucleotide sequence of the VL is selected from the VL sequences presented in Table 1b (CDRs under or according to the Kabat naming system are in bold, and CDRs under or according to the Chothia naming system are underlined).

[0110] [Table 1b]

[0111] Provided herein are anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11, wherein the anti-id antibodies comprise a variable heavy chain (VH) and a variable light chain (VL), and the amino acid sequence or polynucleotide sequence of the VH is selected from the VH sequences presented in Table 1a, and the amino acid sequence or polynucleotide sequence of the VL is selected from the VL sequences presented in Table 1b.

[0112] In some embodiments, anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprise the VH CDR1, CDR2, and CDR3 of the VH sequences listed in Table 1a. In some embodiments, the VH CDR1, CDR2, and CDR3 are selected from the CDR sequences listed in Table 1c.

[0113] [Table 1c]

[0114] In some embodiments, anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprise the VL CDR1, CDR2, and CDR3 sequences presented in Table 1d.

[0115] In some embodiments, anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprise the heavy chain constant region sequences and light chain constant region sequences presented in Table 1e.

[0116] [Table 1d]

[0117] [Table 1e]

[0118] In some of any such embodiments, the targeting antibody or antigen-binding fragment is within or contained in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor, and / or the anti-id antibody or antigen-binding fragment specifically binds to a targeting antibody or antigen-binding fragment contained within or contained in the antigen-binding domain of the extracellular portion of the CAR. In some embodiments, the targeting antibody or antigen-binding fragment is an scFv, and the anti-id antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR. In some of any such embodiments, the antibody or fragment specifically binds to an scFv derived from antibody 4F11 contained in the extracellular portion of the chimeric antigen receptor, optionally, the scFv derived from antibody 4F11 contains the heavy chain variable region set forth in SEQ ID NO: 15 and / or the light chain variable region set forth in SEQ ID NO: 16 (e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 35). In certain embodiments, the anti-id antibody or antigen-binding fragment thereof binds to an epitope of an scFv derived from 4F11, e.g., the scFv of SEQ ID NO: 35, and does not bind to the anti-CD70 antibody clone 4F11 in IgG format. In some embodiments, the anti-id antibody or antigen-binding fragment thereof differentially binds to 4F11 in scFv format and IgG format. In some embodiments, the anti-id antibody or antigen-binding fragment thereof advantageously distinguishes 4F11 in scFv format from IgG format.

[0119] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope within or comprising all or a portion of a complementarity-determining region (CDR) of the target antibody or antigen-binding fragment.

[0120] In some of any such embodiments, the anti-idiotypic antibody or fragment is an antagonist antibody of the CAR containing the targeting antibody or antigen-binding fragment. In some of any such embodiments, the antibody or fragment is an antagonist of the CAR containing the targeting antibody or antigen-binding fragment.

[0121] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is monoclonal.

[0122] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from among a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable fragment (scFv), or a single domain antibody.

[0123] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof contains at least a portion of an immunoglobulin constant region. In some embodiments, the at least a portion of the immunoglobulin constant region contains an Fc region or a portion of an Fc containing a CH2 domain and a CH3 domain. In some aspects, the constant region is derived from human IgG. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is an intact antibody or a full-length antibody.

[0124] Provided herein is a vector containing a nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell containing an anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, or a nucleic acid molecule according to any one of the embodiments described herein.

[0125] The present disclosure encompasses modifications to anti-id antibodies containing the sequences shown in Tables 1a through 1e, including functionally equivalent anti-id antibodies with modifications that do not significantly affect their properties and variants that enhance or decrease activity and / or affinity. For example, amino acid sequences can be mutated to obtain anti-id antibodies with desired binding affinities for anti-CD70 clones 4F11 and 4F11-derived antigen-binding molecules. Polypeptide modifications are routine in the art and therefore need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, deletions or additions of one or more amino acids that do not significantly adversely alter functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides using chemical analogs.

[0126] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0127] Substitutional variants have at least one amino acid residue in the antigen-binding domain removed and a different residue inserted in its place. In some embodiments, the sites of interest for substitutional mutagenesis include the hypervariable regions / CDRs, although FR changes are also contemplated. Conservative substitutions are shown under the heading "conservative substitutions" in Table 2. If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table 2, i.e., as further described below with respect to amino acid classes, may be introduced, and the products screened.

[0128] [Table 2]

[0129] II. Method for producing anti-id antibodies For cloning a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow the vector itself to replicate, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors generally contain sequence components, including but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements can be selected appropriately by those skilled in the art. For example, an origin of replication can be selected to promote autonomous replication of the vector in the host cell.

[0130] In certain embodiments, the present disclosure provides an isolated host cell containing a vector provided herein. Host cells containing the vector may be useful for expressing or cloning a polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria (such as gram-negative or gram-positive organisms), such as enterobacteria such as Escherichia coli (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, as well as Bacillus (e.g., B. subtilis, B. licheniformis), Pseudomonas aeruginosa, and Streptomyces.

[0131] Vectors can be introduced into host cells using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, biolistics, receptor-mediated gene delivery, polylysine, histones, chitosan, and peptide-mediated delivery. Standard methods for transfecting and transforming cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, with each vector encoding a different CAR disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing one or more different CARs.

[0132] In one embodiment, the present disclosure provides a method for evaluating engineered cells expressing a CAR that targets CD70. In some embodiments, the engineered cells are evaluated after thawing of cryopreserved immune cells.

[0133] In some embodiments, cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating them in a medium and container system suitable for administration in therapeutically effective amounts (a "pharmaceutically acceptable" carrier). Suitable infusion media are any isotonic media formulation, typically saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), although 5% dextrose or Ringer's lactate in water can also be utilized. Infusion media can be supplemented with human serum albumin.

[0134] In some aspects, the anti-id antibodies of the present disclosure are used to quantify a desired cell treatment dose in a composition of engineered T cells comprising a 4F11-derived CAR, e.g., an anti-CD70 CAR (such as a CAR comprising a 4F11-derived scFv) or a fragment thereof. In some embodiments, the desired therapeutic dose is generally at least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset), or more typically, 10 cells. 2 More than 10 6 pieces, maximum 10 8 pieces or 10 9 10 pieces 10 The number of cells will depend on the desired use for which the composition is intended and the type of cells contained therein. Desired cell densities are typically between 10 and 10. 6 cells / ml, generally above 10 7 cells / ml, generally above 10 8 cells / ml or more. Clinically significant numbers of immune cells are measured in the range of 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The cells can be distributed over multiple injections that cumulatively equal or exceed 100 cells. In some embodiments of the present disclosure, particularly since all injected cells are reoriented to a specific target antigen (CD70), 6 / kilogram (10 per patient) 6 ~10 11 ) can be administered. CAR therapy can be administered multiple times at doses within these ranges. Cells can be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0135] The CAR-expressing cell populations of the present disclosure can be administered as pharmaceutical compositions, alone or in combination with a diluent and / or in combination with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present disclosure can include a CAR- or TCR-expressing cell population, such as a T cell, described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure can be formulated for intravenous administration. Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: a sterile diluent such as water for injection; saline such as physiological saline, Ringer's solution, isotonic saline; fixed oils such as synthetic monoglycerides or diglycerides that may serve as a solvent or suspending medium; polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, phosphates; and agents for adjusting tonicity such as sodium chloride or glucose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. Injectable pharmaceutical compositions may be sterilized.

[0136] III. Methods for determining the number of cells expressing the CD70 CAR The present disclosure provides methods for determining the number of cells present in a sample that express an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof. For example, it may be desirable to determine the number of immune cells present in a sample obtained from a subject that expresses an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof. Alternatively, it may be desirable to determine the number of cells that have been transfected and express an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof, which can be used as a measure of the level of transfection efficiency. The methods of the present disclosure can be used for these and other applications in which it is desirable to determine the number of cells present in a sample that express a molecule of interest, such as an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0137] Thus, provided is a method for determining the number of cells presenting a molecule in a sample, wherein the molecule comprises a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0138] In some embodiments, a sample is provided that contains cells known or suspected to express a molecule of interest that includes a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0139] The sample is then contacted with an antigen-binding molecule that specifically binds to the molecule of interest under conditions that allow the formation of a binding complex containing cells present in the sample and the antigen-binding molecule. The antigen-binding molecule may be an antigen-binding molecule (or a fragment thereof) disclosed herein, for example, in the figures, sequence listing, or this section of the present disclosure. Any antigen-binding molecule that specifically binds to a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof can be used in the methods of the present disclosure. Several examples of suitable antigen-binding molecules are provided herein, including, for example, those having one or more VH and VL sequences shown in Tables 1a and 1b, or one or more CDRs shown in Tables 1c and 1d, and described herein.

[0140] The cells may be of any type and may be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In some embodiments, the cells are immune cells. The immune cells of the present methods may be of any type (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). In some embodiments, the immune cells are T cells, including T cytotoxic cells, T helper cells, and Treg cells. In certain embodiments, the cells are T cells, which may be obtained as described herein and by methods known in the art. In this embodiment of the disclosed method, any type of immune cell may be used, and the cells may be human or non-human cells (including both prokaryotic and eukaryotic cells). Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. In additional embodiments, the cells are T cells that present a CAR. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the methods of the present disclosure, the T cells can be in vivo T cells or in vitro T cells. Furthermore, the cells can be placed in or isolated from any environment that can maintain the cells in a viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo cultured tissue, a suitable buffer, etc.

[0141] In some embodiments, a sample containing an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof is contacted with an anti-id antibody disclosed herein that specifically binds to a CD70-derived binding molecule. In some embodiments, the anti-id antibody comprises a detectable label. In some embodiments, the anti-ID antibody conjugated to a detectable label is contacted with a sample expressing an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) under conditions that allow the formation of a binding complex comprising cells present in the sample and the anti-id antibody. Any anti-id antibody that specifically binds an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) can be used in the disclosed methods. Examples of suitable anti-id antibodies are provided herein, for example, those having one or more of the CDRs set forth in Table 1c or 1d.

[0142] Any detectable label can be used in the methods described herein, and an appropriate label can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, Texas Red (Pierce). Cy5, Cy5.5, and Cy7 (Amersham Life Science).Suitable optical dyes include fluorescent chromophores and are described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is expressly incorporated herein by reference, and include radiolabels (e.g., isotopic markers such as H, C, C, N, F, S, CU, Y, Tc, In, I, I, I, and I), photochromic compounds, Halo-tags, Atto dyes, and proteinaceous fluorescent labels, including, for example, green fluorescent protein (GFP, Renilla, Ptilosarcus, or Aequorea species) (Chalfie et al., (1994) Science 263:802-805), EGFP (Clontech Labs), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc.; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417), proteinaceous fluorescent labels, magnetic labels (e.g., DYNABEADS), and the like. Strategies for labeling proteins are well known in the art and can be employed in the disclosed methods. See, for example, Obermaier et al., (2015) Methods Mol Biol 1295:153-65; Strack (2016) Nature Methods 13:33; Site-Specific Protein Labeling, Methods and Protocols, (Gautier and Hinner, eds.) 2015, Springer. In some embodiments, the detectable label is a phycoerythrin (PE) or allophycocyanin (APC) fluorescent probe.

[0143] The label can be associated with the anti-id antibody at any position on the molecule, although it may be desirable to associate the label with the antibody at one position (or multiple positions, if multiple labels are used) so that the binding characteristics of the molecule are not altered (unless such altered binding activity is desired). Any antigen-binding molecule that specifically binds to a CD70-derived binding molecule (or fragment thereof) disclosed herein can be used, such as one having one or more of the CDRs set forth in Table 1c or 1d.

[0144] The antigen-binding molecule may be disposed on any surface, or there may be no surface at all. For example, the antigen-binding molecule may be present in a buffer solution, and the buffer-antigen-binding molecule may be contacted with the sample. Alternatively, the antigen-binding molecule may be associated with a surface. Suitable surfaces include agarose beads, magnetic beads such as DYNABEADS®, or plastic, glass, or ceramic plates such as well plates, or bags such as cell culture bags. The surface itself may be disposed in another structure, such as a column.

[0145] Conditions that permit the formation of binding complexes will depend on various factors, but generally, aqueous buffers at physiological pH and ionic strength, such as in phosphate buffered saline (PBS), favor the formation of binding complexes and are desirable in the methods of the present disclosure.

[0146] The number of cells present in the binding complex in the sample is determined. The specific method used to determine the number of cells present in the binding complex will depend on the nature of the selected label. For example, when fluorescent label is selected, FACS can be used; when isotope label is selected, mass spectrometry, NMR or other techniques can be used; when magnetic label is selected, magnetic-based cell sorting can be used, or microscopy can be used. The output of these detection methods can be in the form of cell number, or the output can be in a form that allows calculation of cell number based on the output.

[0147] IV. Methods for determining the presence or absence of 4F11-derived anti-CD70 CAR In some embodiments, knowing whether a molecule comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof is present or absent in a sample is sufficient information. For example, it may be useful to know that such a molecule is expressed, regardless of the level of expression. In other cases, it may be desirable to know whether a purification process or step designed to remove such a molecule is effective. Thus, a qualitative determination of the presence or absence of an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof may be useful in several applications.

[0148] In some embodiments, methods are provided for determining the presence or absence in a sample of a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0149] In some embodiments, the method includes providing a sample known or suspected to contain a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0150] The present disclosure provides antigen-binding molecules that specifically bind to a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof comprising a detectable label. Suitable labels can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, Texas Red (Pierce). Suitable optical dyes, including fluorophores, include those described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is expressly incorporated herein by reference, radiolabels (e.g., Cy5, Cy5.5, Cy7 (Amersham Life Science)). 3 H, 11 C. 14 C. 15 N, 18 F, 35 S, 64 CU, 90 Y, 99 Tc, 111 In, 124 I, 125 I, 131I isotope markers, such as photochromic compounds, halotags, Atto dyes, ackie dyes, proteinaceous fluorescent labels (e.g., proteinaceous fluorescent labels also include, but are not limited to, green fluorescent proteins, including GFP from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., (1994) Science 263:802-805), EGFP (Clontech Labs, Inc., Genbank Accession Number U55762), blue fluorescent proteins (BFP, Quant Biotechnologies, Inc. Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent proteins (Clontech Labs, Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417), magnetic labels (eg, DYNABEADS®), and the like can also be used.

[0151] The sample is then contacted with the antigen-binding molecule under conditions that allow for the formation of binding complexes comprising cells present in the sample and the antigen-binding molecule.

[0152] The sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex between the polypeptide comprising the anti-CD70 CAR (e.g., a CAR comprising 4F11-derived scFv) or a fragment thereof and the antigen-binding molecule and the antigen-binding molecule. The conditions that allow the formation of the binding complex depend on various factors. The component parts of the binding complex can be arranged on a surface as described herein, and therefore the formed binding complex can also be arranged on a surface.

[0153] At this stage, no binding complex may be formed, or multiple binding complexes may be formed, each comprising one or more antigen-binding molecules bound to a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof. Non-binding molecules, including anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof, and / or non-binding antigen-binding molecules, may also be present in the local environment of any formed binding complex.

[0154] Any molecules that are not part of the binding complex are then separated from any formed binding complex. The removal method will depend on the structure of the binding complex and / or the local environment. For example, if the antigen-binding molecule is placed on a bead, plate, or bag, the unbound components of the reaction mixture can be washed away using a solution that leaves the formed binding complex intact. In some embodiments, separation of the binding complex is not required for detection.

[0155] The solution used to induce the formation of the binding complex can be used, for example, as a wash solution to remove unbound components. Any suitable buffer or solution that does not disrupt the formed binding complex can also be used. Typically, high salt buffers at non-physiological pH containing chaotropes or denaturing agents should be avoided when performing this step of the method.

[0156] The presence or absence of a binding complex comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof and a polypeptide comprising an antigen-binding molecule can be detected. The specific method used to detect the presence or absence of a binding complex will typically depend on the nature of the label selected. In some embodiments, the detection method is colorimetric. The result of this method is a qualitative assessment of the presence or absence of an antigen-binding molecule comprising a detectable label, and therefore the presence or absence of its binding partner, a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof.

[0157] As with the methods of the present disclosure, a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof can be placed in any environment. In some embodiments, a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv) or a fragment thereof is expressed on the surface of a cell. In this embodiment, the cell can be any type of cell, and can be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In some embodiments, the cell is an immune cell. The immune cell of the present method can be any type of immune cell (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). T cells (including T cytotoxic cells, T helper cells, and Treg cells) are particularly preferred. In certain embodiments, the cell is a T cell, which can be obtained as described herein and by methods known in the art. In this embodiment of the disclosed method, any type of immune cell can be used, and the cell can be a human cell or a non-human cell. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TILs), NK cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. In additional embodiments, the cells are TCR-presenting T cells. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the disclosed method, the T cells can be in vivo T cells or in vitro T cells. Furthermore, the cells can be derived from stem cells, such as iPSC cells, umbilical cord blood cells, or mesenchymal stem cells.

[0158] In some embodiments, the cells may be placed in or isolated from blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo cultured tissue, a suitable buffer, or any other environment capable of maintaining the cells in a viable form. In some embodiments, the cells are in a formalin-fixed sample. In some embodiments, the sample is formalin-fixed, paraffin-embedded tissue (FFPE).

[0159] V. Antibody Methods and Uses In some embodiments, methods involving the use of one or more anti-idiotype antibodies are provided herein. In some aspects, methods for measuring or detecting a target antibody, such as a CAR or a cell expressing a CAR, and methods for modifying the activity of a target antibody, such as the activity of a CAR or a cell expressing a CAR, are provided herein. In certain embodiments, one or more anti-idiotype antibodies bind to, detect, identify, and / or quantify a CAR and / or a cell expressing a CAR. In some embodiments, the methods provided herein provide one or more steps of contacting and / or incubating one or more anti-idiotype antibodies with cells or a sample containing or suspected of containing a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the anti-idiotype antibody is treated, incubated, and / or contacted with a composition or sample under conditions that allow the formation of a complex between the anti-idiotype antibody and a target antibody, such as a CAR. In some aspects, the complex can be used to detect, isolate, and / or measure a CAR. In some embodiments, the formation of the complex alters the activity of the targeting antibody, e.g., the CAR, such as by stimulating receptor signaling activity, or in some embodiments, antagonizes the activity of the targeting antibody, e.g., the CAR, by preventing association of the CAR with an antigen.

[0160] A. Detection / Isolation Methods In some embodiments, methods are provided that involve the use of one or more anti-idiotype antibodies and / or molecules (such as conjugates and complexes) containing one or more of such anti-idiotype antibodies to detect, bind, and / or isolate antibodies, e.g., target antibodies. In certain embodiments, the methods provide one or more steps of contacting, incubating, and / or exposing one or more anti-idiotype antibodies to a sample and / or composition. In some embodiments, the sample and / or composition has, is likely to have, and / or is suspected of having a target antibody and / or antigen-binding fragment thereof that is bound and / or recognized by one or more anti-idiotype antibodies. In certain embodiments, the antibody or antigen-binding fragment thereof that is bound by or recognized by one or more anti-idiotype antibodies comprises one or more fusion domains and / or is a fusion protein. In certain embodiments, the target antibody and / or antigen-binding fragment thereof is a CAR or is present in a CAR. In certain embodiments, the anti-idiotypic antibody binds to and / or recognizes an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof, including a chimeric molecule or conjugate comprising a CAR containing such an anti-CD70 antibody (e.g., an antibody fragment).

[0161] In some embodiments, the methods involve incubating, treating, and / or contacting a sample and / or composition containing or suspected of containing a target antibody with an anti-idiotype antibody. In certain embodiments, the incubation is performed under conditions that allow binding of the anti-idiotype antibody to the target antibody present in the composition, e.g., to form a complex containing the anti-idiotype antibody and the target antibody.

[0162] In some embodiments, the sample and / or composition contains or is suspected of containing a target antibody, e.g., a CAR. In certain embodiments, the sample and / or composition contains or is suspected of containing a cell expressing a target antibody, e.g., a CAR. In certain embodiments, the sample is a biological sample. In certain embodiments, the sample is a serum sample or a blood sample. In some embodiments, the biological sample contains one or more immune cells. In some embodiments, the biological sample is or is derived from tissue, such as connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In certain embodiments, the biological sample is taken, collected, and / or obtained from a human subject. In certain embodiments, the sample contains live and / or intact cells. In some embodiments, the sample is or contains homogenates and / or cells that have been disrupted and / or lysed. In some embodiments, the biological sample contains proteins and / or antibodies isolated from blood, serum, and / or tissue.

[0163] In certain embodiments, the anti-idiotype antibody forms or can form a complex with a target antibody, e.g., a CAR. In certain embodiments, the complex is detected, measured, quantified, and / or evaluated, e.g., to enable detection, identification, measurement, and / or quantification of the target antibody in a composition or sample. In certain embodiments, the method includes detecting whether a complex has formed between the anti-idiotype antibody and the target antibody in a sample, and / or detecting the presence, absence, or level of such binding. In some embodiments, the complex contains a detectable label. In certain embodiments, the anti-idiotype antibody is an immune complex containing a detectable label. In certain embodiments, the anti-idiotype antibody contains, is conjugated to, bound to, and / or is bound to a detectable label. In some embodiments, the complex contains an antibody that binds to and / or recognizes the anti-idiotype antibody, e.g., a secondary antibody conjugated to, bound to, and / or bound to a detectable label.

[0164] In some embodiments, methods for detecting, quantifying, detecting, and / or evaluating a target antibody, for example, in a sample or composition, include detecting a complex of the target antibody and an anti-idiotype antibody. In some embodiments, the complex contains a detectable label. In certain embodiments, the complex is probed and / or contacted with a detectable label. In some embodiments, the complex is detected by any suitable method or means, such as, but not limited to, flow cytometry, immunocytochemistry, immunohistochemistry, Western blot analysis, and ELISA.

[0165] In some embodiments, the targeting antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell.In certain embodiments, the targeting antibody, for example, CAR, is not bound to or contained within a cell, for example, in some embodiments, the targeting antibody is secreted.In certain embodiments, the antibody is cleaved, removed, and / or dissolved from the surface of a cell.

[0166] In some embodiments, the targeting antibody is an anti-CD70 antibody. In some embodiments, the targeting antibody is or is derived from antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 15 and / or a light chain variable region set forth in SEQ ID NO: 16.

[0167] In some embodiments, a method for detecting a target antibody is provided, comprising contacting, for example, antibody 4F11, or an antigen-binding fragment thereof (and / or a chimeric molecule comprising such an antibody, e.g., an antibody fragment, such as a CAR), a composition comprising the target antibody or antigen-binding fragment, with an anti-idiotype antibody or antigen-binding fragment thereof or an anti-idiotype antibody immune complex described herein, and detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment. In some embodiments, the method further comprises detecting whether a complex has formed between the anti-idiotype antibody and the target antibody in the composition, such as detecting the presence, absence, or level of such binding. In some embodiments, the target antibody or antigen-binding fragment is bound to or expressed on the surface of a cell, and detecting comprises detecting cells bound by the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection. In some embodiments, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 15 and / or a light chain variable region set forth in SEQ ID NO: 16.

[0168] In certain embodiments, the method for detecting a target antibody using an anti-idiotype antibody described herein is used to evaluate a target antibody in a subject. For example, in some embodiments, the present specification provides a method for using an anti-idiotype antibody to evaluate, measure, and / or quantify the in vivo pharmacokinetics, proliferation, and / or persistence of CAR-expressing cells of a therapeutic cell composition. In some embodiments, in the methods provided herein, the in vivo pharmacokinetics, proliferation, and / or persistence of cells, such as CAR-expressing cells administered in immunotherapy, such as CAR-T cell therapy, and / or changes in the phenotype or functional activity of the cells can be measured with the anti-idiotype antibody provided herein. In some embodiments, the pharmacokinetics, proliferation, and / or persistence of CAR-expressing cells are measured and evaluated by detecting the presence and / or amount of CAR-expressing cells in the subject and / or in a sample obtained from the subject during therapy and / or after administration of a therapeutic cell composition using the anti-idiotype antibody provided herein.

[0169] In some aspects, anti-idiotypic antibodies are used in conjunction with flow cytometry to assess the amount of cells expressing the recombinant receptor (e.g., CAR-expressing cells administered for T cell-based therapy) in a subject's blood or serum or organ or tissue sample (e.g., a disease site, e.g., a tumor sample). In some aspects, persistence is quantified, e.g., as the number of CAR-expressing cells per microliter of a sample, such as blood or serum, or the number of CAR-expressing cells per microliter of peripheral blood mononuclear cells (PBMCs) or white blood cells or total number of T cells per microliter of sample. In certain aspects, proliferation is quantified, e.g., as the increase in the number of CAR-expressing cells per microliter between blood or serum samples, or the increase in CAR-expressing cells per microliter of total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells over time. In some embodiments, pharmacokinetics, proliferation, and / or persistence are measured or assessed by detecting the amount of CAR-expressing cells in the subject and / or samples taken from the subject at multiple time points.

[0170] In some embodiments, a method for selecting cells expressing a CAR comprising a targeting antibody, such as antibody 4F11, or an antigen-binding fragment thereof is provided, comprising contacting a cell population comprising cells expressing a CAR with an anti-idiotype antibody or antigen-binding fragment thereof described herein and selecting cells bound by the anti-idiotype antibody. In some embodiments, cells bound by the anti-idiotype antibody are selected by affinity-based separation. In some embodiments, the affinity-based separation is selected from the group consisting of immunoaffinity-based separation, flow cytometry, magnetic-based separation, and affinity chromatography. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof, or the anti-idiotype antibody immune complex is reversibly bound or immobilized to a support or stationary phase. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and / or the light chain variable region amino acid sequence set forth in SEQ ID NO: 16 (e.g., comprises the amino acid sequence of SEQ ID NO: 35).

[0171] In some embodiments, a method for validating a CAR comprising a targeting antibody, e.g., antibody 4F11, or an antigen-binding fragment thereof, is provided, comprising: a) incubating a sample comprising T cells transduced with the CAR with an anti-idiotype antibody or antigen-binding fragment thereof that targets the CAR; b) determining the percentage of cells bound by the anti-idiotype antibody or antigen-binding fragment thereof; and c) validating the CAR based on the percentage of anti-idiotype antibody-binding T cells. In some embodiments, the anti-idiotype antibody is labeled, and the anti-idiotype antibody-binding T cells are assayed by flow cytometry. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and / or the light chain variable region amino acid sequence set forth in SEQ ID NO: 16 (e.g., comprising the amino acid sequence of SEQ ID NO: 35).

[0172] Also provided are methods involving the use of the provided anti-idiotype antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotype antibodies to inform treatment decisions in an individual, such as by detecting a CAR recognized by the anti-idiotype antibody, such as a CAR comprising a targeting antibody, such as an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof. In some embodiments, the method is for informing treatment decisions in an individual related to a therapy comprising administration of CAR T cells, such as anti-CD70 CAR T cells. In some embodiments, the method comprises incubating and / or probing a biological sample with the anti-idiotype antibody and / or administering the anti-idiotype antibody to the individual. In certain embodiments, the biological sample comprises cells or tissue, or portions thereof, such as tumor or cancer tissue or biopsy or sections thereof. In certain embodiments, the incubation is under conditions that allow binding of the anti-idiotype antibody to a CAR present in the sample. In some embodiments, the method further comprises detecting whether a complex has formed between the anti-idiotype antibody and the CAR in the sample, such as detecting the presence or absence or level of such binding. Such methods may be in vitro or in vivo methods.

[0173] B. Use in Cell Stimulation In some embodiments, the provided anti-idiotypic antibodies or antigen-binding fragments thereof are agonistic and / or exhibit specific activity for stimulating cells expressing a targeting antibody, such as an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof, including a conjugate or chimeric receptor containing the same. In some embodiments, methods are provided that involve the use of the provided anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of the anti-idiotypic antibodies for the stimulation or activation of CAR-expressing cells or other chimeric receptor-expressing cells, such as T cells. In some aspects, the CAR or other receptor comprises a targeting antibody, such as an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof.

[0174] In some embodiments, the method can be used in conjunction with methods for preparing engineered T cells, such as methods for expanding engineered T cells or other cells into which a nucleic acid molecule encoding a chimeric receptor, such as a CAR, comprising a targeting antibody has been introduced by a non-viral means of nucleic acid transfer, such as transfection, transduction, or a transposon-based approach. In some aspects, the targeting antibody is an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof. In certain embodiments, the targeting antibody is or contains a CAR, e.g., an anti-CD70 CAR. In certain embodiments, the anti-CD70 CAR is derived from an anti-CD70 antibody, such as antibody 4F11, and / or contains an scFv derived therefrom.

[0175] In some embodiments, the method comprises incubating a sample comprising CAR-transduced T cells with an anti-idiotypic antibody. In certain embodiments, the method further comprises detecting whether the CAR T cells are activated or stimulated, such as by assessing viability, proliferation, and / or expression of activation markers in the CAR T cells. In some embodiments, the targeting antibody is an anti-CD70 antibody. In some embodiments, the targeting antibody is or is derived from antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and / or the light chain variable region amino acid sequence set forth in SEQ ID NO: 16 (e.g., comprises the amino acid sequence of SEQ ID NO: 35).

[0176] In some embodiments, a method of simulating cells is provided, comprising incubating an input composition comprising cells expressing a CAR comprising a targeting antibody, such as antibody 4F11, or an antigen-binding fragment thereof, with an anti-idiotype antibody or antigen-binding fragment thereof described herein, thereby generating an output composition comprising stimulator cells. In some embodiments, the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells in the input composition. In some embodiments, the cells comprise T cells. In some embodiments, the T cells comprise CD4+ and / or CD8+ T cells.

[0177] In certain embodiments, an anti-idiotype antibody is administered to a subject, such as a subject that has previously been administered a therapeutic cell composition containing CAR-expressing cells. In some embodiments, administering the anti-idiotype antibody to a subject promotes the repopulation of CAR-expressing cells in the subject, which may in some cases reach or exceed the initial peak level of proliferation prior to administration of the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is administered to regulate the proliferation and / or persistence of CAR-expressing cells when the level of CAR-expressing cells is reduced or undetectable. In some embodiments, CAR-expressing cells repopulated by the anti-idiotype antibody exhibit increased potency in the subject to which it is administered, for example, compared to potency prior to administration of the anti-idiotype antibody.

[0178] In some embodiments, a method for generating a cell composition is provided, comprising introducing a nucleic acid molecule encoding a CAR into a cell, thereby generating an input composition, and incubating the input composition with an anti-idiotype antibody or antigen-binding fragment thereof specific to the antigen-binding domain of the CAR, thereby generating a cell composition. In some embodiments, the CAR comprises a targeting antibody or antigen-binding fragment thereof that specifically binds to CD70. In some embodiments, the targeting antibody is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an anti-idiotype antibody or antigen-binding fragment thereof described herein. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an agonist of the CAR. In some embodiments, the introducing comprises introducing the nucleic acid molecule into the cell by viral transduction, transduction, electroporation, or chemical transfection. In some embodiments, introducing comprises introducing the nucleic acid molecule into the cell by transduction with a retroviral vector comprising the nucleic acid molecule, transduction with a lentiviral vector comprising the nucleic acid molecule, transduction with a transposon comprising the nucleic acid molecule, or electroporation or transfection of a vector comprising the nucleic acid molecule.

[0179] C. Use in Cell Inactivation / Depletion In some embodiments, the provided anti-idiotypic antibodies or antigen-binding fragments thereof are antagonistic and / or exhibit specific activity to inhibit, ablate, and / or deplete (e.g., kill via antibody-dependent cell-mediated cytotoxicity, ADCC) cells expressing a targeting antibody, such as an anti-CD70 antibody (e.g., antibody 4F11) or an antigen-binding fragment thereof. Also provided are methods involving the use of the provided anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies for the inactivation, ablation, and / or depletion of CAR T cells, wherein the CAR comprises a targeting antibody, such as an anti-CD70 antibody (e.g., antibody clone 4F11) or an antigen-binding fragment thereof.

[0180] In some embodiments, the method comprises treating, contacting, and / or incubating a composition and / or sample comprising the CAR-transduced T cells with an anti-idiotypic antibody. In certain embodiments, the method further comprises detecting whether the CAR T cells are inactivated, such as by assessing viability, proliferation, and / or expression of activation markers in the CAR T cells. In some embodiments, the method relates to a therapy comprising administration of CAR T cells. In some embodiments, the method comprises administering an anti-idiotypic antibody to an individual. In one embodiment, the anti-idiotypic antibody or conjugate is used to ablate and / or deplete (e.g., kill) CAR T cells in an individual. In some embodiments, the targeting antibody is an anti-CD70 antibody. In some embodiments, the targeting antibody is or is derived from the antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 15 and / or the light chain variable region amino acid sequence set forth in SEQ ID NO: 16 (e.g., comprises the amino acid sequence of SEQ ID NO: 35).

[0181] In some embodiments, the anti-idiotype antibody is administered to deplete, reduce, and / or decrease the number of CAR-expressing cells in a subject. In certain embodiments, administration of the anti-idiotype antibody depletes, reduces, and / or decreases the amount of CAR-expressing cells, e.g., circulating CAR-T cells, by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100%, or about 100%. In certain embodiments, the depletion, reduction, and / or decrease refers to the amount of CAR-expressing cells in the subject before administration of the anti-idiotype antibody. In certain embodiments, the depletion, reduction, and / or decrease refers to the amount of CAR-expressing cells in a subject that has not been administered the anti-idiotype antibody. In some embodiments, CAR-expressing cells are not detectable in the subject after administration of the anti-idiotype antibody. In certain embodiments, the anti-idiotype antibody is a human antibody or a humanized antibody.

[0182] In some embodiments, a method of inactivating CAR T cells is provided, wherein the CAR comprises a targeting antibody or antigen-binding fragment thereof, such as antibody 4F11, comprising incubating a sample comprising CAR T cells with an antagonistic anti-idiotypic antibody or antigen-binding fragment thereof that targets the CAR, thereby inactivating the CAR T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to attenuate activation of CAR T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to substantially inactivate CAR T cells in the sample. In some embodiments, incubation with the anti-idiotypic antibody results in ablation and / or depletion of CAR T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to result in clearance of CAR T cells in the sample. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 15 and / or the light chain variable region set forth in SEQ ID NO: 16 (e.g., comprises the amino acid sequence of SEQ ID NO: 35).

[0183] In some embodiments, methods of modulating CAR T-cell therapy in an individual are provided, wherein the CAR contains a targeting antibody, such as antibody 4F11, or an antigen-binding fragment thereof, and the method comprises administering to the individual an anti-idiotype antibody immunoconjugate targeted to the CAR, wherein the anti-idiotype antibody immunoconjugate contains a cytotoxic agent. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to attenuate CAR T-cell therapy in the individual. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to substantially stop CAR T-cell therapy in the individual. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to result in clearance of CAR T cells in the individual. In some embodiments, the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), and a radioactive isotope. In some embodiments, the targeting antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO:15 and / or the light chain variable region amino acid sequence set forth in SEQ ID NO:16.

[0184] D. Use in Binding Assays or Methods Provided herein are methods for assessing the presence or absence of a molecule in a sample that binds to a chimeric antigen receptor (CAR), such as the extracellular domain of a CAR, or a portion thereof containing an antigen-binding domain. In some embodiments, the methods can be used to assess the presence or absence of a humoral or antibody response in a subject to an administered cell therapy comprising a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor comprises a targeting antibody that is the antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises a targeting antibody that is the antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, an anti-idiotypic antibody or antigen-binding fragment thereof specific for the extracellular domain of a CAR, such as any of those described herein, can be used as a positive control in the methods.

[0185] Any anti-idiotypic antibody that specifically binds to a polypeptide comprising the anti-CD70 antibody 4F11, or an anti-CD70 CAR (e.g., a CAR comprising a 4F11-derived scFv), or a fragment thereof, can be used, such as those disclosed herein, including those having one or more of the VH and VL sequences set forth in Tables 1a and 1b, and / or one or more CDRs set forth in Tables 1c and 1d. In some embodiments, binding of the anti-CD70 antibody to the target antigen CD70 does not interfere with binding of the anti-idiotypic antibody to the anti-CD70 antibody, i.e., non-blocking. In some embodiments, binding of the anti-idiotypic antibody to the anti-CD70 antibody does not interfere with binding of the anti-CD70 antibody to the target antigen CD70. In some embodiments, the anti-idiotypic antibody is a non-blocking anti-idiotypic antibody. In some embodiments, binding of the non-blocking anti-idiotypic antibody can more accurately detect the presence of the anti-CD70 antibody in the presence of the target antigen CD70, or quantify the amount or number of either as part of a CAR.

[0186] In some embodiments, the binding of the anti-idiotype antibody interferes with the binding of the anti-CD70 antibody to the target antigen CD70, or prevents the binding of the anti-CD70 antibody to the target antigen CD70 and prevents the binding of the anti-idiotype antibody to the anti-CD70 antibody, i.e., blocking. In some embodiments, the anti-idiotype antibody is a blocking anti-idiotype antibody. In some embodiments, the binding of the blocking anti-idiotype antibody can be used to estimate or determine the level of engagement or occupancy of the target antibody, e.g., anti-CD70 antibody, alone or as part of a CD70-specific CAR, with the target antigen, e.g., CD70.

[0187] In some embodiments, a method is provided that includes contacting or incubating a binding reagent with a sample from a subject administered a cell therapy comprising cells engineered with a chimeric antigen receptor, wherein the binding reagent is a protein comprising the extracellular domain of a CAR or a portion thereof containing a target antibody or antigen-binding fragment thereof. In some embodiments, the method further includes detecting whether a complex is formed between the binding reagent and a molecule, e.g., a binding molecule such as an antibody present in the sample, and / or detecting the presence, absence, or level of such binding. In certain embodiments, the contacting or incubation is performed under conditions that allow binding of the binding reagent to a molecule present in the sample from the subject. In certain aspects, the method may further be performed on a positive control sample containing an anti-idiotypic antibody or antigen-binding fragment thereof specific for a CAR, such as any of those described. In some embodiments, determining the presence, absence, or level of binding of a molecule to the binding reagent may include comparing the binding or detection to the binding or detection of a positive control sample to the binding reagent.

[0188] In some embodiments, the method includes detecting whether a complex is formed between the binding reagent and a molecule, e.g., a binding molecule such as an antibody, present in the sample, and / or detecting the presence, absence, or level of such binding. In certain embodiments, the contacting or incubation is performed under conditions that allow binding of the binding reagent to the molecule present in the sample from the subject. In some aspects, the complex is detected by an immunoassay, optionally a sandwich assay or a bridge assay. For example, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), flow cytometry, or Western blot. In some embodiments, the immunoassay is or includes mesoscale discovery.

[0189] In some aspects, the immunoassay is a sandwich assay or a bridge assay. In a sandwich assay or a bridge assay, the binding reagent is a first binding reagent, and detecting the presence or absence of a molecule or a complex containing a molecule comprises contacting the complex formed between the first binding reagent and the molecule with a second binding reagent, wherein the second binding reagent is an agent capable of binding to the same or similar molecule as the first binding reagent. In some embodiments, the second binding reagent comprises an extracellular domain of a CAR or a portion thereof. In some aspects, the extracellular domains of a CAR of the first binding agent and the second binding agent or portions thereof are identical or substantially identical. [Example]

[0190] Example 1: Generation of anti-idiotypic antibodies against anti-human CD70scFv clone 4F11 Balb / c mice were immunized with an anti-CD70 scFv 4F11 clone human IgG2 Fc fusion protein. The 4F11 scFv was used to generate an anti-CD70 chimeric antigen receptor (CAR) consisting of the 4F11 scFv plus the CD8a hinge / transmembrane domain, CD3z activation domain, and 4-1BB costimulatory domain.

[0191] The 4F11 scFv amino acid sequence is shown in SEQ ID NO: 35. Hybridomas were generated, cloned, and secreted antibodies were screened for binding specificity to 4F11. Antibody clones were identified and selected based on their ability to specifically bind to the 4F11-derived scFv by testing mouse sera by ELISA against the immunogen.

[0192] Seropositive mice were selected to generate hybridoma fusion. The supernatant of hybridoma culture was screened by ELISA using 4F11 scFv10xHis antigen ("10xHis" disclosed as SEQ ID NO: 40) to identify positive hybridoma clones. The supernatant of these clones was then screened by flow cytometry to confirm the binding specificity to anti-CD70 CAR. The positive clones were then subcloned to ensure clonality. The subclones were screened, and the binding specificity was confirmed by flow cytometry.

[0193] Antigen blocking assays were performed to determine whether the anti-id antibodies could bind to their target antibodies in the presence of the target antigen. Blocking of positive clones that bound to the antibody in the presence of CD70 protein was performed by flow cytometry, and the blocking results of the lead clones were confirmed by surface plasmon resonance.

[0194] The full-length or variable region sequences of the lead and backup anti-idiotype clones were determined.

[0195] Recombinant antibodies were produced and the binding of the recombinantly produced antibodies to antigen was indistinguishable from the antibodies purified from the hybridoma.

[0196] The anti-id antibody clones IDA2002-1.60 (clone 60), IDA2002-1.37 (clone 37) were further analyzed and characterized as described herein.

[0197] Example 2: Flow cytometric determination of specificity We next confirmed binding and determined the optimal titer of two phycoerythrin (PE)-conjugated anti-idiotypic antibodies against 4F11 scFv: anti-idiotypic antibody clone 37 (also referred to herein as IDA2002-1.37 or clone 1.37) and anti-idiotypic antibody clone 60 (also referred to herein as IDA2002-1.60 or clone 1.60). Titrations were performed with clones 60 and 37, and binding to the target CD70 CAR was assessed using flow cytometry. CD70-specific (4F11-QR3) CAR T cells and non-transduced T cells (NTD) were generated from PBMC starting material. Cells were thawed, rested for 24 hours, counted, and 1 × 10 5 (1 x 10^5) cells were stained with each pool or fraction starting at 30 μg (micrograms) / mL of anti-idiotypic antibody over a five-point semi-logarithmic titration curve. Cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer (PBS + 2% FBS + 2 mM EDTA), resuspended in buffer, and analyzed on a Cytoflex flow cytometer. As shown in Figure 1A, the first and second columns show the results for clone 1.37. Figure 1A, the third and fourth columns show the results for clone 1.60.

[0198] To determine the specificity of the PE-conjugated anti-idiotype clone (clone 60-PE) for 4F11scFv, several control CAR T cells were used, including anti-CD19 CAR T cells, anti-BCMA CAR T cells, and anti-FLT3 CAR T cells. CD70 (4F11-QR3) CAR T cells (donor A) were included as a positive control, and NTD cells (donor A) were stained as a negative control. Cells were thawed, rested for 48 hours, counted, and collected at 1 × 10 5Cells were stained with 3 μg / mL of clone 60-PE antibody. Because all CAR T cells contain a rituximab mimetope-based off-switch, cells were also stained with 10 μg / mL of rituximab (RTX) antibody as a control. Cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer, resuspended in buffer, and then analyzed on a Cytoflex flow cytometer. Figure 1B shows the staining results for anti-CD19 CAR, anti-BCMA CAR, anti-FLT3 CAR, anti-CD70 CAR, and NTD.

[0199] Example 3: Flow cytometry comparison of recombinant proteins Purified PE-conjugated anti-idiotype clone 60 from Biolegend was compared to freshly purified antibodies purified from recombinant or hybridomas using flow cytometry. CD70 (4F11-QR3) CAR T cells and NTD cells were generated from pan T cell starting material (donor D). Cells were stained with 0.3 μg / mL of PE-conjugated or non-conjugated recombinant or hybridoma versions of clone 60. Cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer, resuspended in buffer, and then analyzed on a Cytoflex flow cytometer. Results are shown in Figure 2A (SSC-A: side scatter-A).

[0200] Separately, cells were stained with 0.3 μg / mL of unconjugated recombinant or hybridoma forms of clone 60. Cells were stained with the primary antibody for 20 minutes at 4°C, washed with flow cytometry buffer, stained with a secondary anti-mouse Fc-specific antibody (Jackson, catalog no. 115-116-071) at a 1:100 dilution for 15 minutes at 4°C, washed with flow cytometry buffer, and resuspended in buffer before analysis on a Cytoflex flow cytometer. The results are shown in Figure 2B. Figure 2A shows the results of staining with the PE-conjugated antibody. Figure 2B shows the results of staining with the unconjugated antibody. The results indicate that PE conjugation did not affect antigen binding.

[0201] Example 4: Flow cytometric demonstration of the effect on binding in the presence of target antigen CD70 To determine whether anti-idiotype clones could bind to 4F11 scFv in the presence of the target antigen CD70, CD70-specific CAR Jurkat cells were incubated and blocked with recombinant human CD70 (rCD70 or hCD70) before staining with anti-idiotype by flow cytometry. CD70 (4F11-QR3) CAR Jurkat cells and NTD cells were generated by transducing Jurkat cells with LVV containing 4F11-QR3 CAR. Cells were thawed, counted, and collected at 1 x 10 5 Cells were incubated with 100 μg / mL hCD70 (Abcam catalog no. ab119815) for 30 minutes. Cells were then stained with various polyclonal supernatants from hybridoma cultures at a 1:10 dilution for 20 minutes and washed with flow cytometry buffer. Cells were stained with a secondary anti-mouse Fc-specific antibody (Jackson, catalog no. 115-116-071) at a 1:100 dilution for 15 minutes at 4°C, washed with flow cytometry buffer, resuspended in buffer, and analyzed on a Cytoflex flow cytometer. Cells were stained with 10 μg / mL PE-conjugated rituximab (RTX) antibody as a positive control (to demonstrate that the cells express the 4F11-QR3 CAR) and with secondary antibody alone as a negative control. The results are shown in the top panel of Figure 3 (NTD control not shown).

[0202] Cells not treated with hCD70 were stained as a positive control (see Figure 3, bottom panel). The results show that when 4F11 scFv formed a complex with its target antigen, CD70, it blocked the binding of the anti-idiotypic antibody of clone 37 to 4F11 scFv, whereas 4F11 scFv-CD70 binding did not block the binding of the anti-idiotypic antibody of clone 60 to 4F11 scFv.

[0203] For confirmation, the experiment was repeated using a purified PE-conjugated clone (clone 60) that was determined to be a non-blocker of the CAR-CD70 interaction. Cells (4F11-QR3 CAR and non-transduced (NTD) cells) were thawed, allowed to settle for 48 hours, counted, and 1 x 10 5 Cells were incubated with hCD70 at 100 μg / mL for 30 minutes. They were then stained with clone 60-PE antibody at 1.5 μg / mL (Figure 4A) or rituximab (RTX) antibody at 5 μg / mL (Figure 4B) for 20 minutes at 4°C, washed with flow cytometry buffer, resuspended in buffer, and analyzed on a Cytoflex flow cytometer. Cells not treated with hCD70 were stained as a positive control. The results confirm that the anti-idiotypic antibody binding of clone 60 to 4F11 scFv was not blocked by the scFv binding to CD70. Therefore, clone 60 is a non-blocking anti-idiotypic antibody. Example 5: Surface Plasmon Resonance ("SPR") Determination of Affinity for Clone 60

[0204] All SPR analyses were performed on a BIACore T200 SPR instrument (Cytiva, Marlborough, MA). For kinetic analysis of CD70 anti-idiotypic antibodies and simultaneous target antigen binding determination, mouse Fc was captured by amine coupling of anti-mouse Fc reagent (Mouse Antibody Capture Kit, Cytiva, BR100838) to a Biacore Series S CM4 sensor chip at 25°C using a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.4. The entire surface of the sensor chip was activated with a 1:1 (v / v) mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) at 10 μL / min for 7 minutes. The anti-mouse Fc reagent was diluted to 30 μg / mL in 10 mM sodium acetate (pH 5.0) and injected over all four flow cells at 10 μL / min for 7 min, after which all flow cells were blocked with 100 mM ethylenediamine in 200 mM borate buffer pH 8.5 at 10 μL / mL for 7 min.

[0205] All interaction experiments were performed at 37°C using the same running buffer as described above, supplemented with 1 mg / mL BSA. The binding affinity of the CD70 anti-idiotype antibody was determined by first capturing 5 μg / mL of CD70 anti-idiotype antibody on an anti-mouse Fc chip surface, followed by injection of buffer and 4F11 scFv 10xHis ("10xHis," disclosed as SEQ ID NO: 40) at concentrations of 1.2, 3.7, 11.1, 33.3, or 100 nM at 30 μL / min for 2 minutes. Dissociation was monitored for 10 minutes, followed by regeneration of all flow cells with 10 mM glycine at pH 1.7 for 3 minutes. The results are shown in Figure 5, and the data are reproduced in Table 3 below.

[0206] [Table 3]

[0207] Example 6: SPR demonstration of simultaneous binding of clone 60 with the target antigen CD70 To determine the simultaneous binding of CD70 anti-idiotype antibodies to the CD70 target antigen using a premix, followed by a secondary detection method, 0.5 μg / ml of CD70 anti-idiotype antibodies were first captured onto the surface of an anti-mouse Fc chip. A premix of 100 nM 4F11 scFv 10xHis (10xHis disclosed as SEQ ID NO: 40) and 100 nM biotin-hCD70 and 100 nM streptavidin (Thermo Fisher Scientific, catalog no. 21125) was injected sequentially at 30 μL / min for 2 minutes using dual injections. Streptavidin, specific for the premixed Biotin-hCD70 target antigen, was used as a secondary detection reagent to detect whether a complex was formed between the premix and the captured CD70 anti-idiotype antibody. Dissociation was monitored for 3 min, followed by regeneration of all flow cells with 10 mM glycine at pH 1.7 for 3 min. Figure 6A shows the assay (left panel) and results (top right and bottom panels for hybridoma-purified and recombinantly produced anti-idiotypic antibodies, respectively).

[0208] To determine simultaneous binding of CD70 anti-idiotypic antibodies to the CD70 target antigen using the classical sandwich assay, 0.5 μg / ml of CD70 anti-idiotypic antibodies was captured onto an anti-mouse Fc chip surface. Buffer, 4F11 scFv 10xHis (10xHis disclosed as SEQ ID NO: 40) at 100 nM, and Biotin-hCD70 at 100 nM were injected sequentially at 30 μL / min for 2 minutes using dual injections. Dissociation was monitored for 3 minutes, followed by regeneration of all flow cells with 10 mM glycine at pH 1.7 for 3 minutes. Figure 6B shows the assay (left panel) and results (top right and bottom panels for hybridoma-purified and recombinantly produced anti-idiotypic antibodies, respectively). The results demonstrate that anti-idiotypic antibody clone 60 can bind to anti-CD70 antibodies in the presence of the target antigen, hCD70, in either premix or sandwich assays.

[0209] All data were evaluated for mass transfer using Biacore T200 evaluation software (version 2.0) and fitted to a 1:1 Langmuir binding model. Figure 6A-B shows that the anti-idiotypic antibody clone 60 can bind to 4F11 scFv in the presence of the target antigen hCD70 protein, either by premix assay or by classical sandwich assay.

[0210] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present disclosure. To the extent that any definitions or terminology provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions control.

[0211] The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the present disclosure. The foregoing and subsequent description and examples detail certain embodiments of the present disclosure and set forth the best mode contemplated by the inventors. It will, of course, be understood, however, that no matter how detailed the foregoing appears in text, the present disclosure can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. An isolated antibody that specifically binds to a molecule comprising an anti-CD70 antibody or antigen-binding fragment thereof, wherein the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO:35, or the molecule comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

34.

2. the isolated antibody (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 4; or (b) an isolated antibody according to claim 1, comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO:

23.

3. the isolated antibody (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 7, or 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9 or 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (b) an isolated antibody according to claim 1, comprising a VH region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, 26 or 27, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28 or 29, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:

30.

4. the isolated antibody (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; or (b) the isolated antibody of claim 1 further comprising a VL region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

33.

5. The isolated antibody of claim 4, wherein the VH region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2 and the VL region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

4.

6. The isolated antibody of claim 5, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 2 and the VL region comprises the amino acid sequence of SEQ ID NO:

4.

7. 5. The isolated antibody of claim 4, wherein the VH region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 21 and the VL region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

23.

8. 8. The isolated antibody of claim 7, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 21 and the VL region comprises the amino acid sequence of SEQ ID NO:

23.

9. 2. The isolated antibody of claim 1, wherein the isolated antibody comprises a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10, the VH CDR3 comprises the amino acid sequence SEQ ID NO:11, the VL CDR1 comprises the amino acid sequence SEQ ID NO:12, the VL CDR2 comprises the amino acid sequence SEQ ID NO:13, and the VL CDR3 comprises the amino acid sequence SEQ ID NO:

14.

10. 2. The isolated antibody of claim 1, wherein the isolated antibody comprises a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29, the VH CDR3 comprises the amino acid sequence SEQ ID NO:30, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:31, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:32, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

33.

11. The isolated antibody of any one of claims 1 to 10, wherein the isolated antibody further comprises a detectable label.

12. 12. The isolated antibody of claim 11, wherein the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a magnetic label, a radiolabel, and a hapten.

13. The fluorescent label may be selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine-rhocamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutant), GFP (Y6 6F mutant), EBFP, EBFP2, Azurite, GFPUv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutant), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutant), Green Cysteine, wild-type GFP, GFP (S65C mutant), TurboGFP, TagGFP, GFP (S65L mutant) mutant), Emerald, GFP (S65T mutant), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (RFP), mStra 13. The isolated antibody of claim 12, wherein the antibody is selected from the group consisting of: Raspberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, peridinin chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

14. A polynucleotide encoding the isolated antibody of any one of claims 1 to 10.

15. A vector comprising the polynucleotide of claim 14.

16. A cell comprising the polynucleotide of claim 14 or the vector of claim 15.

17. (a) an anti-CD70 scFv comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16; (b) an anti-CD70 scFv comprising the amino acid sequence of SEQ ID NO: 35; and (c) an anti-CD70 chimeric antigen receptor ("CAR") comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 34, comprising: A method comprising growing or culturing the cells of claim 16 under suitable conditions.

18. 14. A method for determining the number of cells expressing an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NOs: 15 and 16, the method comprising contacting said cells with the isolated antibody of any one of claims 1 to 13, and determining the number of cells expressing said anti-CD70 antibody among said cells.

19. 14. A method for determining the presence or absence of a cell expressing an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NO: 15 and 16, the method comprising contacting the cell with the isolated antibody of any one of claims 1 to 13, and determining the presence or absence of a cell expressing the anti-CD70 antibody in the cell.

20. The method of claim 18 or 19, wherein the anti-CD70 antibody is an scFv comprising the amino acid sequence of SEQ ID NO:

35.

21. 20. The method of claim 18 or 19, wherein the cell expresses an anti-CD70 chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34, with or without a signal peptide.

22. 22. The method of claim 21, wherein the cell is a CAR T cell.

23. A method for selecting a cell expressing an anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 34, comprising: (a) contacting said cell with the isolated antibody of any one of claims 1 to 13; (b) selecting the cells that are bound to the antibody. Optionally, the isolated antibody further comprises a detectable label.

24. 24. The method of claim 23, wherein the cells bound by the isolating antibody are selected by affinity-based separation.

25. 24. The method of claim 23, wherein the cells bound to the isolated antibody are selected by flow cytometry.

26. 1. A method for purifying an anti-CD70 antibody or antigen-binding fragment thereof, wherein the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NOs: 15 and 16, or the amino acid sequence of SEQ ID NO: 35, the method comprising: (a) contacting a sample containing the anti-CD70 antibody with the isolated antibody of any one of claims 1 to 13 to form a complex; (b) purifying the complex comprising the anti-CD70 antibody and the isolated antibody from the sample.

27. 14. A method of stimulating cells expressing an anti-CD70 chimeric antigen receptor (anti-CD70 CAR T cells), comprising incubating the anti-CD70 CAR T cells with the isolated antibody of any one of claims 1 to 13, wherein the anti-CD70 CAR T cells express an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NOs: 15 and 16, or the anti-CD70 CAR T cells express an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NO: 35.

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