CD70 anti-idiotypic antibodies

Abstract

Provided herein are anti-idiotypic antibodies that specifically recognize anti-CD70 antibody moieties, in particular recombinant receptors, including anti-CD70 antibody moieties present in chimeric antigen receptors (CARs). The disclosure further relates to the use of anti-idiotypic antibodies for the specific identification and / or selection of cells expressing such recombinant receptors, such as anti-CD70 CAR T cells. The disclosure further relates to the use of anti-idiotypic antibodies for the specific activation of such cells.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefit of U.S. Provisional Application No. 63 / 387,334, filed on December 14, 2022, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Reference to sequence listing

[0004] This application contains a sequence listing submitted electronically in XML file format, and the sequence listing is hereby incorporated 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. Technical field

[0005] In some aspects, the present disclosure relates to anti - idiotypic (anti - Id) antibodies that specifically recognize an anti - CD70 antibody moiety, specifically recombinant receptors, including the anti - CD70 antibody moiety present in a chimeric antigen receptor (CAR). The present disclosure further relates to uses of the anti - idiotypic antibodies, for example, for specifically identifying, quantifying, or selecting cells that express such recombinant receptors, such as anti - CD70 CAR T cells. The present disclosure further relates to uses of the anti - idiotypic antibodies for specifically activating such cells. Background art

[0006] Anti - idiotypic antibodies are a subset of antibodies produced against immune antibodies. These anti - idiotypic antibodies exhibit specific binding to the idiotype (unique antigenic determinant on the surface of the antibody) of the immune antibody. Anti - idiotypic antibodies can generally be classified into three different groups: (1) antibodies that recognize an idiotype on the immune antibody that is different from the antigen - binding site (ABS); (2) antibodies that recognize an epitope within the ABS and mimic the structure of the nominal antigen; and (3) antibodies that recognize an epitope within the ABS but do not have a structural similarity to the nominal antigen (see, for example, Pan et al., (1995) The FASEB Journal 9:43 - 49).

[0007] Conventional methods for detecting CAR expression (such as anti - CD70 CAR expression), for example, using soluble human CD70 - Fc fusion proteins, may lack specificity, have inter - batch variability, produce low - intensity signals in analytical methods (such as flow cytometry), or be difficult to prepare in sufficient quantities, and such conventional methods may underestimate the true number of cells expressing the CAR.

[0008] Accordingly, robust reagents are needed to accurately detect anti-CD70 CAR expression on engineered T cells or in in vitro assays. Provided herein are methods and compositions that address this need and other needs. SUMMARY OF THE INVENTION

[0009] Provided herein are agents that specifically bind to antibodies (including antibody fragments such as single-chain variable fragments (scFv)) and chimeric molecules containing them (such as chimeric antigen receptors). Also provided in the examples herein are uses and methods of using such agents, including for detecting, using, manipulating, and / or stimulating cells or therapies containing or suspected of containing the antibody or chimeric molecule, such as in the detection, stimulation, or use of CAR-expressing cells.

[0010] In some aspects, the antibody is or comprises an anti-idiotype (anti-Id) antibody or an antigen-binding fragment thereof that specifically binds to a target antibody that is or contains the variable region of an antibody designated 4F11, and / or specifically binds to a chimeric molecule containing such an antibody fragment, such as a CAR, having a binding domain containing the antibody variable region or a portion thereof derived from 4F11, such as in the form of an scFv.

[0011] In some embodiments, the anti-idiotype antibody is a non-blocking anti-idiotype antibody. For example, the binding of the anti-idiotype antibody to the target antibody (e.g., an anti-CD70 antibody) does not block the binding of the target antibody to the target antigen (e.g., CD70).

[0012] In some embodiments, the anti-idiotype antibody is a blocking anti-idiotype antibody. For example, the binding of the anti-idiotype antibody to the target antibody (e.g., an anti-CD70 antibody) blocks or reduces the binding of the target antibody to the target antigen (e.g., CD70).

[0013] In some embodiments, the anti-CD70 scFv derived from 4F11 comprises an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the isolated antibody does not bind to the framework region of 4F11. In some embodiments, the isolated antibody has a dissociation constant K of no greater than 10 nM, no greater than 5 nM, no greater than 1 nM, no greater than 100 pM, no greater than 90 pM, no greater than 80 pM, no greater than 70 pM, no greater than 60 pM, no greater than 50 pM, no greater than 40 pM, no greater than 30 pM, or no greater than 20 pM DBinding to anti-CD70 scFv derived from 4F11, as determined by Biacore assay at 25°C. In various embodiments, the antigen-binding molecule is selected from the group consisting of: antibody, scFv, Fab, Fab', Fv, F(ab')2, dAb, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, IgE antibody, IgD antibody, IgM antibody, IgG1 antibody, IgG1 antibody having at least one mutation in the hinge region, IgG2 antibody, IgG2 antibody having at least one mutation in the hinge region, IgG3 antibody, IgG3 antibody having at least one mutation in the hinge region, IgG4 antibody, IgG4 antibody having at least one mutation in the hinge region, antibody comprising at least one non-naturally occurring amino acid, and any combination thereof.

[0014] In additional embodiments, the isolated anti-idiotypic antibody comprises a heavy chain (HC), and in a specific embodiment, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:2. In additional specific embodiments of the antibodies provided herein, the heavy chain CDR1 is selected from the group consisting of SEQ ID NO:6, 7, and 8. In additional specific embodiments of the antibodies provided herein, the heavy chain CDR2 is selected from the group consisting of SEQ ID NO:9 and 10. In still other embodiments provided herein, the heavy chain CDR3 is selected from the group consisting of SEQ ID NO:11. In still additional embodiments, the heavy chain of the antibodies provided herein comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each CDR comprising the amino acid sequence shown in Table 1c.

[0015] In some embodiments, the anti-idiotypic 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% identical to the VH of the antigen-binding molecule provided herein.

[0016] In some embodiments, the isolated anti-idiotypic 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 further 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 still 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 light chain CDR1, light chain CDR2, and light chain CDR3, each CDR comprising the amino acid sequence in Table 1d.

[0017] 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% identical to the VL of an antigen-binding molecule.

[0018] In some embodiments, the anti-idiotypic antibodies provided herein comprise (a) a VH that comprises the amino acid sequence of SEQ ID NO:2, and (b) a VL that comprises the amino acid sequence of SEQ ID NO:4. In another embodiment, the antibody provided herein comprises (a) a VH CDR1 that comprises the amino acid sequence of SEQ ID NO:6, 7, or 8; (b) a VH CDR2 that comprises the amino acid sequence of SEQ ID NO:9 or 10; (c) a VH CDR3 that comprises the amino acid sequence of SEQ ID NO:11; (d) a VL CDR1 that comprises the amino acid sequence of SEQ ID NO:12; (e) a VL CDR2 that comprises the amino acid sequence of SEQ ID NO:13; and (f) a VL CDR3 that comprises the amino acid sequence of SEQ ID NO:14.

[0019] In additional embodiments, the isolated anti-idiotypic antibody comprises a heavy chain (HC), and in a specific embodiment, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:21. In additional specific embodiments of the antibodies provided herein, the heavy chain CDR1 is selected from the group consisting of SEQ ID NO:25, 26, and 27. In additional specific embodiments of the antibodies provided herein, the heavy chain CDR2 is selected from the group consisting of SEQ ID NO:28 and 29. In yet other embodiments of the antibodies provided herein, the heavy chain CDR3 is selected from the group consisting of SEQ ID NO:30. In still additional embodiments, the heavy chain of the antibodies provided herein comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, each CDR comprising the amino acid sequence shown in Table 1c.

[0020] In some embodiments, the anti-idiotypic 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% identical to the VH of the antigen-binding molecule provided herein.

[0021] In some embodiments, the isolated anti-idiotypic antibody provided herein comprises 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 additional specific embodiments, the light chain CDR1 of the antibodies provided herein can comprise SEQ ID NO:31. In other embodiments, the light chain CDR2 of the antibodies provided herein can comprise SEQ ID NO:32. In still additional embodiments, the light chain CDR3 of the antibodies provided herein can comprise SEQ ID NO:33. In still additional embodiments, the light chain of the antibodies provided herein comprises light chain CDR1, light chain CDR2, and light chain CDR3, each CDR comprising the amino acid sequence in Table 1d.

[0022] In some embodiments, there is provided a VL 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% identical to the VL of the antigen-binding molecule provided herein.

[0023] In some embodiments, the anti-idiotypic antibodies provided herein comprise (a) a VH that comprises the amino acid sequence of SEQ ID NO:21, and (b) a VL that comprises the amino acid sequence of SEQ ID NO:23. In another embodiment, the antibody provided herein comprises (a) a VH CDR1 that comprises the amino acid sequence of SEQ ID NO:25, 26, or 27; (b) a VH CDR2 that comprises the amino acid sequence of SEQ ID NO:28 or 29; (c) a VH CDR3 that comprises the amino acid sequence of SEQ ID NO:30; (d) a VL CDR1 that comprises the amino acid sequence of SEQ ID NO:31; (e) a VL CDR2 that comprises the amino acid sequence of SEQ ID NO:32; and (f) a VL CDR3 that comprises the amino acid sequence of SEQ ID NO:33.

[0024] In some embodiments, the anti-idiotypic 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% identical to the VH of the antigen-binding molecule provided herein.

[0025] In some embodiments, provided herein is a VL 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% identical to the VL of the antigen-binding molecule.

[0026] In some embodiments, the anti-idiotypic 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% identical to the VH of the antigen-binding molecule provided herein.

[0027] In some embodiments of any such embodiment, the target antibody or antigen-binding fragment is a single-chain fragment. In some aspects, the fragment contains antibody variable regions joined by a flexible linker. In some embodiments of any such embodiment, the fragment contains a scFv.

[0028] In some embodiments of any such embodiment, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope of the target antibody or its antigen-binding fragment, and the epitope is the same as or overlaps with the epitope specifically bound by the anti-id antibody or antigen-binding fragment according to any one of the embodiments described herein.

[0029] In some embodiments of any such embodiment, the target antibody or antigen-binding fragment is within or included 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 target antibody or antigen-binding fragment included within or included in the antigen-binding domain of the extracellular portion of the CAR. In some embodiments, the target antibody or antigen-binding fragment is a scFv, and the anti-id antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR.

[0030] In some embodiments of any such embodiment, the antibody or fragment specifically binds to a scFv derived from antibody 4F11 included within the extracellular portion of the chimeric antigen receptor, optionally wherein the scFv derived from antibody 4F11 contains the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16 and / or a sequence containing the amino acids shown in SEQ ID NO: 35.

[0031] In some embodiments of any such embodiment, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope within or encompassing all or a portion of the complementarity-determining region (CDR) of the target antibody or antigen-binding fragment.

[0032] In some embodiments of any such embodiment, the anti-idiotypic antibody or fragment is an antagonist antibody of the CAR containing the target antibody or antigen-binding fragment. In some embodiments of any such embodiment, the antibody or fragment is an antagonist of the CAR containing the target antibody or antigen-binding fragment.

[0033] In some embodiments of any such embodiment, the anti-idiotypic antibody or its antigen-binding fragment is humanized. In some embodiments of any such embodiment, the anti-idiotypic antibody or its antigen-binding fragment is recombinant. In some embodiments of any such embodiment, the anti-idiotypic antibody or its antigen-binding fragment is monoclonal.

[0034] In some embodiments of any such embodiment, the anti-idiotypic antibody or its antigen-binding fragment is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, single-chain variable fragments (scFv) or single-domain antibodies.

[0035] In some embodiments of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof comprises at least a portion of an immunoglobulin constant region. In some embodiments, at least a portion of the immunoglobulin constant region comprises an Fc region or a portion of the Fc containing the CH2 and CH3 domains. In some aspects, the constant region is derived from human IgG. In some embodiments of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment is a complete antibody or a full-length antibody.

[0036] Provided herein is a vector comprising a nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell comprising an anti-idiotypic 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.

[0037] Provided herein is a method of producing an anti-idiotypic antibody or antigen-binding fragment thereof, the method 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 of producing an anti-idiotypic antibody or antigen-binding fragment comprises culturing a cell according to any one of the embodiments described herein under conditions for expressing the heavy chain and / or the light chain, and recovering or isolating the antibody. Also provided herein is an anti-idiotypic antibody or antigen-binding fragment thereof produced by the method according to any one of the embodiments described herein.

[0038] Also provided herein is a method of preparing an isolated antibody that specifically binds to a molecule disclosed herein, the 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 chimeric antigen receptor ("CAR") comprising the amino acid sequence of SEQ ID NO:1 ("4F11 CAR") or 34 ("4F11-RSRQR" or "4F11-QR3", "R" refers to the rituximab recognition epitope, and "Q" refers to the CD34 epitope recognized by QBEND10), the method comprising growing or culturing a cell comprising a polynucleotide encoding such isolated antibody under suitable conditions.

[0039] Also provided are methods of detecting using any of the provided agents, such as anti-idiotype antibodies. In some embodiments, provided is a method of detecting a target antibody or an antigen-binding fragment thereof (such as a CAR containing the same), the method comprising: (a) contacting a composition containing the target antibody (such as a target antibody having a variable region derived from antibody 4F11 or an antigen-binding fragment derived from 4F11) with an anti-idiotype antibody or an antigen-binding fragment thereof; and (b) detecting the anti-idiotype antibody that binds to the target antibody or antigen-binding fragment and / or detecting the presence or absence of the target antibody or agent.

[0040] In some embodiments, the provided methods relate to detecting a CAR containing a target antibody or an antigen-binding fragment thereof according to any of the embodiments herein, such as a CAR containing a variable domain derived from 4F11. In some aspects, the method comprises: (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or an antigen-binding fragment thereof according to any of the embodiments herein or a conjugate that specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any of the embodiments herein; and (b) detecting the cells that bind to the anti-idiotype antibody. In some embodiments of any such embodiments, the anti-idiotype antibody or an antigen-binding fragment thereof is directly or indirectly labeled for detection.

[0041] In some embodiments, provided is a method of selecting cells from a cell population, the method comprising: (a) contacting a cell population expressing a chimeric antigen receptor (CAR) containing a target antibody or cells that bind to a target antibody with an anti-idiotype antibody or an antigen-binding fragment thereof according to any of the embodiments herein or a conjugate that specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any of the embodiments herein, wherein the target antibody is antibody 4F11 or an antigen-binding fragment thereof; and (b) selecting the cells that bind to the anti-idiotype antibody.

[0042] In some cases, the cells that bind to the anti-idiotype antibody are selected by affinity-based separation. In some aspects, the affinity-based separation is immunoaffinity-based separation. In some embodiments of any such embodiments, the affinity-based separation is performed by flow cytometry.

[0043] Also provided are methods of using an agent to stimulate cells, such as methods of stimulating cells containing a molecule (such as a CAR that is or contains a target antibody recognized by an anti-idiotype antibody). In some aspects, the methods involve incubating an input composition of cells containing a chimeric antigen receptor (CAR) that expresses a target antibody that is antibody 4F11 or an antigen-binding fragment thereof with an anti-idiotype antibody or an antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate that specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any one of the embodiments described herein, thereby generating an output composition containing the stimulated cells.

[0044] In some embodiments, the methods result in the proliferation, activation, stimulation, cytokine release, or other functional outcomes of cells expressing a chimeric receptor (such as a CAR recognized by an anti-id antibody), such as upregulation of activation markers or cytokine release or production.

[0045] In some aspects, the CAR contains a target antibody that specifically binds to CD70. In some embodiments, the target antibody is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments of any such embodiment, the anti-idiotype antibody or an antigen-binding fragment thereof is an anti-idiotype antibody or an antigen-binding fragment thereof that specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any one of the embodiments described herein.

[0046] In some embodiments of any such embodiment, the target antibody or antigen-binding fragment contains a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:15 and / or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:16.

[0047] In some embodiments, provided is a method of purifying an antibody or an antigen-binding fragment thereof, the method comprising (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 an antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate that specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any one of the embodiments described herein; and (b) separating 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 an antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate that specifically binds to the target antibody that is antibody 4F11 or an antigen-binding fragment thereof according to any one of the embodiments described herein; and (b) separating the complex comprising the anti-idiotype antibody.

[0048] In some embodiments of any such embodiment, the antigen-binding fragment comprises 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 a scFv. In some embodiments of any such embodiment, the antigen-binding fragment is located within or included in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR).

[0049] In some embodiments of any such embodiment, the target antibody binds to CD70. In some embodiments, the antigen-binding fragment of the target antibody is derived from antibody 4F11, optionally wherein the antigen-binding fragment of the target antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:15 and / or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:16. In some embodiments of any such embodiment, the antigen-binding fragment of the target antibody is a single-chain variable fragment (scFv) derived from antibody 4F11, optionally wherein the scFv comprises the sequence of the amino acids shown in SEQ ID NO:35.

[0050] In some embodiments, there is provided a method of depleting cells, the method comprising administering to a subject a composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate that specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof, wherein the subject has been administered cells expressing a chimeric antigen receptor (CAR) comprising a 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 an antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate that specifically binds to a target antibody that is antibody 4F11 or an antigen-binding fragment thereof, wherein the subject has been administered cells expressing a chimeric antigen receptor (CAR) comprising a target antibody that contains antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, depletion occurs via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0051] In various embodiments, the antibodies provided herein further comprise a detectable label, and the detectable label can be selected from the group consisting of: fluorescent labels, photochromic compounds, protein fluorescent labels, magnetic labels, radioactive labels, and haptens. When a fluorescent label is desired, the fluorescent label can 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 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 rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), 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"), 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 a specific embodiment, the fluorescent label can be R-phycoerythrin (PE) or allophycocyanin (APC).

[0052] The present invention also provides a composition comprising the antibody provided herein and optionally a pharmaceutically acceptable carrier or vehicle.

[0053] The present invention provides polynucleotides encoding the heavy chain of the isolated antibody of the antibody provided herein. In addition, polynucleotides encoding the light chain of the isolated antibody of the antibody provided herein are also provided. Vectors comprising polynucleotides encoding the heavy chain of the isolated antibody of the antibody provided herein and encoding the light chain of the isolated antibody of the antibody provided herein are also provided. Cells comprising such vectors are also provided, and in various embodiments, the cells comprise cells selected from the group consisting of CHO cells, Sp2 / 0 cells, rabbit cells, and Escherichia coli (E. coli) cells.

[0054] A method for preparing the isolated antibody provided herein is also provided and may comprise incubating the cells provided herein under suitable conditions.

[0055] A method for determining the number of cells expressing the scFv derived from 4F11 is provided and may comprise contacting a sample with an isolated antibody that specifically binds to the scFv derived from 4F11 conjugated to a detectable label, and determining the number of cells expressing the scFv derived from 4F11 in the sample. In some embodiments, the isolated antibody that specifically binds to the scFv derived from 4F11 is the antibody provided herein or a humanized form thereof.

[0056] A method for determining the number of cells presenting a polypeptide comprising an anti-CD70 scFv derived from 4F11 is also provided, wherein the method comprises: (a) providing a sample comprising 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 permit binding of the polypeptide to the antigen-binding molecule; and (c) determining the number of cells presenting the polypeptide in the sample.

[0057] The present disclosure provides a method for determining the presence or absence of a polypeptide comprising an anti-CD70 scFv derived from 4F11, wherein the method comprises: (a) providing a sample known or suspected to comprise 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 permit binding of the polypeptide to the antigen-binding molecule; and (c) detecting the presence or absence of a polypeptide:antigen-binding molecule complex. In an embodiment of the method, the sample is a formalin-fixed sample. In another embodiment, the scFv derived from 4F11 is a component of a chimeric antigen receptor (CAR), and in a further embodiment, the cell expressing the scFv CAR derived from 4F11 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 can be selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.When the detectable label is a fluorescent label, the fluorescent label can be selected from the group consisting of: Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, cascade blue, pacific blue, pacific orange, fluorescein 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 rhodamine B, Texas red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, 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 a specific embodiment, the fluorescent label is R-phycoerythrin (PE) or allophycocyanin (APC). In some embodiments, the cell expressing the scFv CAR derived from 4F11 is an immune cell, wherein the immune cell is a T cell, and the immune cell can be placed in vitro or in vivo. In some embodiments, the T cell is placed in blood, an extracted tissue, an ex vivo grown tissue, or a cell culture medium. In one embodiment, the T cell is an autologous T cell.In another embodiment, the T cells are allogeneic T cells.

[0058] In another 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 expressing an anti-CD70 antibody comprising the amino acid sequences of SEQ ID NOs: 15 and 16, the method comprising contacting the cells with an anti-idiotype antibody disclosed herein and determining the number of cells expressing the anti-CD70 antibody in the cells.

[0059] In another aspect, the present disclosure provides a method of determining the presence or absence of cells expressing an anti-CD70 antibody comprising the amino acid sequence of SEQ ID NO: 15 and / or 16, the method comprising contacting the cells with an isolated antibody disclosed herein and determining the presence or absence of cells expressing the anti-CD70 antibody in the cells. In yet another aspect, the present disclosure provides a method of purifying an anti-CD70 antibody or an 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-idiotype antibody disclosed herein to form a complex; and (b) separating or purifying the complex comprising the anti-CD70 antibody and the isolated antibody from the sample. In some embodiments, the anti-CD70 antibody is a scFv comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the cells express 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 cells are CAR T cells.

[0060] Also provided is a method of 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 the cells that bind to the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody further comprises a detectable label. In some embodiments, the cells that bind to the anti-idiotype antibody are selected by affinity-based separation.

[0061] Also provided is a method for stimulating anti-CD70 CAR T cells, the method comprising contacting the anti-CD70 CAR T cells with an antibody described herein. In some embodiments, the antibody is conjugated to beads or a solid surface. In certain embodiments, the anti-CD70 CAR T cells are present in a drug substance or a medicinal product. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A and 1B : Flow cytometry determination of the specificity of anti-idiotypic antibody clone 37 ( Figure 1A , first and second row of small panels from the top) and clone 60 ( Figure 1A , third and fourth row of small panels from the top, and Figure 1B ).

[0063] Figure 2A and 2B : Flow cytometry comparison of anti-idiotypic antibody clone 60, the clone conjugated with PE (phycoerythrin, Figure 2A ) or unconjugated ( Figure 2B ), recombinantly produced or purified from hybridoma cultures.

[0064] Figure 3 : Flow cytometry determination of the simultaneous binding of anti-Id antibody to CD70 CAR or the blocking of the binding of anti-Id antibody to CD70 CAR in the presence (top small panels) or absence (bottom small panels) of the target antigen CD70 protein.

[0065] Figures 4A - 4B : Flow cytometry determination of the simultaneous binding or blocking to the target antigen. NTD, untransduced.

[0066] Figure 5 : Surface plasmon resonance determination of affinity.

[0067] Figures 6A - 6B : Surface plasmon resonance determination of the simultaneous binding to the target antigen hCD70. Detailed Description

[0068] The present disclosure provides agents, such as anti-idiotypic (anti-Id) antibodies and antigen-binding fragments (such as Fv, Fab, F(ab')2, single-domain Abs, VHHs, or single-chain fragments, including scFv), that specifically recognize an anti-CD70 antibody moiety (such as an anti-CD70 antibody moiety present in a recombinant receptor, including a chimeric antigen receptor). Also provided are uses and methods of use thereof, as well as compositions and articles comprising such agents, including for specifically identifying, quantifying, selecting, isolating, purifying, and / or stimulating and / or activating cells expressing or comprising a target antibody or fragment, such as anti-CD70 CAR T cells. In some embodiments, the provided antibodies can be used for the specific identification, quantification, and / or selection of various anti-CD70 CARs, such as CARs that bind to or are expressed on the cell surface, and can also be used to specifically activate cells expressing a target CAR, such as CAR T cells. In some embodiments, antibodies are provided that are specific for an anti-CD70 antibody designated 4F11 or antibody fragments derived therefrom, including antibodies and CARs containing variable regions derived from such antibodies, and / or antibodies containing an idiotype contained therein.

[0069] In some aspects, compared to conventional reagents, the provided anti-idiotypic antibodies have advantages in detecting, identifying, manipulating, and / or influencing and / or engineering cells expressing a CAR (and specifically a CAR containing an anti-CD70 antibody scFv extracellular domain or one containing a recognized idiotype). In certain available methods, the detection (and / or stimulation or manipulation) of the presence, absence, or amount of a CAR or cells expressing a CAR in a sample is performed by assessing the presence, absence, or amount of a surrogate molecule (such as a surrogate molecule included in a construct encoding a CAR and thus serving as an indirect or surrogate marker of its expression).

[0070] In some embodiments, the provided anti-idiotypic antibodies and antigen-binding fragments overcome the challenges of low binding affinity associated with a target antibody ligand and non-specific binding associated with antibody reagents directed against the constant region of a target antibody, thereby providing reagents with high affinity and specificity for their target antibody or antigen-binding fragment. In some embodiments, compared to CD70-Fc and other reagents currently available for detecting or identifying a CAR comprising an anti-CD70 antibody, the provided antibodies exhibit higher specificity and binding affinity for their target antibody or antigen-binding fragment (such as an anti-CD70 antibody designated 4F11).

[0071] An "antibody" is an immunoglobulin molecule that is capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through 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 configurations of immunoglobulin molecules that contain an antigen recognition site, such as, including but not limited to, single-chain (scFv) and domain antibodies (such as shark and camel antibodies), and fusion proteins that contain an antibody. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not need to have any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0072] 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 the intact antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab; Fab'; F(ab')2; the Fd fragment consisting of the VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single-domain antibody (dAb) fragments (see, for example, Ward et al., Nature 341:544-546, 1989) and isolated complementarity-determining regions (CDRs).

[0073] Antibodies, antibody conjugates, or polypeptides that "specifically bind" to a target are well-known terms 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 more frequently, more rapidly, for a longer duration, and / or with greater affinity with a particular cell or substance compared to alternative cells or substances. An antibody "specifically binds" to a target if it binds to the target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. It should also be understood from this definition that, for example, an antibody (or portion 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.

[0074] A chimeric antigen receptor (CAR) can refer to a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to the target antigen, the CAR expressed on the surface of an immune cell can activate the immune cell to attack and destroy cells carrying the antigen (e.g., cancer cells). The CAR can also incorporate co-stimulatory or signaling domains to increase its potency. See Krause et al., J. Exp. Med., Vol. 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.

[0075] CARs can be expressed on the surface membrane of cells. Thus, CARs can include a transmembrane domain. Suitable transmembrane domains for the CARs disclosed herein have the ability to: (a) be expressed on the surface of a cell (e.g., an immune cell), such as, but not limited to, a lymphocyte (e.g., a T cell) or a natural killer (NK) cell; and (b) interact with a ligand-binding domain and an intracellular signaling domain to direct an immune cell's cellular response to a predetermined target cell. The transmembrane domain can be derived from a natural or a synthetic source. 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 α, β, γ, or δ; a polypeptide that constitutes the CD3 complex; an IL-2 receptor, such as p55 (α chain), p75 (β chain or γ chain); an Fc receptor, specifically a subunit chain of Fcγ receptor III; or a CD protein. Alternatively, the transmembrane domain can be synthetic and can primarily comprise hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1). The transmembrane domain can further include a stalk domain located between the extracellular ligand-binding domain and the transmembrane domain. The stalk domain can comprise up to 300 amino acids, such as 10 to 100 amino acids or 25 to 50 amino acids. The stalk region can be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stalk domain can be a synthetic sequence corresponding to a naturally occurring stalk sequence, or can be a completely synthetic stalk sequence. In some embodiments, the stalk domain is part of the human CD8α chain (e.g., NP_001139345 and its isotypes). In another specific embodiment, the transmembrane domain comprises a part of the human CD8α chain. In some embodiments, the CAR can be introduced into an immune cell as a transgene via a vector (e.g., a plasmid vector). In some embodiments, the vector (e.g., a plasmid vector) can also contain, for example, a selection marker that provides for the identification and / or selection of cells that have received the vector.

[0076] The intracellular (cytoplasmic) domain of the CARs disclosed herein can provide activation of at least one of the normal effector functions of the immune cells that contain the CARs, such as signal 1 / activation and / or signal 2 / corestimulation. For example, the effector functions of a T cell can refer to cytolytic activity or helper activity, including the secretion of cytokines.

[0077] In some embodiments, the intracellular signaling domain used in CARs can be, for example but not limited to, the cytoplasmic sequences of the T cell receptor and co-receptors that cooperate upon antigen receptor engagement to initiate signal transduction, and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0078] It should be understood that suitable (e.g., activating) intracellular domains include but are not limited to signaling domains derived from (or corresponding to) the following: CD3ζ, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine 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, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, 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, a ligand that specifically binds to CD83, or any combination thereof.

[0079] In addition to the activation domain described above, the intracellular domain of the CAR of the present disclosure may incorporate a co-stimulatory signaling domain (which may be interchangeably referred to herein as a co-stimulatory molecule or co-stimulatory domain) to increase its potency. The co-stimulatory domain may provide a signal in addition to the primary signal provided by an activating molecule as described herein.

[0080] As used herein, "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as but not limited to proliferation. Co-stimulatory molecules include but are not limited to MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc. In certain embodiments, the anti-idiotypic antibody is multi-specific. Among multi-specific binding molecules are multi-specific antibodies, including for example bispecific antibodies. A multi-specific binding partner, such as an antibody, has binding specificity for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is directed against an anti-CD70 antibody portion, and the other is directed against another antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of the anti-CD70 antibody portion. Bispecific antibodies can also be used to localize a cytotoxic agent to cells that express the anti-CD70 antibody portion on their surface, such as anti-CD70 CAR T cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Among bispecific antibodies are multi-specific single-chain antibodies, such as diabodies, tribodies, and tetra-bodies, tandem di-scFv, and tandem tri-scFv.

[0081] In addition, in certain embodiments, anti-idiotypic antibodies and antigen-binding fragments can be selected as agonists or antagonists of chimeric receptors (comprising their target antibodies or antigen-binding fragments), thereby allowing for the selective detection, isolation, ablation, and / or depletion (e.g., via antibody-dependent cell-mediated cytotoxicity (ADCC) killing) and / or stimulation or activation of cells having such chimeric receptors bound to or expressed on their surface. In some embodiments, the anti-idiotypic antibodies can be humanized or fully human antibodies. Anti-idiotypic antibody agonists are provided herein that exhibit activity stimulating (such as activating) CARs containing extracellular binding domains derived from an anti-CD70 antibody named 4F11. In some aspects, such antibodies can be used in methods for stimulating and expanding cells expressing a particular CAR, the methods being included in the process for generating and preparing cells expressing the CAR. In some embodiments, the methods for stimulation and expansion can be in vitro methods. In some embodiments, the methods for stimulation and expansion can be incorporated into the CAR T preparation process. In some embodiments, the methods for stimulation and expansion can be in vivo methods. In some embodiments, the anti-idiotypic antibodies can be humanized or fully human antibodies.

[0082] Nucleic acids encoding the provided anti-idiotypic antibodies and fragments are also provided, as well as cells for expressing and for generating these anti-idiotypic antibodies and fragments, such as recombinant cells. Methods for preparing and using the anti-idiotypic antibodies and fragments are also provided, as well as cells expressing or containing the anti-idiotypic antibodies and fragments.

[0083] The scFv portion of some chimeric antigen receptors (CARs) is derived from a fully human antibody clone 4F11 that has high affinity for CD70. The present disclosure provides reagents for detecting anti-CD70 CARs comprising an scFv portion derived from antibody clone 4F11. Anti-idiotypic antibodies are disclosed herein that specifically bind to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11. The anti-id antibodies from molecules derived from 4F11 disclosed herein exhibit specific high-affinity binding to chimeric antigen receptors (CARs) comprising an scFv derived from 4F11. A non-limiting example of an scFv derived from 4F11 comprises the amino acid sequence of SEQ ID NO:35.

[0084] When conjugated to a bright fluorophore, the anti-id antibodies disclosed herein stain cells expressing a chimeric antigen receptor (CAR) comprising a scFv derived from 4F11 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, for example, in the context of non-clinical study learning to prepare immune cells comprising a scFv derived from 4F11 as a clinical flow-based pharmacokinetic reagent, and for clinical immunogenicity studies.

[0085] 4F11 is a CD70 monoclonal antibody that recognizes human CD70. The single-chain variable fragment (scFv) formed from 4F11 comprises a targeting component of some chimeric antigen receptors (CARs). In some embodiments, the scFv derived from the CD70 monoclonal antibody 4F11 comprises a portion of the immunoglobulin gamma-1 heavy chain of the CD70 monoclonal antibody 4F11 (SEQ ID NO: 15) and a portion of the immunoglobulin kappa light chain of the anti-CD70 monoclonal antibody 4F11 (SEQ ID NO: 16) linked together by a flexible linker. In some embodiments, the scFv comprises the variable fragment of the immunoglobulin gamma-1 heavy chain of the anti-CD70 monoclonal antibody 4F11 and the variable fragment of the immunoglobulin kappa light chain of the anti-CD70 monoclonal antibody 4F11 linked together by a flexible linker.

[0086] The variable domain of the immunoglobulin gamma-1 heavy chain of the anti-CD70 monoclonal antibody 4F11 comprises the amino acid sequence (“4F11VH”), bold residues indicating the CDR sequences according to the Kabat nomenclature system, and underlined residues indicating the CDR sequences according to the Chothia nomenclature system:

[0087]

[0088] The variable domain of the immunoglobulin kappa light chain of the anti-CD70 monoclonal antibody 4F11 comprises the amino acid sequence (“4F11VL”), bold residues indicating the CDR sequences according to the Kabat nomenclature system, and underlined residues indicating the CDR sequences according to the Chothia nomenclature system:

[0089]

[0090] An exemplary chimeric antigen receptor (“CAR”) derived from 4F11 comprises the following amino acid sequence or alternatively consists of the following amino acid sequence (underlined is the exemplary signal peptide MALPVTALLLPLALLLHAARP SEQ ID NO: 39):

[0091] MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGFSLS

[0092] NARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQV

[0093] VLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSG

[0094] GGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKP

[0095] GKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLN

[0096] SFPFTFGGGTKVEINTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT

[0097] RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV

[0098] QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG

[0099] RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG

[0100] MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:1) or the mature protein without a signal peptide:

[0101] QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWL

[0102] AHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIR

[0103] DYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMS

[0104] ASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFS

[0105] GSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINTTTPAPRPP

[0106] TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL

[0107] LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0108] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

[0109] PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT

[0110] KDTYDALHMQALPPR(SEQ ID NO:37)。

[0111] The second exemplary chimeric antigen receptor (“CAR”) derived from 4F11 comprises the following amino acid sequence or alternatively consists of the following amino acid sequence (underlined is an exemplary signal peptide, e.g., derived from CD8a):

[0112] MALPVTALLLPLALLLHAARP GGGGSCPYSNPSLCSGGGGSGGGGSQVT

[0113] LKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIF

[0114] SNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYY

[0115] DISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASV

[0116] GDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSG

[0117] SGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINGSGGGGSCPYS

[0118] NPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCTTTPAPRP

[0119] PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL

[0120] LLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

[0121] LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG

[0122] KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA

[0123] Mature protein with or without signal peptide: TKDTYDALHMQALPPR (SEQ ID NO:34)

[0124] GGGGSCPYSNPSLCSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINGSGGGGSCPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:38).

[0125] 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 comprises 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 with the amino acid sequence of SEQ ID NO:15 and / or SEQ ID NO:16 and / or with SEQ ID NO:35.

[0126] Exemplary scFv derived from 4F11 comprises the following amino acid sequence or alternatively consists of the following amino acid sequence:

[0127] QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWL

[0128] AHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIR

[0129] DYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMS

[0130] ASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEIN(SEQ ID NO:35)

[0131] An exemplary 4F11-derived scFv with a His tag comprises the following amino acid sequence: QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVTWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKTQVVLTMTNMDPVDTATYYCARIRDYYDISSYYDYWGQGTLVSVSSGGGGSGGGGSGGGGSDIQMTQSPSAMSASVGDRVTITCRASQDISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLLPEDFATYYCLQLNSFPFTFGGGTKVEINGGSGHHHHHHHHHH(SEQ ID NO:36)

[0132] In some embodiments, the scFv comprises 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 to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:34. Antigen-binding molecules are disclosed herein that include an antibody that specifically binds to a 4F11-derived anti-CD70 scFv, as well as molecules comprising these sequences and cells presenting such molecules. A humanized form of the antigen-binding molecule also forms one aspect of the present disclosure. The use and applications of these antigen-binding molecules are also disclosed.

[0133] The subsection headings used herein are for purposes of arrangement only and should not be construed as limiting the subject matter described.

[0134] I. Antigen-Binding Molecules and Polynucleotides Encoding Them

[0135] As used herein, "antigen-binding domain" means any polypeptide that binds to a specific target antigen, for example, the specific target antigen can be the CD70 protein or a fragment thereof (which may be interchangeably referred to herein as "CD70 antigen", "CD70 target antigen" or "CD70 target"). In the context of the anti-idiotypic 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 scFv).

[0136] In some embodiments, the antigen-binding domain binds to the CD70 antigen on tumor cells. In some embodiments, the antigen-binding domain binds to the CD70 antigen on cells involved in hyperproliferative diseases or cells involved in viral or bacterial antigens.

[0137] The antigen-binding domain includes, but is not limited to, antibody-binding regions that are immunofunctional fragments. The term "immunofunctional fragment" (or "fragment") of an antigen-binding domain is an antigen-binding domain that contains a portion of an antibody 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 (regardless of how the portion is obtained or synthesized). Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding domains, including full 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., blocking). In some embodiments, the fragment can antagonize the activity of an anti-CD70 CAR.

[0138] In a specific embodiment, the anti-id antibody of the present disclosure is the antibody identified herein as clone 60 or clone 37 and contains heavy and light chain amino acids, variable regions, CDR sequences, and nucleotide sequences encoding such sequences, as provided and labeled herein.

[0139] Immunofunctional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity-determining regions ("CDR"), diabodies (where the heavy chain variable domain and the light chain variable domain are on the same polypeptide, linked by 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 (containing two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments can be derived from any mammalian source, including but not limited to human, mouse, rat, camel, or rabbit. As will be appreciated by those skilled in the art, the antigen-binding domain can include non-protein components.

[0140] Variable regions generally exhibit the same general structural pattern of relatively conserved framework regions (FRs) joined by three hypervariable regions (CDRs). The CDRs from each pair of chains are typically aligned by the framework regions, which can bind to a specific epitope. Both the light chain variable region and the heavy chain variable region generally contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. 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.

[0141] In some embodiments, the antigen-binding domain comprises one or more complementary binding regions (CDRs) present in the full-length light or heavy chain of an antibody and, in some embodiments, comprises a single heavy chain and / or light chain or a portion thereof. These fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of the antigen-binding domain, including the intact antibody.

[0142] In some embodiments, the antigen-binding domain is an antibody that is a fragment thereof, including one or more of its complementarity-determining regions (CDRs). In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) that comprises the light chain CDRs CDR1, CDR2, and CDR3, and the heavy chain CDRs CDR1, CDR2, and CDR3.

[0143] Assignment of amino acids to each of the framework, CDR, and variable domains is typically made according to the following numbering schemes: Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia and 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. Patent No. 7,709,226), contact numbering, or the AbM scheme (Antibody Modeling program, Oxford Molecular).

[0144] Thus, in some embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the Kabat numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the Chothia numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the contact numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the AbM numbering scheme.

[0145] 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 can comprise the antigen-binding site of a murine or rabbit monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for producing engineered monoclonal antibodies include those described in, for example, 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, chimeric antibodies are CDR-grafted antibodies. Techniques for humanizing antibodies are discussed in, for example, 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.

[0146] Variants of the anti-idiotypic antibodies are also within the scope of the present disclosure, such as variants comprising variable light and / or variable heavy domains or regions, each of which has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or higher than 99% identity with the amino acid sequence of the antigen-binding domain sequence described herein. In some embodiments, the anti-idiotypic 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% identical to the heavy chain variable region sequence provided in Table 1a and / or the 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% identical to the heavy chain constant region sequence provided in Table 1e and / or the light chain constant region sequence provided in Table 1e.

[0147] In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant forms contain two variable light chains and two variable heavy chains (or sub-parts thereof). Those skilled in the art will be able to determine suitable variants of the anti-idiotypic antibodies as described herein using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions not considered important for activity.

[0148] The anti-id antibodies of the present disclosure can also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any number of techniques familiar to those of ordinary skill in the art. Such methods include, but are 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 spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V region phage library, or other procedures known in the art and based on the present disclosure.

[0149] An anti-id antibody that specifically binds to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11 is considered "selective" when its binding to one target is tighter than its binding to a second target.

[0150] When the dissociation constant (Kd) is about 1 nM, an anti-idiotype antibody that specifically binds to anti-CD70 clone 4F11 and anti-CD70 molecules derived from 4F11 is described as "specifically binding" to its target antigen (e.g., murine 4F11 and molecules derived from 4F11). When the Kd is 1 - 5 nM, the antigen-binding domain specifically binds to the antigen with "high affinity", and when the Kd is 0.1 - 0.5 nM, the antigen-binding domain specifically binds to the antigen with "extremely high affinity". In one embodiment, the Kd of the antigen-binding domain is about 1 nM. In one embodiment, the off-target rate is <1×10 -5 . In other embodiments, the antigen-binding domain will bind to murine 4F11 and molecules derived from 4F11 with a Kd of about 1x10 -7 M to 1x10 -12 M, and in yet another embodiment, the antigen-binding domain will bind with a Kd of about 1x10 -5 to 1x10 -12 .

[0151] As provided herein, the anti-idiotype antibodies of the present disclosure specifically bind to 4F11 and molecules derived from 4F11 (e.g., CARs derived from 4F11). In certain embodiments, the anti-idiotype antibodies of the present disclosure bind to 4F11 and molecules derived from 4F11 with a KD of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, or less than 1×10 -9 M. In a specific embodiment, the anti-idiotype antibody binds to 4F11 and molecules derived from 4F11 with a KD of less than 1×10 -7 M. In another embodiment, the anti-idiotype antibody binds to 4F11 and molecules derived from 4F11 with a KD of less than 1×10 -8 M. In some embodiments, the anti-idiotype antibody binds with a KD of about 1×10 -7 M, about 2×10 -7 M, about 3×10 -7 M, about 4×10 -7 M, about 5×10 -7 M, about 6×10 -7 M, about 7×10 -7 M, about 8×10 -7 M, about 9×10 -7 M, about 1x10 -8 M, about 2×10 -8 M, about 3×10 -8 M, about 4×10 -8 M, about 5×10 -8 M, about 6×10 -8 M, about 7×10 -8 M, about 8×10 -8 M, about 9×10 -8 M, about 1×10 -9 M, about 2×10 -9 M, about 3×10 -9 M, about 4×10 -9 M, about 5×10 -9 M, about 6×10 -9 M, about 7×10 -9 M, about 8×10 -9 M, about 9×10 -9 M, about 1×10 -10 M or about 5×10 -10 The Kd of M binds to 4F11 and molecules derived from 4F11. In certain embodiments, Kd is calculated as K off / K on The quotient of K on and K off Using monovalent antibodies (such as Fab fragments) to determine, for example, by In other embodiments, Kd is calculated as K off / K on The quotient of K on and K off Using a bivalent antibody (such as a Fab fragment) to determine, for example, by Measured by surface plasmon resonance technique.

[0152] In some embodiments, the anti-ID antibody is present in an amount less than 1×10 -4 M -1 s -1 , less than 2×10 -4 M -1 s -1 , less than 3×10 -4 M -1 s -1 , less than 4×10 -4 M -1 s -1 , less than 5×10 -4 M -1 s -1 , less than 7×10 -4 M -1 s -1 , less than 8×10 -4 M -1 s -1 , less than 9×10 -4 M -1 s -1 , less than 1×10-5 M -1 s -1 、 less than 2×10 -5 M -1 s -1 、 less than 3×10 -5 M -1 s -1 、 less than 4×10 -5 M -1 s -1 、 less than 5×10 -5 M -1 s -1 、 less than 6×10 -5 M -1 s -1 、 less than 7×10 -5 M -1 s -1 、 less than 8×10 -5 M -1 s -1 、 less than 9×10 -5 M - 1 s -1 、 less than 1×10 -6 M -1 s -1 、 less than 2×10 -6 M -1 s -1 、 less than 3×10 -6 M -1 s -1 、 less than 4×10 -6 M -1 s -1 、 less than 5×10 -6 M -1 s -1 、 less than 6×10 -6 M -1 s -1 、 less than 7×10 -6 M -1 s -1 、 less than 8×10 -6 M -1 s -1 、 less than 9×10 -6 M -1 s -1 or less than 1×10 -7 M -1 s -1 of the association rate (k on ) binds to 4F11 and molecules derived from 4F11. In certain embodiments, k on is determined using a monovalent antibody (such as a Fab fragment), such as by, for example, Measured by surface plasmon resonance technology. In other embodiments, k on Determined using a bivalent antibody, such as by, for example Measured by surface plasmon resonance technology.

[0153] In some embodiments, the anti - id antibody is at less than 1×10 -2 s -1 、less than 2×10 -2 s -1 、less than 3×10 -2 s -1 、less than 4×10 -2 s -1 、less than 5×10 -2 s -1 、less than 6×10 -2 s -1 、less than 7×10 -2 s -1 、less than 8×10 -2 s -1 、less than 9×10 -2 s -1 、less than 1×10 -3 s -1 、less than 2×10 -3 s -1 、less than 3×10 -3 s -1 、less than 4×10 -3 s -1 、less than 5×10 -3 s -1 、less than 6×10 -3 s -1 、less than 7×10 -3 s -1 、less than 8×10 -3 s -1 、less than 9×10 -3 s -1 、less than 1×10 -4 s -1 、less than 2×10 -4 s -1 、less than 3×10 -4 s -1 、less than 4×10 -4 s -1 、less than 5×10 -4 s -1 、less than 6×10 -4 s -1 、less than 7×10 -4 s -1 、less than 8×10 -4s -1 and less than 9×10 -4 s -1 and less than 1×10 -5 s -1 or less than 5×10 -5 s -1 dissociation rate (k off ) binds to 4F11 and molecules derived from 4F11. In certain embodiments, k off is determined using a monovalent antibody (such as a Fab fragment), such as by, for example surface plasmon resonance technology. In other embodiments, k off is determined using a bivalent antibody, such as by, for example surface plasmon resonance technology.

[0154] Provided herein are anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11, which comprise 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 are bolded in the Kabat nomenclature system or according to the system, and CDRs are underlined in the Chothia nomenclature system or according to the nomenclature system).

[0155] Table 1a: Heavy chain variable region (VH)

[0156]

[0157] Provided herein are anti-idiotypic antibodies that specifically bind to anti-CD70 clone 4F11, which comprise 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 are bolded in the Kabat nomenclature system or according to the system, and CDRs are underlined in the Chothia nomenclature system or according to the nomenclature system).

[0158] Table 1b: Light chain variable region

[0159]

[0160] 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 antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the amino acid sequence or polynucleotide sequence of the VH is selected from the VH sequences presented in Table 1a; and wherein the amino acid sequence or polynucleotide sequence of the VL is selected from the VL sequences presented in Table 1b.

[0161] In some embodiments, an anti-idiotypic antibody that specifically binds to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprises VH CDR 1, CDR2, and CDR3 of the VH sequences presented in Table 1a. In some embodiments, VH CDR1, CDR2, and CDR3 are selected from the CDR sequences presented in Table 1c.

[0162] Table 1c: Heavy chain CDRs

[0163]

[0164]

[0165] In some embodiments, an anti-idiotypic antibody that specifically binds to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprises the VL CDR 1, CDR2, and CDR3 sequences presented in Table 1d.

[0166] In some embodiments, an anti-idiotypic antibody that specifically binds to anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11 comprises the heavy and light chain constant region sequences listed in Table 1e.

[0167] Table 1d: Light chain CDRs

[0168]

[0169] Table 1e: 1.60 Constant regions

[0170]

[0171]

[0172] In some embodiments of any such embodiments, the target antibody or antigen-binding fragment is within or included in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor; and / or the anti-idiotypic antibody or antigen-binding fragment specifically binds to the target antibody or antigen-binding fragment that is within or included in the antigen-binding domain of the extracellular portion of the CAR. In some embodiments, the target antibody or antigen-binding fragment is a scFv, and the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR. In some embodiments of any such embodiments, the antibody or fragment specifically binds to a scFv derived from antibody 4F11 that is included within the extracellular portion of the chimeric antigen receptor, optionally wherein the scFv derived from antibody 4F11 contains the heavy chain variable region shown in SEQ ID NO:15 and / or the light chain variable region shown in SEQ ID NO:16 (e.g., a scFv comprising the amino acid sequence of SEQ ID NO:35). In certain embodiments, the anti-idiotypic antibody or its antigen-binding fragment binds to an epitope in the scFv derived from 4F11, such as 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-idiotypic antibody or its antigen-binding fragment binds differentially to 4F11 in scFv format and IgG format. In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment advantageously differentiates 4F11 in scFv format from IgG format.

[0173] In some embodiments of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope within or comprising all or a portion of the complementarity determining region (CDR) of the target antibody or antigen-binding fragment.

[0174] In some embodiments of any such embodiments, the anti-idiotypic antibody or fragment is an antagonist antibody of the CAR containing the target antibody or antigen-binding fragment. In some embodiments of any such embodiments, the antibody or fragment is an antagonist of the CAR containing the target antibody or antigen-binding fragment.

[0175] In some embodiments of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment is humanized. In some embodiments of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment is recombinant. In some embodiments of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment is monoclonal.

[0176] In some embodiments of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, single-chain variable fragments (scFv) or single-domain antibodies.

[0177] In some embodiments of any such embodiment, the anti-idiotypic antibody or antigen-binding fragment thereof comprises at least a portion of an immunoglobulin constant region. In some embodiments, at least a portion of the immunoglobulin constant region comprises an Fc region or a portion of Fc containing the CH2 and CH3 domains. In some aspects, the constant region is derived from human IgG. In some embodiments of any such embodiment, the anti-idiotypic antibody or antigen-binding fragment is a whole antibody or a full-length antibody.

[0178] The present disclosure provides a vector comprising a nucleic acid molecule according to any one of the embodiments described herein. The present disclosure also provides a cell comprising an anti-idiotypic 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.

[0179] The present disclosure encompasses modifications of anti-id antibodies directed to sequences shown in Tables 1a to 1e, including functionally equivalent anti-id antibodies having modifications that do not significantly affect their properties and variants having enhanced or reduced activity and / or affinity. For example, the amino acid sequence can be mutated to obtain an anti-id antibody having the desired binding affinity for anti-CD70 clone 4F11 and antigen-binding molecules derived from 4F11. Modification of polypeptides is a routine practice in the art and thus does not need to be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly and adversely alter functional activity or maturation (enhancement) of the polypeptide's affinity for its ligand, or use of chemical analogs.

[0180] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging from one residue to the length of a polypeptide containing one hundred or more residues, as well as internal insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include antibodies having an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion-type variants of antibody molecules include fusions of enzymes or polypeptides that extend the half-life of the antibody in the bloodstream to the N-terminus or C-terminus of the antibody.

[0181] Substitution 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, sites of interest for substitution-type mutations include hypervariable regions / CDRs, but FR alterations are also encompassed. Conservative substitutions are shown under the heading "Conservative Substitutions" in Table 2. If such substitutions cause a change in biological activity, then more substantial changes named in Table 2 as "Exemplary Substitutions" or further described below with respect to amino acid classes can be introduced and the products screened.

[0182] Table 2: Amino Acid Substitutions

[0183]

[0184]

[0185] II. Method for Preparing Anti-Id Antibodies

[0186] For cloning a polynucleotide, a vector can be introduced into a host cell (an isolated host cell) to permit replication of the vector itself and thereby amplification of the copies of the polynucleotide contained therein. A cloning vector can contain sequence elements, typically including but not limited to an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and an optional marker. These elements can be selected by one of ordinary skill in the art as appropriate. For example, an origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0187] In certain embodiments, the present disclosure provides isolated host cells containing the vectors provided herein. Host cells containing the vector can be used for expression or cloning of the polynucleotide contained in the vector. Suitable host cells can 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, for example, members of the family Enterobacteriaceae such as Escherichia, for example, Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescens; and Shigella; and Bacilli such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas such as Pseudomonas aeruginosa; and Streptomyces.

[0188] 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 methods, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, gene gun methods, receptor-mediated gene delivery, and delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfecting and transforming cells for expressing the vector of interest are well known in the art. In additional embodiments, mixtures of different expression vectors can be used to genetically modify a donor population of immune effector cells, where each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, where a proportion of the engineered cells express more than one different CAR.

[0189] In one embodiment, the present disclosure provides a method for evaluating genetically engineered cells expressing a CAR targeting CD70. In some embodiments, the engineered cells are evaluated after thawing cryopreserved immune cells.

[0190] In some embodiments, the cells are formulated by first collecting the cells from their medium and then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount ("pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic media formulation, typically Normal Saline Normosol TM R (Abbott) or Plasma-Lyte TM A (Baxter), but a 5% dextrose aqueous solution or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin.

[0191] In some aspects, the anti-idiotype antibodies of the present disclosure are used to quantify the therapeutically required amount of cells in a composition of engineered T cells comprising a CAR derived from 4F11, such as an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof. In some embodiments, the therapeutically required amount is typically at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset), or more typically greater than 10 2 cells, and at most 10 6 cells, up to and including 10 8 or 10 9 cells, and can be greater than 10 10 cells. The number of cells will depend on the intended use of the composition and the type of cells included therein. The density of the required cells is typically greater than 10 6 cells / ml, and is typically greater than 107 cells / ml, typically 10 8 cells / ml or greater. Clinically relevant amounts of immune cells can be administered as multiple infusions that cumulatively equal or exceed 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 or 10 12 cells. In some aspects of the present disclosure, specifically, since all infused cells will be redirected to a specific target antigen (CD70), lower amounts of cells in the range of 10 6 / kg (10 6 -10 11 ) per patient can be administered. CAR therapy can be administered in multiple doses within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient receiving the therapy.

[0192] The CAR-expressing cell populations of the present disclosure can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present disclosure can comprise a cell population expressing a CAR or TCR as described herein, such as a T cell, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline, etc.; 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. The pharmaceutical compositions (solutions, suspensions, etc.) can include one or more of the following: sterile diluents such as water for injection, saline solutions (such as physiological saline), Ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic mono- or diglycerides that can act as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral preparations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions can be sterile.

[0193] III. Methods for Determining the Number of Cells Expressing Anti-CD70 CAR

[0194] The present disclosure provides a method for determining the number of cells expressing an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof present in a sample. For example, it may be desirable to determine the number of immune cells expressing an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof present in a sample obtained from a subject. Alternatively, it may be desirable to determine the number of cells transfected and expressing an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof, which can be used as a measure of the level of transfection efficiency. The disclosed method can be used in these and other applications where it is desired to determine the number of cells present in a sample expressing a molecule of interest, such as an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof.

[0195] Accordingly, a method is provided for determining the number of cells in a sample presenting a molecule, wherein the molecule comprises a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof.

[0196] In some embodiments, a sample is provided that comprises cells known or suspected of expressing a molecule of interest, the molecule of interest comprising a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising a scFv derived from 4F11) or a fragment thereof.

[0197] The sample is then contacted with an antigen-binding molecule that specifically binds to the molecule of interest under conditions that permit the formation of a binding complex comprising the cells present in the sample and the antigen-binding molecule. The antigen-binding molecule can be an antigen-binding molecule (or a fragment thereof) disclosed herein, e.g., in the figures, sequence listing, or examples 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 scFv derived from 4F11) or a fragment thereof can be employed in the disclosed method. Multiple examples of suitable antigen-binding molecules are provided herein, e.g., examples having one or more of the VH and VL shown in Table 1a and 1b or one or more of the CDRs shown in Table 1c and 1d and described herein.

[0198] The cells can be of any type and can 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 method can be any type of immune cells (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 cytotoxic T cells, T helper cells, and Treg cells. In a specific embodiment, the cells are T cells, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be employed in this embodiment of the disclosed method, and the cells can 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 (TIL), 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 CAR-expressing T cells. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are employed in the disclosed method, the T cells can be in vivo T cells or in vitro T cells. Additionally, the cells can be placed in or separated from any environment capable of maintaining the cells in a viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, a suitable buffer, etc.

[0199] In some embodiments, a sample comprising an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof is contacted with an anti-idiotype antibody that specifically binds to a binding molecule derived from CD70 as disclosed herein. In some embodiments, the anti-idiotype antibody comprises a detectable label. In some embodiments, the anti-idiotype antibody conjugated with a detectable label is contacted with a sample expressing an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) under conditions that permit the formation of a binding complex comprising the cells present in the sample and the anti-idiotype antibody. Any anti-idiotype antibody that specifically binds to an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) can be employed in the disclosed method. Multiple examples of suitable anti-idiotype antibodies are provided herein, e.g., examples having one or more of the CDRs shown in Table 1c or 1d.

[0200] Any detectable label can be employed in the methods described herein, and a suitable label can be selected using the desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein yellow, cascade blue, Texas red, IAEDANS, EDANS, BODIPYFL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexafluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680), cascade blue, cascade yellow, and R-phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas red (Pierce), Cy5, Cy5.5, Cy7 (Amersham LifeScience)). Suitable optical dyes, including fluorophores, are described in Johnson, "Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques", 11th Edition Methods Mol Biol Site - Specific Protein Labeling: , Life Technologies, (2010), which is hereby incorporated by reference; radiolabels (e.g., isotope markers such as 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); photochromic compounds, Halo tags, Atto dyes, Tracy dyes, protein fluorescent labels (e.g., protein fluorescent labels also include, but are not limited to, green fluorescent proteins, including Renilla, Ptilosarcus, or jellyfish luminescent protein species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clontech Labs., Inc., Genbank accession number U55762), 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) Journal of Immunology 150:5408-5417)), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be used in the methods of the present disclosure. See, for example, Obermaier et al., (2015) Methods in Molecular Biology Methods and Protocols ) 1295:153-65; Strack (2016) Nature Methods 13:33; Site-Specific Protein Labeling: Methods and Protocols "Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques", 11th Edition Figure 1A ) (edited by Gautier and Hinner) 2015, Springer. In some embodiments, the detectable label is a phycoerythrin (PE) or allophycocyanin (APC) fluorescent probe.

[0201] The label can be associated with the anti-id antibody at any position in the molecule, but it may be necessary to associate the label at one position (or multiple positions if multiple labels are used) at a point where the binding properties of the molecule are not altered (unless such altered binding activity is desired). Any antigen-binding molecule that specifically binds to the CD70-binding molecule (or a fragment thereof) can be used, such as the antigen-binding molecules disclosed herein, e.g., an antigen-binding molecule having one or more of the CDRs shown in Table 1c or 1d.

[0202] The antigen-binding molecule can be disposed on any surface or not disposed on a surface at all. For example, the antigen-binding molecule can be present in a buffer and the buffer-antigen-binding molecule can contact the sample. Alternatively, the antigen-binding molecule can associate with a surface. Suitable surfaces include agarose beads, magnetic beads (such as ) or plastic, glass, or ceramic plates (such as well plates), bags (such as cell culture bags), etc. The surface can be disposed in another structure (such as a column) by itself.

[0203] The conditions that allow the formation of the binding complex will depend on various factors. However, an aqueous buffer at physiological pH and ionic strength (such as in phosphate-buffered saline (PBS)) will generally favor the formation of the binding complex and is desirable in the disclosed methods.

[0204] Determine the number of cells present in the binding complex in the sample. The specific method for determining the number of cells present in the binding complex will depend on the nature of the selected label. For example, FACS can be employed when a fluorescent label is selected; when an isotope label is selected, mass spectrometry, NMR, or other techniques can be used; when a magnetic label is selected, magnetic-based cell sorting can be employed; microscopy can also be used. The output of these detection methods can be in the form of the number of cells, or the output can be in a form that allows the calculation of the number of cells based on the output.

[0205] IV. Methods for determining the presence or absence of an anti-CD70 CAR derived from 4F11

[0206] In some embodiments, knowing the presence or absence in a sample of a molecule comprising an anti-CD70 CAR (such as a CAR comprising an scFv derived from 4F11) or a fragment thereof is sufficient information. For example, it may be advantageous to know that such a molecule is to be expressed regardless of the expression level. 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 (such as a CAR comprising an scFv derived from 4F11) or a fragment thereof can be used in a variety of applications.

[0207] In some embodiments, provided is a method for determining the presence or absence in a sample of a polypeptide comprising an anti-CD70 CAR (such as a CAR comprising an scFv derived from 4F11) or a fragment thereof.

[0208] In some embodiments, the method comprises providing a sample known or suspected to comprise a polypeptide comprising an anti-CD70 CAR (such as a CAR comprising an scFv derived from 4F11) or a fragment thereof.

[0209] The present disclosure provides an antigen-binding molecule that specifically binds to a polypeptide comprising an anti-CD70 CAR (such as a CAR comprising an scFv derived from 4F11) or a fragment thereof, the antigen-binding molecule comprising a detectable label. Appropriate 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, erythrosin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein yellow, cascade blue, Texas red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexafluor 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, and Texas red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Biosciences)). Suitable optical dyes, including fluorophores, are described in Johnson, Figure 1A , Life Technologies, (2010), which is hereby incorporated by reference; radiolabels (e.g., isotope markers such as 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). Photochromic compounds, Halo tags, Atto dyes, Tracy dyes, protein fluorescent labels (e.g., protein fluorescent labels also include but are not limited to green fluorescent protein, including Renilla, sea pen or GFP jellyfish luminescent protein species (Chalfie et al., (1994) Science 263:802 - 805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc.; Stauber, (1998) Biotechnology 24:462 - 471; Heim et al., (1996) Current Biology 6:178 - 182), enhanced yellow fluorescent protein (Clontech Laboratories, Inc.), luciferase (Ichiki et al., (1993) Journal of Immunology 150:5408 - 5417)), magnetic labels (e.g., ) etc. Strategies for labeling proteins are well known in the art and can be used in the methods of the present disclosure. The label can be associated with the antigen - binding molecule at any position in the molecule, but may need to be associated with the molecule at a position (or multiple positions if multiple labels are used) such that the binding properties of the molecule are not altered (unless such altered binding activity is desired). Any antigen - binding molecule that specifically binds to a polypeptide comprising an anti - CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof can be used, such as the antigen - binding molecules disclosed herein, e.g., an antigen - binding molecule having one or more of the VH and VL sequences described in Tables 1a and 1b and / or one or more CDRs described in Tables 1c and 1d.

[0210] Next, the sample is contacted with the antigen - binding molecule under conditions that permit the formation of a binding complex comprising the cells present in the sample and the antigen - binding molecule.

[0211] Then the sample is contacted with the antigen - binding molecule under conditions that permit the formation of a binding complex between a polypeptide comprising an anti - CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof and the antigen - binding molecule. The conditions that permit the formation of the binding complex will depend on a variety of factors. Since the components of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on the surface.

[0212] At this stage, binding complexes may not have formed yet, or multiple binding complexes may have formed that include one or more antigen-binding molecules that bind to a polypeptide that includes an anti-CD70 CAR (e.g., a CAR that includes an scFv derived from 4F11) or a fragment thereof. Unbound molecules that include an anti-CD70 CAR (e.g., a CAR that includes an scFv derived from 4F11) or a fragment thereof and / or unbound antigen-binding molecules may also be present in the local environment of any formed binding complexes.

[0213] Any molecules that are not part of the binding complex are then separated from any formed binding complexes. The method of removal will depend on the structure of the binding complex and / or the local environment. For example, if the antigen-binding molecule is disposed on a bead, plate, or pouch, unbound components of the reaction mixture can be washed away using a solution that leaves the formed binding complex intact. In some embodiments, it is not necessary to separate the binding complex for detection.

[0214] The solution used to induce binding complex formation 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. Generally, when performing this step of the method, buffers with high salt concentrations, chaotropic agents or denaturing agents, or non-physiological pH should be avoided.

[0215] The presence or absence of a binding complex can be detected, which will include a polypeptide that includes an anti-CD70 CAR (e.g., a CAR that includes an scFv derived from 4F11) or a fragment thereof and an antigen-binding molecule. The specific method for detecting the presence or absence of a binding complex will generally depend on the nature of the label selected. In some embodiments, the detection method is by colorimetric assay. The result of the method is a qualitative assessment of the presence or absence of an antigen-binding molecule that includes a detectable label and thus the presence or absence of its binding partner, i.e., a polypeptide that includes an anti-CD70 CAR (e.g., a CAR that includes an scFv derived from 4F11) or a fragment thereof.

[0216] As in the case of the disclosed methods, a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof can be placed in any context. In some embodiments, a polypeptide comprising an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof is expressed on the surface of a cell. In this embodiment, the cell can be of any type 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 cells of the 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 cytotoxic T cells, T helper cells, and Treg cells) are particularly suitable. In a specific embodiment, the cell is a T cell, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be employed in this embodiment of the disclosed method, and the cell can be a human or non-human cell. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TIL), NK cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. In additional embodiments, the cell is a TCR-presenting T cell. The T cell can be a CD4+ T cell or a CD8+ T cell. When T cells are employed in the disclosed method, the T cells can be in vivo T cells or in vitro T cells. Additionally, the cell can be derived from stem cells, such as iPSC cells, umbilical cord blood cells, or mesenchymal stem cells.

[0217] In some embodiments, the cell can be placed in or separated from any context capable of maintaining the cell in a viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, a suitable buffer, etc. In some embodiments, the cell is in a formalin-fixed sample. In some embodiments, the sample is formalin-fixed paraffin-embedded tissue (FFPE).

[0218] V. Methods and Uses of Antibodies

[0219] In some embodiments, methods involving the use of one or more anti-idiotypic 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 are provided herein, as well as methods for altering the activity of a target antibody (such as the activity of a CAR) or the activity of a cell expressing a CAR. In certain embodiments, the one or more anti-idiotypic antibodies bind, detect, identify, and / or quantify a CAR and / or a cell expressing a CAR. In some embodiments, the methods provided herein include one or more steps of contacting and / or incubating one or more anti-idiotypic antibodies with a cell or a sample containing or suspected of containing a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the anti-idiotypic antibodies are treated, incubated, and / or contacted with a composition or sample under conditions that permit the formation of a complex between the anti-idiotypic antibody and the target antibody (e.g., a CAR). In some aspects, the complex can be used for the purpose of detecting, isolating, and / or measuring the CAR. In some embodiments, the formation of the complex alters the activity of the target antibody (e.g., a CAR), such as by stimulating receptor signaling activity, or in some embodiments, antagonizes the activity of the target antibody (e.g., a CAR) by preventing the association of the CAR with an antigen.

[0220] A. Detection / Isolation Methods

[0221] In some embodiments, methods are provided that involve the use of one or more of the anti-idiotypic antibodies and / or molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies for detecting, binding, and / or isolating an antibody (e.g., a target antibody). In certain embodiments, the methods include one or more steps of contacting, incubating, and / or exposing one or more anti-idiotypic antibodies with a sample and / or a composition. In some embodiments, the sample and / or composition has, may have, and / or is suspected of having a target antibody and / or an antigen-binding fragment thereof that is bound and / or recognized by one or more anti-idiotypic antibodies. In certain embodiments, the antibody or antigen-binding fragment thereof that is bound or recognized by one or more anti-idiotypic antibodies contains one or more fusion domains and / or is a fusion protein. In certain embodiments, the target antibody and / or an antigen-binding fragment thereof 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 containing a CAR that includes such an anti-CD70 antibody (e.g., an antibody fragment).

[0222] In some embodiments, the method includes 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 carried out under conditions that permit the anti-idiotype antibody to bind to the target antibody present in the composition, for example to form a complex containing the anti-idiotype antibody and the target antibody.

[0223] In some embodiments, the sample and / or composition contains or is suspected of containing a target antibody, such as a CAR. In certain embodiments, the sample and / or composition contains or is suspected of containing cells that express the target antibody (e.g., a CAR). In certain embodiments, the sample is a biological sample. In a particular embodiment, 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, nerve tissue, or epithelial tissue. In a particular embodiment, the biological sample is taken from, collected from, 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 a homogenate and / or cells that have been disrupted and / or lysed. In some embodiments, the biological sample contains proteins and / or antibodies that have been isolated from blood, serum, and / or tissue.

[0224] In a particular embodiment, the anti-idiotype antibody forms or is capable of forming a complex with the target antibody (e.g., a CAR). In a particular embodiment, the complex is detected, measured, quantified, and / or evaluated, for example to permit the detection, identification, measurement, and / or quantification of the target antibody, such as in a composition or sample. In certain embodiments, the method includes detecting whether a complex forms between the anti-idiotype antibody and the target antibody in the sample, and / or detecting the presence or absence or level of such binding. In some embodiments, the complex contains a detectable label. In a particular embodiment, the anti-idiotype antibody is an immunoconjugate containing a detectable label. In certain embodiments, the anti-idiotype antibody contains a detectable label, is conjugated to, bound to, and / or attached to the detectable label. In some embodiments, the complex contains an antibody that binds to and / or recognizes the anti-idiotype antibody, such as a secondary antibody, which is conjugated to, bound to, and / or attached to the detectable label.

[0225] In some embodiments, a method for detecting, quantifying, detecting, and / or evaluating a target antibody, such as in a sample or composition, includes detecting a complex of the target antibody and the anti-idiotype antibody. In some embodiments, the complex contains a detectable label. In certain embodiments, the complex is probed with and / or contacted with the 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.

[0226] In some embodiments, the target antibody or antigen-binding fragment binds to a cell or is expressed on the surface of a cell. In certain embodiments, the target antibody (e.g., a CAR) does not bind to a cell or is intracellular, e.g., in some embodiments, the target antibody is secreted. In some embodiments, the antibody has been separated, removed, and / or lysed from the surface of the cell.

[0227] In some embodiments, the target antibody is an anti-CD70 antibody. In some embodiments, the target antibody is or derived from antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy-chain variable region shown in SEQ ID NO:15 and / or the light-chain variable region shown in SEQ ID NO:16.

[0228] In some embodiments, a method for detecting a target antibody (such as antibody 4F11) or its antigen-binding fragment (and / or a chimeric molecule comprising such an antibody (e.g., an antibody fragment), such as a CAR) is provided, the method comprising contacting a composition comprising the target antibody or antigen-binding fragment with an anti-idiotypic antibody or its antigen-binding fragment or an anti-idiotypic antibody immunoconjugate described herein, and detecting the anti-idiotypic antibody that binds to the target antibody or antigen-binding fragment. In some embodiments, the method further comprises detecting whether a complex between the anti-idiotypic antibody and the target antibody is formed in the composition, such as detecting the presence, absence, or level of such binding. In some embodiments, the target antibody or antigen-binding fragment binds to a cell or is expressed on the surface of a cell, and the detection comprises detecting a cell that binds to the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment is directly or indirectly labeled for detection. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy-chain variable region shown in SEQ ID NO:15 and / or the light-chain variable region shown in SEQ ID NO:16.

[0229] In certain embodiments, methods for detecting a target antibody with an anti-idiotypic antibody described herein are used to evaluate the target antibody in a subject. For example, in some embodiments, methods are provided herein for using an anti-idiotypic antibody to evaluate, measure, and / or quantify the in vivo pharmacokinetics, expansion, and / or persistence of CAR-expressing cells of a therapeutic cell composition. In some embodiments, in the methods provided herein, changes in the in vivo pharmacokinetics, expansion, and / or persistence of cells, such as CAR-expressing cells administered for immunotherapy (e.g., CAR-T cell therapy), and / or the cell phenotype or functional activity of the cells can be measured with the anti-idiotypic antibodies provided herein. In some embodiments, the pharmacokinetics, expansion, and / or persistence of CAR-expressing cells are measured and evaluated by detecting the presence and / or amount of CAR-expressing cells in a sample obtained from and / or in a subject after administering a therapeutic cell composition during and / or after administration of the anti-idiotypic antibody provided herein.

[0230] In some aspects, the anti-idiotypic antibody is used in conjunction with flow cytometry to evaluate the amount of cells expressing a recombinant receptor (e.g., CAR-expressing cells administered for T cell-based therapy) in a blood or serum or organ or tissue sample of a subject (e.g., a disease site, such as a tumor sample). In some aspects, persistence is quantified as the number of CAR-expressing cells per microliter of sample (e.g., a sample of blood or serum), or the total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of sample. In certain aspects, expansion is quantified as the increase over time in the number of CAR-expressing cells per microliter of sample (e.g., a sample of blood or serum), or the total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of sample. In some embodiments, the pharmacokinetics, expansion, and / or persistence are measured or evaluated by detecting the amount of CAR-expressing cells in samples obtained from and / or collected from a subject at multiple time points.

[0231] In some embodiments, provided is a method of selecting cells that express a chimeric antigen receptor (CAR) comprising a target antibody (such as antibody 4F11) or an antigen-binding fragment thereof, the method comprising contacting a cell population comprising cells expressing the CAR with an anti-idiotypic antibody or an antigen-binding fragment thereof described herein, and selecting cells that bind to the anti-idiotypic antibody. In some embodiments, the cells that bind to the anti-idiotypic 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-idiotypic antibody or an antigen-binding fragment thereof or an anti-idiotypic antibody immunoconjugate is reversibly bound to a support or solid phase or immobilized on the support or solid phase. In some embodiments, the target antibody or an antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:15 and / or the light chain variable region amino acid sequence shown in SEQ ID NO:16 (e.g., comprises the amino acid sequence of SEQ ID NO:35).

[0232] In some embodiments, provided is a method of validating a chimeric antigen receptor (CAR) comprising a target antibody (such as antibody 4F11) or an antigen-binding fragment thereof, the method comprising: a) incubating a sample comprising T cells transduced with the CAR with an anti-idiotypic antibody or an antigen-binding fragment thereof that targets the CAR; b) determining the percentage of cells that bind to the anti-idiotypic antibody or an antigen-binding fragment thereof; and c) validating the CAR based on the percentage of T cells that bind to the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody is labeled and the T cells that bind to the anti-idiotypic antibody are assayed by flow cytometry. In some embodiments, the target antibody or an antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:15 and / or the light chain variable region amino acid sequence shown in SEQ ID NO:16 (e.g., comprises the amino acid sequence of SEQ ID NO:35).

[0233] 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 to provide therapeutic decision - making information in an individual, such as by detecting a CAR recognized by the anti - idiotypic antibody, such as a CAR comprising a target antibody (such as an anti - CD70 antibody (e.g., antibody 4F11)) or an antigen - binding fragment thereof. In some embodiments, the methods are used to provide therapeutic decision - making information in an individual, where the therapeutic decisions are related to a therapy involving the administration of CAR T cells (such as anti - CD70 CAR T cells). In some embodiments, the methods include incubating and / or probing a biological sample with the anti - idiotypic antibody and / or administering the anti - idiotypic antibody to an individual. In certain embodiments, the biological sample comprises cells or tissue or a portion thereof, such as a tumor or cancer tissue or a biopsy or section thereof. In certain embodiments, the incubation is carried out under conditions that permit the anti - idiotypic antibody to bind to the CAR present in the sample. In some embodiments, the method further comprises detecting whether a complex between the anti - idiotypic antibody and the CAR is formed in the sample, such as detecting the presence or absence or level of such binding. Such methods can be in vitro or in vivo methods.

[0234] B. Use in cell stimulation

[0235] In some embodiments, the provided anti - idiotypic antibody or its antigen - binding fragment is an agonist and / or exhibits specific activity in stimulating cells expressing the target antibody, including conjugates or chimeric receptors containing the target antibody, such as an anti - CD70 antibody (e.g., antibody 4F11) or an antigen - binding fragment thereof. In some embodiments, methods are provided 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 to stimulate or activate cells (such as T cells) expressing a CAR or other chimeric receptor. In some aspects, the CAR or other receptor comprises a target antibody, such as an anti - CD70 antibody (e.g., antibody 4F11) or an antigen - binding fragment thereof.

[0236] In some embodiments, the methods can be used in combination with methods for preparing genetically engineered T cells, such as in methods for expanding genetically engineered T cells or other cells in which a nucleic acid molecule encoding a chimeric receptor (such as a CAR) comprising a target antibody has been introduced, for example, by transfection, transduction, or non - viral methods of nucleic acid transfer (such as transposon - based methods). In some aspects, the target antibody is an anti - CD70 antibody (e.g., antibody 4F11) or an antigen - binding fragment thereof. In certain embodiments, the target antibody is or contains a CAR, such as an anti - CD70 CAR. In certain embodiments, the anti - CD70 CAR contains a scFv derived from and / or originating from an anti - CD70 antibody (such as antibody 4F11).

[0237] In some embodiments, the method comprises incubating a sample comprising T cells transduced with a CAR with an anti-idiotype antibody. In certain embodiments, the method further comprises detecting whether the CAR T cells are activated or stimulated, such as by assessing the viability, proliferation, and / or expression of activation markers in the CAR T cells. In some embodiments, the target antibody is an anti-CD70 antibody. In some embodiments, the target antibody is or derived from antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:15 and / or the amino acid sequence of the light chain variable region shown in SEQ ID NO:16 (e.g., comprising the amino acid sequence of SEQ ID NO:35).

[0238] In some embodiments, a method of mimicking cells is provided, the method comprising incubating an input composition comprising cells expressing a CAR (comprising a target antibody, such as antibody 4F11 or an antigen-binding fragment thereof) with an anti-idiotype antibody or an antigen-binding fragment thereof described herein, thereby generating an output composition comprising stimulated cells. In some embodiments, the incubation is carried out under conditions in which the anti-idiotype antibody or its antigen-binding fragment binds to the CAR, thereby inducing or modulating a signal in one or more cells of the input composition. In some embodiments, the cells comprise T cells. In some embodiments, the T cells comprise CD4+ and / or CD8+ T cells.

[0239] In certain embodiments, the anti-idiotype antibody is administered to a subject, such as a subject who has previously received a therapeutic cell composition comprising cells expressing a CAR. In some embodiments, administering the anti-idiotype antibody to the subject promotes re-amplification of the CAR-expressing cells in the subject, and in some cases, the cells may reach or exceed the initial peak amplification level prior to administration of the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is administered to modulate the amplification and / or persistence of the CAR-expressing cells when the level of the CAR-expressing cells has decreased or is undetectable. In some embodiments, for example, the CAR-expressing cells re-amplified by the anti-idiotype antibody exhibit increased potency in the subject to whom it is administered compared to the potency prior to administration of the anti-idiotype antibody.

[0240] In some embodiments, a method of generating a cell composition is provided, the method 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-idiotypic antibody or an antigen-binding fragment thereof that is specific for the antigen-binding domain of the CAR, thereby generating a cell composition. In some embodiments, the CAR comprises a target antibody or an antigen-binding fragment thereof that specifically binds to CD70. In some embodiments, the target antibody is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the anti-idiotypic antibody or an antigen-binding fragment thereof is the anti-idiotypic antibody or an antigen-binding fragment thereof described herein. In some embodiments, the anti-idiotypic antibody or an 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, transposition, electroporation, or chemical transfection. In some embodiments, the introducing comprises introducing the nucleic acid molecule into the cell by transduction with a retroviral vector comprising the nucleic acid molecule, by transduction with a lentiviral vector comprising the nucleic acid molecule, by transposition with a transposon comprising the nucleic acid molecule, or by electroporation or transfection with a vector comprising the nucleic acid molecule.

[0241] C. Use in cell inactivation / depletion

[0242] In some embodiments, the provided anti-idiotypic antibody or an antigen-binding fragment thereof is an antagonist and / or exhibits specific activity for inhibiting, ablating, and / or depleting (e.g., killing via antibody-dependent cell-mediated cytotoxicity (ADCC)) cells expressing the target 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 target antibody, such as an anti-CD70 antibody (e.g., antibody clone 4F11) or an antigen-binding fragment thereof.

[0243] In some embodiments, the method includes treating, contacting, and / or incubating a composition and / or sample comprising T cells transduced with a CAR with an anti-idiotype antibody. In certain embodiments, the method further includes detecting whether the CAR T cells are inactivated, such as by assessing the viability, proliferation, and / or expression of activation markers in the CAR T cells. In some embodiments, the method is associated with a therapy comprising administering CAR T cells. In some embodiments, the method includes administering an anti-idiotype antibody to an individual. In one embodiment, the anti-idiotype antibody or conjugate is used to ablate and / or deplete (such as kill) CAR T cells in an individual. In some embodiments, the target antibody is an anti-CD70 antibody. In some embodiments, the target antibody is or derived from antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:15 and / or the amino acid sequence of the light chain variable region shown in SEQ ID NO:16 (e.g., comprising the amino acid sequence of SEQ ID NO:35).

[0244] In some embodiments, an anti-idiotype antibody is administered to deplete, reduce, and / or decrease the number of CAR-expressing cells in a subject. In certain embodiments, administering 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 is related to the amount of CAR-expressing cells in the subject prior to administration of the anti-idiotype antibody. In certain embodiments, the depletion, reduction, and / or decrease is related to the amount of CAR-expressing cells in a subject that has not received the anti-idiotype antibody. In some embodiments, no CAR-expressing cells are detectable in the subject after administration of the anti-idiotype antibody. In certain embodiments, the anti-idiotype antibody is a human or humanized antibody.

[0245] In some embodiments, a method of inactivating CAR T cells is provided, wherein the CAR comprises a target antibody, such as antibody 4F11 or an antigen-binding fragment thereof, and the method comprises incubating a sample comprising CAR T cells with an anti-idiotypic antibody or an 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 the activation of CAR T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to substantially inactivate the CAR T cells in the sample. In some embodiments, incubating with the anti-idiotypic antibody results in ablation and / or depletion of the CAR T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to eliminate the CAR T cells in the sample. In some embodiments, the target antibody or an antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:15 and / or the light chain variable region amino acid sequence shown in SEQ ID NO:16 (e.g., comprising the amino acid sequence of SEQ ID NO:35).

[0246] In some embodiments, a method of modulating CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as antibody 4F11 or an antigen-binding fragment thereof, and the method comprises administering to the individual an anti-idiotypic antibody immunoconjugate that targets the CAR, wherein the anti-idiotypic antibody immunoconjugate comprises a cytotoxic agent. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to attenuate the CAR T cell therapy in the individual. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to substantially halt the CAR T cell therapy in the individual. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to effect elimination of the CAR T cells in the individual. In some embodiments, the cytotoxic agent is selected from the group consisting of: chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin), and radioisotopes. In some embodiments, the target antibody or an antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:15 and / or the light chain variable region amino acid sequence shown in SEQ ID NO:16.

[0247] D. Use or method in a binding assay

[0248] The present disclosure provides methods for assessing the presence or absence in a sample of a molecule that binds to an extracellular domain of a chimeric antigen receptor (CAR), such as a CAR, or a portion thereof that contains an antigen-binding domain. In some embodiments, the methods can be used to assess the presence or absence in a subject of a humoral or antibody response to a administered cell therapy comprising a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor comprises a target antibody that is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises a target antibody that is antibody 4F11 or an antigen-binding fragment thereof. In some embodiments, an anti-idiotypic antibody or an antigen-binding fragment thereof that is specific for the extracellular domain of the CAR, such as any antibody or antigen-binding fragment thereof described herein, can be used as a positive control in the methods.

[0249] Any anti-idiotypic antibody that specifically binds to a polypeptide comprising an anti-CD70 antibody 4F11 or an anti-CD70 CAR (e.g., a CAR comprising an scFv derived from 4F11) or a fragment thereof, such as the anti-idiotypic antibodies disclosed herein, can be used. For example, an anti-idiotypic antibody having one or more of the VH and VL sequences described in Tables 1a and 1b and / or one or more of the CDRs described in Tables 1c and 1d. In some embodiments, the binding of the anti-CD70 antibody to the target antigen CD70 does not interfere with the binding of the anti-idiotypic antibody to the anti-CD70 antibody, i.e., it is non-blocking. In some embodiments, the binding of the anti-idiotypic antibody to the anti-CD70 antibody does not interfere with the 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, in the presence of the target antigen CD70, the binding of the non-blocking anti-idiotypic antibody can more accurately detect the presence of or quantify the amount or quantity of the anti-CD70 antibody, either alone or as part of a CAR.

[0250] In some embodiments, the binding of the anti-idiotypic antibody interferes with the binding of the anti-CD70 antibody to the target antigen CD70, or the binding of the anti-CD70 antibody to the target antigen CD70 interferes with the binding of the anti-idiotypic antibody to the anti-CD70 antibody, i.e., it is blocking. In some embodiments, the anti-idiotypic antibody is a blocking anti-idiotypic antibody. In some embodiments, the binding of the blocking anti-idiotypic antibody can be used to deduce or determine the level of engagement or occupancy of the target antibody (e.g., an anti-CD70 antibody), either alone or as part of a CD70-specific CAR, with the target antigen (e.g., CD70).

[0251] In some embodiments, methods are provided that involve contacting or incubating a binding reagent with a sample from a subject who has received a cell therapy comprising cells engineered with a chimeric antigen receptor, wherein the binding reagent is a protein comprising an extracellular domain or a portion thereof of a CAR that includes a target antibody or an antigen-binding fragment thereof. In some embodiments, the method further comprises detecting whether a complex is formed between the binding reagent and a molecule present in the sample (e.g., a binding molecule such as an antibody), and / or detecting the presence, absence, or level of such binding. In certain embodiments, the contacting or incubating is carried out under conditions that permit binding of the binding reagent to a molecule present in the sample from the subject. In certain aspects, the method can further be carried out on a positive control sample that contains an anti-idiotypic antibody or an antigen-binding fragment thereof that is specific for the CAR, such as any of the antibodies or antigen-binding fragments described. In some embodiments, determining the presence, absence, or level of binding of the molecule to the binding reagent can include comparing the binding or detection to the binding or detection of the positive control sample with the binding reagent.

[0252] In some embodiments, the method comprises detecting whether a complex is formed between the binding reagent and a molecule present in the sample (e.g., a binding molecule such as an antibody), and / or detecting the presence, absence, or level of such binding. In certain embodiments, the contacting or incubating is carried out under conditions that permit binding of the binding reagent to a molecule present in the sample from the subject. In some aspects, the complex is detected by an immunoassay, optionally a sandwich assay or a bridging assay. For example, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, a surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), flow cytometry, or a western blot. In some embodiments, the immunoassay is or includes Meso Scale Discovery.

[0253] In some aspects, the immunoassay is a sandwich assay or a bridging assay. In a sandwich or bridging assay, the binding reagent is a first binding reagent, and detecting the presence or absence of a molecule or a complex comprising the molecule includes: 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 the same or a similar molecule as the first binding reagent. In some embodiments, the second binding reagent comprises an extracellular domain or a portion thereof of a CAR. In some aspects, the extracellular domain or a portion thereof of the CAR of the first binding reagent and the second binding reagent are the same or substantially the same.

[0254] Examples

[0255] Example 1: Generation of an anti-idiotypic antibody against the anti-human CD70 scFv clone 4F11

[0256] 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”) which, in addition to the 4F11 scFv, also contains a CD8a hinge / transmembrane domain, a CD3z activation domain, and a 4-1BB co-stimulatory domain.

[0257] The 4F11 scFv amino acid sequence is shown in SEQ ID NO:35. Hybridomas were generated, cloned, and the secreted antibodies were screened based on their binding specificity to 4F11. Antibody clones were identified and selected based on their ability to specifically bind to the scFv derived from 4F11 by ELISA testing mouse sera against the immunogen.

[0258] Mice with positive sera were selected for hybridoma fusion. The supernatants of hybridoma cultures were screened by ELISA with 4F11 scFv 10xHis antigen (“10xHis” is disclosed as SEQ ID NO:40) to identify positive hybridoma clones. Then the supernatants of these clones were screened by flow cytometry to confirm the binding specificity to the 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.

[0259] Antigen blocking assays were performed to determine whether anti-idiotypic antibodies could bind to their target antibodies in the presence of the target antigen. Blocking of positive clones that bind 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.

[0260] The full-length or variable region sequences of the lead and backup anti-idiotypic clones were determined.

[0261] Recombinant antibodies were produced, and the binding of the recombinantly produced antibodies to the antigen was indistinguishable from that of the antibodies purified from the hybridomas.

[0262] As described herein, the anti-idiotypic antibody clones IDA2002-1.60 (clone 60), IDA2002-1.37 (clone 37) were further analyzed and characterized.

[0263] Example 2: Specificity determination by flow cytometry

[0264] Next, confirm the combination and determine the optimal titers of two phycoerythrin (PE)-conjugated anti-idiotypic antibodies (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")) with 4F11scFv. Titrate clones 60 and 37 and evaluate binding to the target CD70 CAR using flow cytometry. CD70-specific (4F11-QR3) CAR T cells and untransduced T cells (NTD) were generated from PBMC starting material. The cells were thawed, allowed to rest for 24 hours, counted, and 1x10 5 (1×10^5) cells were stained for each pool or fraction starting with 30 ug (micrograms) / mL anti-idiotypic antibody for a 5-point semi-logarithmic titration curve. The cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer (PBS + 2% FBS + 2 mM EDTA), and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. Figure 1B , the first and second rows show the results for clone 1.37. Figure 2A , the third and fourth rows show the results for clone 1.60.

[0265] To determine the specificity of the PE-conjugated anti-idiotypic clone (clone 60-PE) for 4F11 scFv, several control CAR T cells, including anti-CD19 CAR T cells, anti-BCMACAR T cells, and anti-FLT3 CAR T cells, CD70 (4F11-QR3) CAR T cells (donor A) were included as positive controls, and NTD cells (donor A) were stained as a negative control. The cells were thawed, allowed to rest for 48 hours, counted, and 1x10 5 cells were stained with 3 ug / mL of clone 60-PE antibody. The cells were also stained with 10 ug / mL of rituximab (RTX) antibody as a control since all CAR T cells contain a rituximab-based switch. The cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer, and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. Figure 2B The staining results for anti-CD19 CAR, anti-BCMACAR, anti-FLT3CAR, anti-CD70 CAR, and NTD are shown.

[0266] Example 3: Flow Cytometry Comparison of Recombinant Proteins

[0267] Purified PE-conjugated anti-idiotype clone 60 from Biolegend was compared to newly purified antibodies generated by recombinant or purified from 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 ug / mL of the PE-conjugated or unconjugated recombinant or hybridoma form of clone 60. Cells were stained for 20 minutes at 4°C, washed with flow cytometry buffer, and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. The structure is shown in Figure 2A (SSC-A: side scatter-A).

[0268] Individually, cells were stained with 0.3 ug / mL of the unconjugated recombinant or hybridoma form of clone 60. Cells were stained with the primary antibody for 20 minutes at 4°C, washed with flow cytometry buffer, stained with the secondary anti-mouse Fc-specific antibody (Jackson, catalog number 115-116-071) at a 1:100 dilution for 15 minutes at 4°C, washed with flow cytometry buffer, and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. The structure is shown in Figure 2B in. Figure 3 The results of staining with the PE-conjugated antibody are shown. Figure 3 The results of staining with the unconjugated antibody are shown. The results show that PE conjugation does not affect antigen binding.

[0269] Example 4: Flow cytometry demonstration of the effect on binding in the presence of the target antigen CD70

[0270] To determine whether the anti-idiotype clone can bind to 4F11scFv in the presence of the target antigen CD70, CD70-specific CAR Jurkat cells were incubated and blocked with recombinant human CD70 (rCD70 or hCD70) prior to anti-idiotype staining 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 1x10 5Cells were incubated with hCD70 at 100 μg / mL (Abcam catalog number ab119815) for 30 minutes. The cells were then stained with various polyclonal supernatants from hybridoma cultures diluted 1:10 for 20 minutes and washed with flow cytometry buffer. The cells were stained with a secondary anti-mouse Fc-specific antibody (Jackson ImmunoResearch, catalog number 115-116-071) diluted 1:100 for 15 minutes at 4°C, washed with flow cytometry buffer, and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. Cells were also stained with a PE-conjugated rituximab (RTX) antibody at 10 μg / mL as a positive control (to show that the cells express 4F11-QR3CAR) and with the secondary antibody only as a negative control. The constructs are shown in Figure 4A , top row (NTD control not shown).

[0271] Cells not treated with hCD70 were stained as a positive control (see Figure 4B , bottom row). The results showed that when the 4F11 scFv formed a complex with its target antigen CD70, binding of the anti-idiotypic antibody of clone 37 to the 4F11 scFv was blocked, while binding of the anti-idiotypic antibody of clone 60 to the 4F11 scFv was not blocked by 4F11 scFv-CD70 binding.

[0272] For confirmation, the experiment was repeated using purified PE-conjugated clone (clone 60) which was determined to be a non-blocker of CAR-CD70 interaction. Cells (4F11-QR3 CAR and untransduced ("NTD") cells) were thawed, allowed to sit for 48 hours, counted, and 1 x 10 5 cells were incubated with hCD70 at 100 μg / mL for 30 minutes. The cells were then stained with clone 60-PE antibody at 1.5 μg / mL ( Figure 5 ) or with rituximab (RTX) antibody at 5 μg / mL ( Figure 6A ) at 4°C for 20 minutes, washed with flow cytometry buffer, and resuspended in buffer prior to analysis on a Cytoflex flow cytometer. Cells not treated with hCD70 were stained as a positive control. The results confirmed that binding of the anti-idiotypic antibody of clone 60 to the 4F11 scFv was not blocked by binding of the scFv to CD70. Thus, clone 60 is a non-blocking anti-idiotypic antibody.

[0273] Example 5: Surface Plasmon Resonance ("SPR") Determination of the Affinity of Clone 60

[0274] All SPR analyses were determined on a BIACore T200 SPR instrument (Cytiva, Marlborough, MA). For kinetic analysis and determination of simultaneous target antigen binding of the CD70 anti-idiotypic antibody, mouse Fc was captured on a Biacore S Series CM4 sensor chip by amine coupling of an anti-mouse Fc reagent (Mouse Antibody Capture Kit, Cytiva, BR100838) to the Biacore S Series CM4 sensor chip using an electrophoresis buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20 at pH 7.4 at 25 °C. All surfaces of the sensor chip were activated for 7 minutes at 10 μL / min with a 1:1 (v / v) mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS). The anti-mouse Fc reagent was diluted to 30 μg / mL in 10 mM sodium acetate (pH 5.0) and injected at 10 μL / min onto all four flow cells for 7 minutes, followed by blocking of all flow cells for 7 minutes with 100 mM ethylenediamine in 200 mM borate buffer at pH 8.5 at 10 μL / mL.

[0275] All interaction experiments were performed at 37 °C using the same electrophoresis buffer supplemented with 1 mg / mL BSA as described above. The binding affinity of the CD70 anti-idiotypic antibody was determined by first capturing 5 μg / mL of the CD70 anti-idiotypic antibody on the anti-mouse Fc chip surface and then buffering and injecting the 4F11 scFv 10xHis (where "10xHis" is 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 6B and the data are reproduced in Table 3 below.

[0276] Table 3.

[0277]

[0278] Example 6: SPR Demonstration of Simultaneous Binding of Target Antigen CD70 and Clone 60

[0279] To use the premix and subsequently determine the simultaneous binding of the CD70 anti-idiotypic antibody to the CD70 target antigen using a secondary detection method, 0.5 μg / ml of the CD70 anti-idiotypic antibody was first captured on the surface of an anti-mouse Fc chip. A premix of 100 nM 4F11 scFv 10xHis (“10xHis”, disclosed as SEQ ID NO:40), 100 nM biotin-hCD70 complex, and 100 nM streptavidin (ThermoFisher, catalog number 21125) was sequentially injected at 30 μl / min for 2 minutes using dual injections. Streptavidin is specific for the premixed biotin-hCD70 target antigen and is used as a secondary detection reagent to detect whether a complex is formed between the premix and the captured CD70 anti-idiotypic antibody. 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 6A The assay (left inset) and results (top right and bottom right insets for hybridoma-purified and recombinantly produced anti-idiotypic antibodies, respectively) are shown.

[0280] To use the classical sandwich method to determine the simultaneous binding of the CD70 anti-idiotypic antibody to the CD70 target antigen, 0.5 μg / ml of the CD70 anti-idiotypic antibody was captured on the surface of an anti-mouse Fc chip. A 100 nM 4F11 scFv 10xHis (“10xHis”, disclosed as SEQ ID NO:40) and 100 nM biotin-hCD70 buffer were sequentially injected 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. ​ The assay (left inset) and results (top right and bottom right insets for hybridoma-purified and recombinantly produced anti-idiotypic antibodies, respectively) are shown. The results show that in the premix assay or the sandwich assay, in the presence of the target antigen hCD70, the anti-idiotypic antibody clone 60 was able to bind to the anti-CD70 antibody.

[0281] The data were fit to a 1:1 Langmuir binding model with mass transport evaluation using Biacore T200 evaluation software (version 2.0). ​ The data in -B show that by the premix assay or the classical sandwich assay, in the presence of the target antigen hCD70 protein, the anti-idiotypic antibody clone 60 can bind to the 4F11 scFv.

[0282] Incorporated by reference

[0283] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of references herein shall not be construed as an admission that such references are prior art to the present disclosure. To the extent that any definition or term provided in the references incorporated by reference differs from the terms and discussions provided herein, the terms and definitions herein shall control.

[0284] The foregoing written specification is considered to be sufficient to enable one of ordinary skill in the art to practice the present disclosure. The foregoing description and examples detail certain embodiments of the present disclosure and describe the best mode contemplated by the inventors. However, it should be understood that, regardless of the level of detail in the foregoing text, the present disclosure may be practiced in many ways and the present disclosure 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 an 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 according to claim 1, wherein the isolated antibody comprises (a) a heavy chain variable (VH) region and a light chain variable (VL) region, 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; or (b) a VH region and a VL region, 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.

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

30.

4. The isolated antibody according to claim 3, wherein the isolated antibody further comprises a VL region that comprises (a) light chain CDR1, light chain CDR2, and light chain CDR3, wherein light chain CDR1 comprises the amino acid sequence of SEQ ID NO:12, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:13, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:14; or (b) light chain CDR1, light chain CDR2, and light chain CDR3, wherein light chain CDR1 comprises the amino acid sequence of SEQ ID NO:31, light chain CDR2 comprises the amino acid sequence of SEQ ID NO:32, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO:

33.

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

23.

8. The isolated antibody according to 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. The isolated antibody according to claim 1, wherein the isolated antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, the light chain variable region comprises VL CDR1, VL CDR2, and 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, and the VH CDR3 comprises the amino acid sequence SEQ ID NO: 11, and wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

14.

10. The isolated antibody according to claim 1, wherein the isolated antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3, the light chain variable region comprises VL CDR1, VL CDR2, and 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, and the VH CDR3 comprises the amino acid sequence SEQ ID NO: 30, and wherein 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 according to any one of the preceding claims, wherein the isolated antibody further comprises a detectable label.

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

13. The isolated antibody according to claim 12, wherein the fluorescent label is 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 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 rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, KusabiraOrange, 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 protein (PerCP), mKate (TagFP635), TurboFP635, mPlum and mRaspberry.

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

15. A vector comprising the polynucleotide according to claim 14.

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

17. A method for preparing an isolated antibody that specifically binds to a molecule, the molecule comprising: (a) an anti-CD70 scFv, the anti-CD70 scFv comprising VH and VL, the VH comprising the amino acid sequence of SEQ ID NO:15, and the VL comprising the amino acid sequence of SEQ ID NO:16, (b) an anti-CD70 scFv, the anti-CD70 scFv comprising the amino acid sequence of SEQ ID NO:35, or (c) an anti-CD70 chimeric antigen receptor ("CAR"), the anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:34, the method comprising growing or culturing the cell according to claim 16 under suitable conditions.

18. A method for determining the number of cells expressing an anti-CD70 antibody, the anti-CD70 antibody comprising the amino acid sequences of SEQ ID NO:15 and 16, the method comprising contacting the cell with the isolated antibody according to any one of claims 1 to 13, and determining the number of cells expressing the anti-CD70 antibody in the cell.

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

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

35.

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

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

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

24. The method according to claim 23, wherein the cells that bind to the isolated antibody are selected by affinity-based separation.

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

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

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