Anti-CD19 Antibodies Anti-idiotypical Antibodies

Description

Field

[0001] The present disclosure relates in some aspects to anti-idiotype antibodies that specifically recognize anti-CD19 antibody moieties, in particular, anti-CD19 antibody moieties present in recombinant receptors, including chimeric antigen receptors (CARs). The disclosure further relates to uses of anti-idiotype antibodies for specifically identifying or selecting cells expressing such recombinant receptors, such as anti-CD19 CAR T cells. The disclosure further relates to uses of anti-idiotype antibodies for specifically activating such cells.Background

[0002] Methods are available for adoptive cell therapy using engineered cells expressing recombinant receptors, such as chimeric antigen receptor (CARs) containing extracellular antibody antigen-binding domains. Various strategies are available to assess activity of such cells either in vitro or upon in vivo to a subject. Improved methods are needed to specifically assess activity of CAR-expressing cells. Provided are reagents, compositions, and articles of manufacture that meet such needs.

[0003] Bipulendu Jena et al., describes chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

[0004] WO2014 / 190273A1 describes chimeric antigen receptor-targeting monoclonal antibodies.Summary

[0005] The present invention is set out in the appended set of claims.

[0006] The present invention provides an anti-idiotype antibody or antigen-binding fragment thereof comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 89 or 90, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, 92 or 93, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 94; and a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 95, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 96, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 97, wherein: (i) the anti-idiotype antibody or antigen-binding fragment thereof specifically binds to a target antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31; and (ii) the anti-idiotype antibody or antigen-binding fragment thereof is an agonist antibody of a chimeric antigen receptor (CAR) comprising the target antibody or antigen-binding fragment.

[0007] The present invention also provides a conjugate, comprising the anti-idiotype antibody or antigen-binding fragment of the present invention and a heterologous molecule or moiety, optionally wherein the heterologous molecule or moiety is: i) a label, optionally wherein the label is selected from a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a streptavidin, a biotin, a luminescent compound or an oligonucleotide; or ii) a protein, peptide, nucleic acid or small molecule, which optionally is or comprises a toxin or a Strep-Tag.

[0008] The present invention also provides a nucleic acid molecule(s) encoding the heavy chain and light chain of the anti-idiotype antibody or antigen-binding fragment thereof of the present invention.

[0009] The present invention also provides a vector, comprising the nucleic acid molecule(s) of the present invention.

[0010] The present invention also provides a cell, comprising the anti-idiotype antibody or antigen-binding fragment thereof of the present invention or the nucleic acid molecule of the present invention.

[0011] The present invention also provides a method of producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising expressing the heavy and light chain encoded by the nucleic acid molecule of the present invention or the vector of the present invention in a suitable host cell and recovering or isolating the antibody.

[0012] The present invention also provides a composition comprising the anti-idiotype antibody or antigen-binding fragment thereof of the present invention, conjugate of the present invention, or the cell of the present invention, optionally further comprising a pharmaceutically acceptable excipient.

[0013] The present invention also provides a method of detecting a target antibody or antigen-binding fragment thereof, comprising: (a) contacting a composition comprising a target antibody or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of the present invention or the conjugate of the present invention that specifically binds to the target antibody or antigen-binding fragment thereof, wherein the target antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31; and (b) detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment, optionally wherein the target antibody or antigen-binding fragment is expressed on the surface of a cell, wherein the cell expresses on its surface a CAR comprising the target antibody or antigen-binding fragment, and detecting in (b) comprises detecting cells bound with the anti-idiotype antibody.

[0014] The present invention also provides an in vitro or ex vivo method of stimulating cells, comprising incubating an input composition comprising cells expressing a chimeric antigen receptor (CAR) comprising a target antibody or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of the present invention or the conjugate of the present invention that specifically binds to the target antibody or antigen-binding fragment thereof, thereby generating an output composition comprising stimulated cells, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31.

[0015] Provided herein are agents that specifically bind to antibodies, including antibody fragments such as scFvs, and chimeric molecules containing the same, such as chimeric antigen receptors. Also provided are compositions and articles of manufacture containing such agents, including those including a surface to which the agent is bound, such as a solid surface, e.g. a plate or bead. Also among the embodiments provided herein are uses and methods of using such agents, compositions and articles, including for detection, use, manipulation and / or stimulation of 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.

[0016] In some embodiments, the agent is an anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody that is or contains variable region(s) of the antibody designated FMC63 or an antigen-binding fragment thereof and specifically binds to a chimeric molecule containing such an antibody fragment, in particular a CAR with a binding domain containing antibody variable regions derived from FMC63, such as in the form of an scFv. In some embodiments, the agent, in particular the anti-idiotype antibody or antigen-binding fragment contains a light chain variable (VL) region containing at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 40; and / or a heavy chain variable (VH) region containing at least 90% sequence identity (and / or at least 95 % or 99 % sequence identity, or 100 % identity) to the VH region amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the antibody or antigen-binding fragment contains a VL region comprising at least 90% sequence identity (and / or at least 95 % or 99 % sequence identity, or 100 % identity) to the VL region amino acid sequence set forth in SEQ ID NO: 40; and / or a VH region comprising at least 90 % sequence identity(and / or at least 95 % or 99 % sequence identity, or 100 % identity) to the VH region amino acid sequence set forth in SEQ ID NO: 36.

[0017] In the invention, the complementarity determining region 3 (CDR-H3) contains the amino acid sequence set forth in SEQ ID NO: 94 and contains a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 36; and the light chain complementarity determining region 3 (CDR-L3) contains the amino acid sequence set forth in SEQ ID NO: 97 and containing a CDR-L3 contained within the VL sequence set forth in SEQ ID NO: 40.

[0018] In the invention, the VH region contains a CDR-H1 and a CDR-H2, respectively, including the amino acid sequences of CDR-H1 and CDR-H2 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 36; and / or the VL region contains a CDR-L1 and CDR-L2, respectively, including the amino acid sequences of CDR-L1 and CDR-L2 sequences contained within the VL region amino acid sequence set forth in SEQ ID NO: 40.

[0019] In the invention, the VH region contains a CDR-H1 set forth in SEQ ID NO: 89 or 90, a CDR-H2 set forth in SEQ ID NO: 91, 92 or 93 and a CDR-H3 set forth in SEQ ID NO: 94; and the VL region contains a CDR-L1 set forth in SEQ ID NO: 95, a CDR-L2 set forth in SEQ ID NO: 96, and a CDR-L3 set forth in SEQ ID NO: 97.

[0020] In the invention, the anti-idiotype antibody or antigen-binding fragment thereof contains a CDR-H1, a CDR-H2, and a CDR-H3, respectively, containing the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 36; and a CDR-L1, a CDR-L2, and a CDR-L3, respectively, including the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the VL region amino acid sequence set forth in SEQ ID NO: 40.

[0021] In the invention, the anti-idiotype antibody or antigen-binding fragment thereof comprises a CDR-H1 including the amino acid sequence of SEQ ID NO: 89 or 90, a CDR-H2 including the amino acid sequence of SEQ ID NO: 91, 92 or 93, and a CDR-H3 including the amino acid sequence set forth as SEQ ID NO: 94; and a CDR-L1 including the amino acid sequence of SEQ ID NO: 95, a CDR-L2 including the amino acid sequence of SEQ ID NO: 96, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 97.

[0022] In some of any such embodiments, the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 36; and / or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH region of the antibody or fragment includes the amino acid sequence of SEQ ID NO: 36 and the VL region of the antibody or fragment includes the amino acid sequence of SEQ ID NO: 40.

[0023] In the invention, the VH region contains a CDR-H1 set forth in SEQ ID NO: 89, or 90, a CDR-H2 set forth in SEQ ID NO: 91, 92, or 93 and a CDR-H3 set forth in SEQ ID NO: 94; and the VL region contains a CDR-L1 set forth in SEQ ID NO: 95, a CDR-L2 set forth in SEQ ID NO: 96, and a CDR-L3 set forth in SEQ ID NO: 97.

[0024] In the invention, the VH region contains a CDR-H1, CDR-H2 and CDR-H3 including the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 36; and the VL region contains a CDR-L1, a CDR-L2, and a CDR-L3, respectively, including the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the VL region amino acid sequence set forth in SEQ ID NO: 40.

[0025] In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 36; and / or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 40.

[0026] In some of any such embodiments, 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 of any such embodiments, the fragment contains an scFv.

[0027] In the invention, the target antibody or antigen-binding fragment contains a heavy chain variable region set forth in SEQ ID NO: 30 and a light chain variable region set forth in SEQ ID NO: 31; and optionally is an scFv comprising the sequence of amino acids set forth in SEQ ID NO: 34.

[0028] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to the same or an overlapping epitope of a target antibody or antigen-binding fragment thereof as the epitope specifically bound by the anti-idiotype antibody or antigen-binding fragment according to any one of the embodiments described herein.

[0029] In some 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 a chimeric antigen receptor (CAR); and / or the anti-idiotype antibody or antigen-binding fragment specifically binds the target antibody or antigen-binding fragment contained within or included in the antigen-binding domain of the extracellular portion of a CAR. In some embodiments, the target antibody or antigen-binding fragment is an scFv and the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR.

[0030] In some of any such embodiments, the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from antibody FMC63 comprised in the extracellular portion of a chimeric antigen receptor, wherein the scFv derived from antibody FMC63 contains a heavy chain variable region set forth in SEQ ID NO: 30 and a light chain variable region set forth in SEQ ID NO: 31; and optionally contains the sequence of amino acids set forth in SEQ ID NO: 34.

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

[0032] In some of any such embodiments, the CAR further contains a transmembrane domain linked to the antigen-binding domain via a spacer. In some embodiments, the spacer contains an extracellular portion from CD28, which optionally is human CD28. In some aspects, the extracellular portion from CD28 contains the sequence of amino acids set forth in SEQ ID NO: 27. In some of any such embodiments, the transmembrane domain contains a transmembrane portion of CD28, which optionally is human CD28. In some of any such embodiments, the antibody or fragment does not bind to an epitope in the spacer domain of the CAR.

[0033] In some of any such embodiments, the antibody or fragment does not bind or does not specifically bind to CD28 or a portion thereof, which optionally is human CD28, which optionally contains an extracellular portion of CD28, which optionally contains the sequence of amino acids set forth in SEQ ID NO: 27. In some of any such embodiments, the antibody or fragment does not bind to an epitope in an Fc domain, which optionally is a human IgG1 Fc domain.

[0034] In some of any such embodiments, the target antibody or antigen-binding fragment specifically binds to human CD19. In some of any such embodiments, the anti-idiotype antibody or fragment does not cross-react with another anti-CD 19 antibody, which optionally is contained in the extracellular antigen-binding domain of another CAR. In some of any such embodiments, the anti-idiotype antibody or fragment does not cross-react with another CAR.

[0035] In the invention, the anti-idiotype antibody or fragment is an agonist antibody of a CAR containing the target antibody or antigen-binding fragment.

[0036] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is monoclonal.

[0037] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from among fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, a single chain variable fragment (scFv) or a single domain antibody.

[0038] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof contains at least a portion of an immunoglobulin constant region. In some embodiments, the at least a portion of an immunoglobulin constant region contains 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 of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is an intact antibody or full-length antibody.

[0039] In some of any such embodiments, provided is a conjugate containing the anti-idiotype antibody or antigen-binding fragment according to any one of the embodiments described above and a heterologous molecule or moiety. In some embodiments, the heterologous molecule or moiety is a label. In some aspects, the label is selected from a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a streptavidin, a biotin, a luminescent compound or an oligonucleotide. In some instances, the heterologous molecule or moiety is a protein, peptide, nucleic acid or small molecule, which optionally is or contains a toxin, Strep-Tag.

[0040] In some embodiments, provided is a nucleic acid molecule(s) encoding the heavy chain and light chain of the anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein.

[0041] In some embodiments, the nucleic acid molecule contains a sequence of nucleotides encoding (i) the heavy chain variable region set forth in SEQ ID NO: 50, (ii) a sequence of nucleotides that has at least 90% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 50; or (iii) a degenerate sequence of (i) or (ii); and / or a sequence of nucleotides encoding (iv) the light chain variable region set forth in SEQ ID NO: 54, (v) a sequence of nucleotides that has at least 90% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 54; or (vi) a degenerate sequence of (iv) or (v). In some embodiments, the nucleic acid molecule contains a sequence of nucleotides encoding (i) the heavy chain set forth in SEQ ID NO: 52, (ii) a sequence of nucleotides that has at least 90% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 52; or (iii) a degenerate sequence of (i) or (ii); and / or a sequence of nucleotides encoding (iv) the light chain set forth in SEQ ID NO: 56, (v) a sequence of nucleotides that has at least 90% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 56; or (vi) a degenerate sequence of (iv) or (v). In some embodiments, the nucleotide sequence encoding the heavy chain and / or light chain contains a signal sequence.

[0042] Provided herein is a vector containing the nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell containing the anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein or the nucleic acid molecule according to any one of the embodiments described herein.

[0043] Provided herein is a method of producing an anti-idiotype antibody or antigen-binding fragment thereof, including expressing the heavy and / or light chain encoded by the nucleic acid molecule according to any one of the embodiments described herein or the 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-idiotype antibody or antigen-binding fragment includes culturing the cell according to any one of the embodiments described herein under conditions in which the heavy chain and / or light chain is expressed and recovering or isolating the antibody. Also provided herein is an anti-idiotype antibody or antigen-binding fragment thereof produced by the method according to any one of the embodiments described herein.

[0044] In some embodiments, provided is a composition containing the anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, the conjugate according to any one of the embodiments described herein, or the cell according to any one of the embodiments described herein. In some of any such embodiments, the composition further contains a pharmaceutically acceptable excipient.

[0045] In some embodiments, provided is a kit containing one or more of the anti-idiotype antibody or antigen-binding fragment thereof according to any one of the embodiments described herein, the conjugate according to any one of the embodiments described herein, the nucleic acid according to any one of the embodiments described herein, and, optionally, instructions for use. In some instances, the kit further contains a reagent or support for immobilizing the anti-idiotype antibody or antigen-binding fragment thereof or conjugate, wherein said reagent or support is a bead, a column, a microwell, a stick, a filter, a strip or a soluble oligomeric streptavidin mutein reagent.

[0046] Also provided are methods of detection using any of the provided agents, such as the anti-idiotype antibodies. In some embodiments, provided is a method of detecting a target antibody or antigen-binding fragment thereof, such as a CAR containing the same, including (a) contacting a composition containing a target antibody with variable regions derived from an antibody FMC63, or from an antigen-binding fragment thereof, with the anti-idiotype antibody or antigen-binding fragment thereof of the invention; and (b) detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment and / or detecting the presence or absence of the target antibody or agent.

[0047] In some embodiments, the method includes (a) contacting a composition containing or suspected of containing a target antibody that is the antibody FMC63 or an antigen-binding fragment with the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described or the conjugate of any of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof; and (b) detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment and / or detecting the presence or absence of the target antibody or agent..

[0048] In some aspects, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell and detecting in (b) includes detecting cells bound with the anti-idiotype antibody. In some embodiments, the cell expresses on its surface a CAR containing the target antibody or target antigen-binding fragment.

[0049] In some embodiments, the provided methods involve detecting a CAR containing a target antibody or antigen-binding fragment thereof of any of the embodiments, such as a CAR containing variable domains derived from FMC63. In some embodiments, the method includes (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described or the conjugate of any one of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof; and (b) detecting cells bound with the anti-idiotype antibody. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection.

[0050] In some the disclosure, provided is a method of selecting cells from a cell population, including (a) contacting a cell population expressing a chimeric antigen receptor (CAR) comprising a target antibody or a cell bound to a target antibody with the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described or conjugate of any one of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof, wherein the target antibody is the antibody FMC63 or an antigen-binding fragment thereof; and (b) selecting cells bound with the anti-idiotype antibody.

[0051] In some instances, the cells bound with the anti-idiotype antibody are selected by affinity-based separation. In some aspects, the affinity-based separation is immunoaffinity-based separation. In some of any such embodiments, the affinity-based separation is by flow cytometry. In some embodiments, the affinity-based separation is by magnetic activated cell sorting. In some aspects, the affinity-based separation contains affinity chromatography. In some of any such embodiments, the anti-idiotype antibody is reversibly bound or immobilized to a support or a stationary phase.

[0052] Also among the provided methods are methods for stimulating cells using the agents, such as stimulating cells containing a molecule such as a CAR that is or contains the target antibody recognized by the anti-idiotype antibody. In some embodiments, the method includes incubating an input composition containing cells expressing a chimeric antigen receptor (CAR) containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described or the conjugate of any one of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof, thereby generating an output composition containing stimulated cells.

[0053] In some embodiments, the methods result in proliferation, activation, stimulation, cytokine release, or other functional outcome such as upregulation of an activation marker or cytokine release or production, of cells expressing the chimeric receptor such as the CAR recognized by the anti-Id antibody. In some aspects, such proliferation or other functional response or readout is induced in such cells to a degree that is similar to or greater than that induced by incubation of the cells with an agent and / or conditions that stimulates proliferation of T cells, such as anti-CD3 / CD28 beads and / or crosslinked anti-CD3. In some aspects, the methods do not involve crosslinking of the anti-idiotype antibody. In some aspects of any of the embodiments, the anti-idiotype agents are capable of inducing the specified proliferation or functional outcome or degree thereof, without crosslinking of the anti-idiotype antibody. In some aspects, anti-idiotype agents herein are advantageous in their ability to stimulate or cause a particular functional outcome of T cells or other immune cells expressing the target receptor, without the need to crosslink the anti-Id antibody or use a secondary agent. In some aspects, the result is achieved with soluble or plate-bound form of the anti-idiotype antibody. In some aspects, the result is achieved with the anti-idiotype antibody coupled to a bead.

[0054] In some instances of the disclosure, provided is a method of producing a cell composition, including (a) introducing into cells a nucleic acid molecule encoding a chimeric antigen receptor (CAR), thereby generating an input composition; and (b) incubating the input composition with an anti-idiotype antibody or antigen-binding fragment thereof specific for the antigen receptor of the CAR, thereby producing the cell composition.

[0055] In some aspects, the CAR contains a target antibody that specifically binds to CD19. In some cases, the target antibody is the antibody FMC63 or an antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof specifically binds to a target antibody that is antibody FMC63 of any one of embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof.

[0056] In some of any such instances, the introducing in (a) includes introducing the nucleic acid molecule into the cells by viral transduction, transposition, electroporation, or chemical transfection. In some instances, the introducing in (a) includes introducing the nucleic acid molecule in the cells by transduction with a retroviral vector containing the nucleic acid molecule, optionally wherein the viral vector is a retroviral vector or a lentiviral vector. In some aspects, the introducing in (a) includes introducing the nucleic acid molecule in the cells by transposition with a transposon containing the nucleic acid molecule. In some instances, the introducing in (a) includes introducing the nucleic acid molecule in the cells by electroporation or transfection of a vector containing the nucleic acid molecule.

[0057] In some of any such instances, the method further includes a step of activating the cells prior to step (a). In some aspects, the step of activating the cells includes contacting the cells with an agonist of CD3 and optionally an agonist of CD28. In some instances, the step of activating the cells includes contacting the cells with a reagent containing agonistic anti-CD3 and anti-CD28 antibodies.

[0058] In some of any such embodiments, the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells in the input composition. In any of such embodiments, the cells contain T cells. In some instances, the T cells contain CD4+ and / or CD8+ T cells.

[0059] In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, which optionally contains or is conjugated to a reagent containing a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble reagent, which optionally is or contains a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some aspects, the reagent contains a streptavidin mutein.

[0060] In some of any such embodiments, the incubation is for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36, 48 hours, 72 hours or 96 hours.

[0061] In some of any such embodiments, the input composition contains less than or less than about 60%, less than or less than about 50%, less than or less than about 40%, less than or less than about 30%, less than or less than about 20% or less than or less than about 10% CAR-expressing cells as a percentage of the total cells in the composition.

[0062] In some of any such embodiments, the number of CAR-expressing cells in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold or more compared to the number of CAR-expressing cells in the input composition; and / or the percentage of CAR-expressing in the output composition compared to the total cells in the composition is increased by greater than 10 %, 20 %, 40 %, 50 %, 60 %, 70 %, 80 % or more.

[0063] In some of any such embodiments, prior to the introducing and / or incubating the cells are not selected or enriched for CAR-expressing cells.

[0064] In the invention, the target antibody or antigen-binding fragment contains a heavy chain variable region set forth in SEQ ID NO: 30 and a light chain variable region set forth in SEQ ID NO: 31.

[0065] In some instances of the disclosure, provided is a method of purifying an antibody or antigen-binding fragment thereof, including (a) contacting a composition containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described or the conjugate of any one of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof; and (b) isolating complexes comprising the anti-idiotype antibody.

[0066] In some instances, the complexes containing the anti-idiotype antibody are isolated by affinity-based separation. In some aspects, the affinity-based separation is immunoaffinity-based separation. In some instances, the affinity-based separation is magnetic-based separation. In some instances, the affinity-based separation includes affinity chromatography.

[0067] In some instances of the disclosure, provided is a method of identifying an anti-idiotype antibody or antigen-binding fragment including (a) introducing into a subject a soluble immunization reagent containing an antigen-binding fragment of the target antibody fused to a solubilizing moiety; and (b) identifying an antibody from the subject that specifically binds to the target antibody or the antigen-binding fragment thereof.

[0068] In some of any such instances, the antigen-binding fragment contains the variable heavy chain region and / or variable light chain region of the target antibody. In some instances, the antigen-binding fragment is a single chain fragment. In some aspects, the antigen-binding fragment is an scFv. In some of any such instances, the antigen-binding fragment is within or included in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR).

[0069] In some of any such instances, the solubilizing moiety is an Fc domain or fragment thereof, which optionally is a human IgG1 Fc. In some aspects, the solubilizing moiety is an Fc domain lacking the hinge region. In some instances, the solubilizing moiety contains the amino acid sequence set forth in SEQ ID NO: 32.

[0070] In some of any such instances, identifying the antibody includes (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) screening the hybridomas for production of antibodies that specifically bind the target antibody or the antigen-binding fragment thereof or a chimeric antigen receptor containing the antigen-binding fragment; and (iii) sequencing an antibody from a hybridoma producing an antibody that specifically binds, thereby identifying the anti-idiotype antibody.

[0071] In some of any such instances, the target antibody binds to CD19. In some instances, the antigen-binding fragment of the target antibody is derived from antibody FMC63, optionally wherein the antigen-binding fragment of the target antibody contains a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31. In some instances, the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody FMC63, optionally wherein the scFv contains the sequence of amino acids set forth in SEQ ID NO: 34.

[0072] In some instances of the disclosure, there is provided a method of depleting cells, comprising administering, to a subject, a composition comprising the anti-idiotype antibody or antigen-binding fragment thereof of any one of the embodiments described herein or conjugate of any one of the embodiments described that specifically binds to a target antibody that is antibody FMC63 or an antigen-binding fragment thereof, wherein the subject has been administered a cell expressing a chimeric antigen receptor (CAR) containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof. In some instances, the depletion occurs via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0073] Disclosed herein is a method of determining the presence or absence of a molecule that binds to a chimeric antigen receptor (CAR), the method including (a) contacting a binding reagent with a sample from a subject having been administered a cell therapy containing cells engineered with a CAR containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof under conditions to form a complex comprising the binding reagent and a molecule from the sample that binds to the binding reagent, wherein the binding reagent contains the extracellular domain of the CAR or a portion of the extracellular domain comprising the target antibody or the antigen-binding fragment thereof; and (b) detecting the presence or absence of the complex. In some instances, the method further includes carrying out steps (a) and (b) on a positive control sample and, optionally, determining the presence or absence of the molecule by comparison to the positive control, wherein the positive control sample contains any of the anti-idiotype antibody or antigen-binding fragment thereof as described herein or any of the conjugates described herein that specifically binds to the target antibody or an antigen-binding fragment thereof.

[0074] In some of any such instances, the molecule that binds to the binding reagent is or contains an antibody. In some instances, the binding reagent is detectably labeled or is capable of producing a detectable signal. In some instances, the binding reagent is bound to a solid support or is soluble.

[0075] In some of any such instances, the complex is detected by an immunoassay. In some example, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR)-based biosensor (e.g. , BIAcore), flow cytometry, or Western blot. In some instances, the immunoassay comprises meso scale discovery. In some cases, the immunoassay is a sandwich assay or bridge assay.

[0076] In some of any such instances, the binding reagent is a first binding reagent and detecting the presence or absence of the complex includes:(i) contacting the complex formed in step (a) with a second binding reagent, wherein the second binding reagent (1) contains the extracellular domain of the CAR or a portion thereof comprising the target antibody or the antigen-binding fragment thereof, and (2) is detectably labeled or is capable of producing a detectable signal; and (ii) assessing the presence or absence of the detectable signal. In some aspects, the first binding reagent is bound to a solid support, optionally wherein first binding reagent is linked, directly or indirectly, to a biotin and / or bound to a solid support through a streptavidin; and / or the second binding reagent is soluble. In some cases, the extracellular domain of the CAR or portion thereof of the first and second binding reagent is the same.

[0077] In some of any such instances, the detectable label is or contains a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label or a radiolabel; and / or the detectable signal is or contains a fluorescent signal, chemiluminescent signal, electroluminescent signal, colorimetric signal, a bioluminescent signal or a radioactive signal. In some of any such instances, the detectable label is or contains a SULFO-TAG.

[0078] In some of any such instances, the antigen-binding fragment of the target antibody contains the variable heavy chain region and / or variable light chain region of the target antibody. In some of any such instances, the antigen-binding fragment of the target antibody is a single chain fragment. In some instances, the antigen-binding fragment of the target antibody is an scFv.

[0079] In some of any such instances, the sample comprises whole blood, serum or plasma.

[0080] Provided herein is an article of manufacture containing any of the anti-idiotype antibodies or antigen-binding fragment thereof as described herein or any of the conjugates described herein, and instructions for using the anti-idiotype antibody to detect an FMC63 antibody or antigen-binding fragment thereof or a chimeric antigen receptor containing the FMC63 antibody or antigen-binding fragment thereof; to select or enrich, from a population of cells, engineered cells expressing a chimeric antigen receptor (CAR) comprising the antibody FMC63 or an antigen-binding fragment thereof; to stimulate an input composition comprising cells expressing a chimeric antigen receptor containing the FMC63 antibody or antigen-binding fragment thereof.

[0081] Disclosed herein is an article of manufacture containing a binding reagent including the extracellular domain of a chimeric antigen receptor (CAR) containing a target antibody that is antibody FMC63 or an antigen-binding fragment thereof, said extracellular domain or portion thereof containing the target antibody or antigen-binding fragment thereof; and an anti-idiotype antibody or antigen-binding fragment described herein or the any of the conjugates described herein. In some instances, the binding reagent is a first binding reagent and the article of manufacture further includes a second binding reagent containing the extracellular domain or portion thereof of the CAR.

[0082] In some of any such instances, the extracellular domain of the CAR or portion thereof of the first and second binding reagent is the same.

[0083] In some of any such instances, the article of manufacture further includes instructions for using the binding reagent, optionally the first and second binding reagent, for assaying a sample for the presence or absence of a molecule that binds to the binding reagent using an immunoassay, optionally wherein the immunoassay is a bridge or sandwich immunoassay, optionally wherein the sample is from a subject having been administered a cell therapy including cells engineered with a CAR containing a target antibody that is the antibody FMC63 or an antigen-binding fragment thereof.

[0084] In some instances, the binding reagent, optionally the first and / or second binding reagent, is detectably labeled or capable of producing a detectable signal. In some cases, one of the first and second binding reagent is attached to a solid support of is capable of being attached to a solid support and the other of the first and second binding reagent is detectable label or is capable of producing a detectable signal. In some instances, the article of manufacture further includes a solid support, optionally wherein the one of the first and second binding reagent is linked, directly or indirectly to biotin, and the solid support comprises a streptavidin-coated surface.Brief Description of the Drawings

[0085] FIG. 1 shows the results of flow cytometry to assess the functional activity of an SJ25C1-derived scFv-specific anti-idiotype antibody clone A-1 (anti-ID A-1) to stimulate Erk1 / 2 phosphorylation in Jurkat cells engineered with an SJ25C1-derived CAR. Activation with an anti-CD3 antibody was included as a positive control. Unstimulated or isotype control stimulated cells were included as negative controls. FIG. 2A-B shows results from an assay for the proliferation of T cell expressing a CAR containing either an SJ25C1-derived binding domain (FIG. 2A) or an FMC63-derived binding domain (FIG. 2B), as assessed by dye dilution using flow cytometry following stimulation with an anti-CD3 antibody (OKT3), anti-ID A-1, or anti-ID B-1 anti-idiotype antibody. Unstimulated cells were included as a negative control. FIG. 2C shows results from an assay for the mock transduced and CAR transduced proliferation of T cells (labeled with dye), following culture in the presence of stimulated by plate-bound anti-idiotype antibody recognizing the binding domain of the CAR as assessed by dye dilution using flow cytometry. FIG. 3 shows results for an assessment of the expression of two markers of T cell activation, CD69 and CD25, in CD4 +< or CD8 +< T cells expressing a CAR with variable regions derived from SJ25C1 as assessed by flow cytometry following stimulation with plate-bound anti-CD3 antibody (OKT3), anti-ID A-1 or anti-ID B-1. FIG. 4 shows results for an assessment of the expression of two markers of T cell activation, CD69 and CD25, in CD4 +< or CD8 +< T cells expressing a CAR with a binding domain having variable regions derived from FMC63, as assessed by flow cytometry following stimulation with plate-bound anti-CD3 antibody (OKT3), anti-ID A-1 or anti-ID B-1, or a negative control non-target anti-idiotype antibody. Unstimulated cells were included as a negative control. FIG. 5 shows results from a bridge ELISA for detecting anti-CAR antibodies using anti-ID B-1 and anti-ID B-2 antibodies recognizing the CAR binding domain, at a range of concentrations, as positive controls. FIG. 6 and FIG. 7 shows fold expansion and cumulative cell numbers of EGFRt+ / CD4+ T cells or EGFRt+ / CD8+ T cells, respectively, stimulated with the indicated ratio of beads coated with anti-idiotype antibody (anti-ID B-1) or control CD3 / CD28 antibody coated beads in the presence or absence of cytokines. FIG. 8 shows PD-1 expression levels of CD4+ T cells positive for staining with the anti-EGFR antibody, and thus positive for the transduction marker EGFRt, after stimulation with the indicated ratio of beads coated with anti-idiotype antibody (anti-ID B-1) or control CD3 / CD28 antibody coated beads in the presence or absence of cytokines as assessed by flow cytometry at days 3, 7, 10 and 14 of culture. FIG. 9 shows the viability of CD4 +< or CD8 +< T cells expressing an FMC63-derived CAR as assessed by flow cytometry following stimulation with the indicated ratio of beads coated with anti-idiotype antibody (anti-ID B-1) or control CD3 / CD28 antibody coated beads in the presence or absence of cytokines as assessed by flow cytometry at days 3, 7, 10 and 14 of culture. FIG. 10A shows intracellular cytokine staining for IL-2, TNFα, and IFNγ of T cells expressing an FMC63-derived CAR following stimulation with FMC63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads. Shown are results for CD8+ T cells positive or negative for the EGFRt surrogate transduction marker (EGFR+ or EGFRt-). FIG. 10B shows intracellular cytokine staining for IL-2, TNFα, and IFNγ of T cells expressing an FMC63-derived CAR following stimulation with antigen-expressing K562-CD19 cells. Shown are results of CD8+ T cells positive for the anti-EGFR antibody as a surrogate for CAR expression. FIG. 11 shows the number of population doublings in a serial stimulation assay over a 14 day culture period of T cells expressing an FMC63-derived scFv-derived CAR following stimulation with the indicated ratio of beads coated with anti-idiotype antibody (anti-ID B-1) or control anti-CD3 / anti-CD28 antibody coated beads in the presence or absence of cytokines. Shown are results for CD4+ T cells positive for the EGFRt surrogate transduction marker (EGFRt+ / CD4+) or CD8+ T cells positive for EGFRt (EGFRt+ / CD8+). FIG. 12A-12C show results following stimulation of CD4+ or CD8+ T cells expressing an FMC63-derived CAR, cultured alone or as a co-culture, with FMC63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads. Results are shown for two different donors. FIG. 12A depicts the fold-expansion of CD4+ T cells or CD8+ T cells in the cultures that were positive for the EGFRt surrogate transduction marker (EGFRt + / CD4+ or EGFRt+ / CD8+). FIG. 12B shows the frequency of CD4+ T cells or CD8+ T cells in the cultures that were positive for EGFRt (EGFRt+ / CD4+ or EGFRt + / CD8+). FIG. 12C shows the viability of CD4+ T cells or CD8+ T cells in the cultures. FIG. 13A and 13B show flow cytometry results for T cell surface markers at days 5, 7 and 9 of culture following stimulation of CD4+ or CD8+ T cells expressing an FMC63-derived CAR, cultured alone or as a co-culture, with FMC63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads. FIG. 13A shows surface expression of PD-1 on CD4+ T cells or CD8+ T cells in the cultures that were positive for the EGFRt surrogate transduction marker (EGFRt+ / CD4+ or EGFRt+ / CD8+). FIG. 13B shows surface expression of CD25 on CD4+ T cells or CD8+ T cells in the cultures that were positive for the anti-EGFR antibody as a surrogate for CAR expression (EGFRt+ / CD4+ or EGFRt+ / CD8+). FIG. 14A shows intracellular cytokine levels of TNFα, IFNγ, and IL-2 as assessed by flow cytometry of CD4+ or CD8+ T cells present in a thawed composition containing T cells expressing an FMC63-derived CAR that had been expanded in culture either with CD19 expressing K562 cells or with PMA / Ionomycin. Shown are the level of the cytokines in CD4+ and CD8+ T cells, alone or as a co-culture, at thaw (d=0) or after a further culture for an additional 9 days in the presence of anti-ID B-1 conjugated beads. FIG. 14B shows the frequency of cells positive for CD25 or Ki67 as assessed by flow cytometry of CD4+ or CD8+ T cells present in a thawed composition containing T cells expressing an FMC63-derived CAR that had been expanded in culture either with CD19 expressing K562 cells or with PMA / Ionomycin. Shown are the level of the markers in CD4+ and CD8+ T cells, alone or as a co-culture, at thaw (d=0) or after a further culture for an additional 9 days in the presence of anti-ID B-1 conjugated beads. FIG. 15A and 15B show a graph depicting results of staining cells with anti-EGFR antibody or FMC63-derived scFv-specific anti-idiotype antibodies (anti-ID B-1 and anti-ID B-2). FIG. 15A shows a graph depicting mean fluorescent intensity of cells that were stained with different concentrations of the antibodies. Cells included a mixture of PBMCs and CAR expressing cells. FIG. 15B shows a graph depicting the percentage of CAR expressing cells in de FMC63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) detected in cells that were stained with different concentrations of antibodies. The cells included a mixture of PBMCs and CAR expressing cells and PBMCs alone. Detailed Description

[0086] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy (or for diagnosis).

[0087] Disclosed herein are agents such as anti-idiotype antibodies and antigen-binding fragments (such as single chain fragments, including scFvs) that specifically recognize anti-CD19 antibody moieties (such as anti-CD19 antibody moieties present in recombinant receptors, including chimeric antigen receptors). Also disclosed are uses and methods of use thereof, and compositions and articles of manufacture including such agents, including for specifically identifying, selecting, and / or stimulating and / or activating cells expressing or including the target antibodies or fragments such as anti-CD19 CAR T cells. In some instances, the disclosed antibodies can be used for specific identification and / or selection of various anti-CD19 CARs, such as CARs bound to or expressed on a cell surface, and can also be used to specifically activate cells expressing target CARs, such as CAR T cells. In some instances, disclosed are antibodies that are specific to the anti-CD19 antibody designated SJ25C1 or FMC63, or an antibody fragment derived therefrom, including antibodies and CARs containing variable regions derived from such antibodies, and / or an antibody containing an idiotope contained therein.

[0088] In some aspects, the disclosed anti-idiotype antibodies offer advantages compared to conventional reagents for detecting, identifying, manipulating and / or affecting and / or engineering cells that express a CAR, and in particular a CAR containing an anti-CD19 antibody scFv extracellular domain or one containing the recognized idiotype. In certain available methods detection of the presence or absence or amount of CAR or CAR-expressing cells (and / or stimulation or manipulation of the CAR), in a sample, is carried out by assessing the presence or absence or amount of a surrogate molecule, such as one included in the construct encoding the CAR and thus serving as an indirect or surrogate marker for its expression. In certain available methods, detection is carried out using a generic antibody reagent and / or a reagent that is not specific for the particular CAR assessed, e.g., as compared to other CARs that may have similar or identical domains other than the antigen-binding region; for example, such antibodies may include anti-species antibodies recognizing spacer or other domains from the species from which a CAR domain was derived, and / or antibodies recognizing particular components used in spacer regions of the target and also other chimeric receptors. In certain available methods designed to detect the presence or absence of CARs, detection is carried out using an agent recognizing a CAR constant region. In certain available methods, CAR cells are stimulated through the use of general reagents, such as anti-CD3 / CD28 recognizing agents. Certain methods use a recombinant ligand of the CAR (e.g., CD19-Fc). Such methods in certain contexts may not be entirely satisfactory and / or have certain limitations. In some cases, CAR ligands, such as CD19, may not always be entirely effective, e.g., for use in complex flow cytometry panels. Improved methods and agents are needed, including those providing improved sensitivity and / or selectivity. Disclosed herein are instances meeting such needs.

[0089] The disclosed anti-idiotype antibodies and antigen-binding fragments in some instances overcome challenges of low binding affinity associated with target antibody ligands and non-specific binding associated with antibody reagents directed to target antibody constant regions, providing a reagent with both high affinity and specificity for its target antibody or antigen binding fragment thereof. In some instances, the disclosed antibodies exhibit greater specificity and binding affinity for their target antibodies or antigen-binding fragments, such as the anti-CD19 antibody designated SJ25C1 or FMC63, compared to CD19-Fc and other reagents currently available for detecting or identifying the CAR.

[0090] Furthermore, in certain instances, anti-idiotype antibodies and antigen-binding fragments that may be selected as agonists or antagonists of chimeric receptors comprising their target antibodies or antigen-binding fragments, allowing for selective detection, isolation, ablation and / or depletion (for example, killing via antibody-dependent cell-mediated cytotoxicity, ADCC), and / or stimulation or activation of cells with such chimeric receptors bound to or expressed on their surface. Disclosed herein are anti-idiotype antibody agonists that exhibit activity to stimulate, such as activate, a CAR containing an extracellular binding domain derived from anti-CD19 antibody designated SJ25C1 or FMC63. In some aspects, such antibodies can be used in methods of stimulating and expanding specific CAR-expressing cells, including in processes for generating and preparing the CAR-expressing cells.

[0091] Also disclosed herein are nucleic acids encoding the disclosed anti-idiotype antibodies and fragments, and cells, such as recombinant cells, expressing and for production of these anti-idiotype antibodies and fragments. Also disclosed are methods of making and using the anti-idiotype antibodies and fragments, as well as cells expressing or containing the anti-idiotype antibodies and fragments.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. ANTI-IDIOTYPE ANTIBODIES

[0093] Disclosed in some aspects are binding molecules, such as anti-idiotype antibodies or antigen-binding fragments ("anti-IDs") that specifically recognize a target anti-CD19 antibody moiety. In some instances, the disclosed antibodies recognize a target anti-CD 19 antibody that is SJ25C1 or an antigen-binding fragment thereof or is an antibody or antigen-binding fragment derived from SJ25C1. In some instances, the disclosed antibodies recognize a target anti-CD19 antibody that is FMC63 or an antigen-binding fragment thereof is an antibody or antigen-binding fragment derived from FMC63.

[0094] SJ25C1 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody comprises CDRH1, H2 and H3 set forth in SEQ ID NOS: 114-116, respectively, and CDRL1, L2 and L3 sequences set forth in SEQ ID NOS: 117-119, respectively. The SJ25C1 antibody comprises the heavy chain variable region (V H ) comprising the amino acid sequence of SEQ ID NO: 23 and the light chain variable region (V L ) comprising the amino acid sequence of SEQ ID NO: 24.

[0095] In some instances, the target antibody is SJ25C1 or an antibody-derived from SJ25C1. In some instances, the antibody derived from SJ25C1is an antibody or antigen-binding fragment that comprises the V H and / or V L of SJ25C1, the idiotype of SJ25C1, the paratope of SJ25C1, or one or more complementarity determining regions (CDRs) of SJ25C1. In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprising the V H of SJ25C1 set forth in SEQ ID NO:23, or a variant thereof having at least 90% sequence identity to SEQ ID NO:23 , and / or the V L of SJ25C1 set forth in SEQ ID NO:24, or a variant thereof having at least 90% sequence identity to SEQ ID NO:24. In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprising the V H of SJ25C1 set forth in SEQ ID NO:23, or a variant thereof having at least 90% sequence identity to SEQ ID NO:23, and the V L of SJ25C1 set forth in SEQ ID NO:24, or a variant thereof having at least 90% sequence identity to SEQ ID NO:24. In some instances, the antibody or antigen-binding fragment comprises the V H and V L of SJ25C1 set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. In some instances, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23 and / or SEQ ID NO:24.

[0096] In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprising one or more heavy chain CDRs (CDR-H) of SJ25C1 V H set forth in SEQ ID NO:23, such as set forth in SEQ ID NOS: 114-116 and / or one or more light chain CDRs (CDR-Ls) of SJ25C1 V L set forth in SEQ ID NO:24, such as set forth in SEQ ID NOS: 117-119. In some instances, the antibody or antigen-binding fragment comprises CDR-H3 of SJ25C1 (e.g. set forth in SEQ ID NO: 116) and / or CDR-L3 of SJ25C1 (e.g. set forth in SEQ ID NO: 119). In some instances, the antibody or antigen-binding fragment comprises CDR-H3 and CDR-L3 of SJ25C1 (e.g. set forth in SEQ ID NO:116 and 119, respectively). In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprises one or more of CDR-H1, CDR-H2, and CDR-H3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 114, 115, 116, respectively) and / or one or more of CDR-L 1, CDR-L2, and CDR-L3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 117, 118, 119, respectively). In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 114, 115, 116, respectively) and / or CDR-L1, CDR-L2, and CDR-L3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 117, 118, 119, respectively). In some instances, the target antibody that is SJ25C1 or an antibody-derived from SJ25C1 is an antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 114, 115, 116, respectively) and CDR-L1, CDR-L2, and CDR-L3 of SJ25C1 (e.g. set forth in SEQ ID NOs: 117, 118, 119, respectively). In some instances, the antibody or antigen-binding fragment comprises an antigen-binding fragment, such as a fragment antigen-binding (Fab), a F(ab')2, a Fab', a fragment variable (Fv), or a single chain Fv (scFv). See for example Bejcek, B. E., et al. (1995). Cancer research. 55(11): 2346-2351.

[0097] FMC63 is a mouse monoclonal IgG1 antibody raised against JVM3 cells expressing CD19 of human origin (Nicholson et al. (1997). Molecular Immunology. 34(16-17):1157-1165). The FMC63 antibody comprises CDRH1, H2 and H3 set forth in SEQ ID NOS: 120-122, respectively, and CDRL1, L2 and L3 sequences set forth in SEQ ID NOS: 123-125, respectively. The FMC63 antibody comprises the heavy chain variable region (V H ) comprising the amino acid sequence of SEQ ID NO: 30 and the light chain variable region (V L ) comprising the amino acid sequence of SEQ ID NO: 31.

[0098] In some instances, the target antibody is FMC63 or an antibody-derived from FMC63. In some instances, the antibody derived from FMC63, is an antibody or antigen-binding fragment that comprises the V H and / or V L of FMC63, the idiotype of FMC63, the paratope of FMC63, or one or more complementarity determining regions (CDRs) of FMC63. In some instances, the target antibody that is FMC63 or an antibody-derived from FMC63 is an antibody or antigen-binding fragment comprising the V H of FMC63 set forth in SEQ ID NO: 30, or a variant thereof having at least 90% sequence identity to SEQ ID NO:30, and / or the V L of FMC63 set forth in SEQ ID NO:31, or a variant thereof having at least 90% sequence identity to SEQ ID NO:31. In some instances, the target antibody that is FMC63 or an antibody-derived from FMC63 is an antibody or antigen-binding fragment comprises the V H of FMC63 set forth in SEQ ID NO:30, or a variant thereof having at least 90% sequence identity to SEQ ID NO:30, and the V L of FMC63 set forth in SEQ ID NO:31, or a variant thereof having at least 90% sequence identity to SEQ ID NO:31. In some instances, the antibody or antigen-binding fragment comprises the V H and V L of FMC63 set forth in SEQ ID NO:30 and SEQ ID NO:31, respectively. In some instances, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:30 and / or SEQ ID NO:31.

[0099] In some instances, the target antibody that is FMC63 or an antibody-derived from FMC63 is an antibody or antigen-binding fragment comprises one or more heavy chain CDRs (CDR-H) of FMC63 V H set forth in SEQ ID NO:30, such as set forth in SEQ ID NOS: 120-122 and / or one or more light chain CDRs (CDR-Ls) of FMC63 V L set forth in SEQ ID NO:31, such as set forth in SEQ ID NOS: 123-125. In some instances, the antibody or antigen-binding fragment comprises CDR-H3 of FMC63 (e.g. set forth in SEQ ID NO: 122) and / or CDR-L3 of FMC63 (e.g. set forth in SEQ ID NO: 125). In some instances, the antibody or antigen-binding fragment comprises CDR-H3 (e.g. set forth in SEQ ID NO: 122) and CDR-L3 of FMC63 (e.g. set forth in SEQ ID NO: 125). In some instances, the antibody or antigen-binding fragment comprises one or more of CDR-H1, CDR-H2, and CDR-H3 of FMC63 (set forth in SEQ ID NOs: 120, 121, 122, respectively) and / or one or more of CDR-L1, CDR-L2, and CDR-L3 of FMC63 (set forth in SEQ ID NOs: 123, 124, 125, respectively). In some instances, the antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 of FMC63 (set forth in SEQ ID NOs: 120, 121, 122, respectively) and / or CDR-L1, CDR-L2, and CDR-L3 of FMC63 (set forth in SEQ ID NOs: 123, 124, 125, respectively). In some instances, the antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 of FMC63 (set forth in SEQ ID NOs: 120, 121, 122, respectively) and CDR-L1, CDR-L2, and CDR-L3 of FMC63 (set forth in SEQ ID NOs: 123, 124, 125, respectively). In some instances, the antibody or antigen-binding fragment comprises an antigen-binding fragment, such as a fragment antigen-binding (Fab), a F(ab')2, a Fab', a fragment variable (Fv), or a single chain Fv (scFv).

[0100] In some instances, the disclosed anti-idiotype antibodies include antibodies that specifically bind to a variable domain (Fv), such as a single chain Fv (scFv), derived from SJ25C1 or FMC63. In some instances, the anti-idiotype antibodies specifically bind to a particular epitope or region of an Fv, generally an epitope or region comprising one or more complementarity determining regions. In some instances, the anti-idiotype antibodies specifically bind to an epitope or region overlapping an Fv paratope.

[0101] In some instances, the disclosed anti-idiotype antibodies include those that specifically bind to an anti-CD 19 moiety derived from SJ25C1 or FMC63 that is contained as part of the extracellular domain of a target chimeric antigen receptor (CAR). In some instances, the target CAR contains an antigen-binding portion that contains the SJ25C1 or FMC63 antibody molecule or antigen-binding fragment or portion of the SJ25C1 or FMC63 antibody. In some instances, the target CAR includes an antigen-binding domain that is an scFv derived from the VH and VL chains of the antibody SJ25C1 or FMC63. In some instances, there is disclosed an anti-idiotype antibody that specifically binds to an anti-CD 19 CAR that contains an scFv derived from antibody SJ25C1 or FMC63. Exemplary features of CARs are described further below.

[0102] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0103] The term "anti-idiotype antibody" refers to an antibody, including antigen-binding fragments thereof, that specifically recognizes, is specifically targeted to, and / or specifically binds to an idiotope of an antibody, such as an antigen-binding fragment. The idiotopes of an antibody may include, but are not necessarily limited to, residues within one or more of complementarity determining region(s) (CDRs) of the antibody, variable regions of the antibody, and / or partial portions or portions of such variable regions and / or of such CDRs, and / or any combination of the foregoing. The CDR may be one or more selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The variable regions of the antibody may be heavy chain variable regions, light chain variable regions, or a combination of the heavy chain variable regions and the light chain variable regions. The partial fragments or portions of the heavy chain variable regions and / or the light chain variable regions of the antibody may be fragments including 2 or more, 5 or more, or 10 or more contiguous amino acids, for example, from about 2 to about 100, from about 5 to about 100, from about 10 to about 100, from about 2 to about 50, from about 5 to about 50, or from about 10 to about 50 contiguous amino acids within the heavy chain variable regions or the light chain variable regions of the antibody; the idiotope may include multiple non-contiguous stretches of amino acids. The partial fragments of the heavy chain variable regions and the light chain variable regions of the antibody may be fragments including 2 or more, 5 or more, or 10 or more contiguous amino acids, for example, from about 2 to about 100, from about 5 to about 100, from about 10 to about 100, from about 2 to about 50, from about 5 to about 50, or from about 10 to about 50 contiguous amino acids within the variable regions, and in some instances contain one or more CDRs or CDR fragments. The CDR fragments may be consecutive or nonconsecutive 2 or more, or 5 or more amino acids within the CDR. Therefore, the idiotopes of the antibody may be from about 2 to about 100, from about 5 to about 100, from about 10 to about 100, from about 2 to about 50, from about 5 to about 50, or from about 10 to about 50 contiguous amino acids containing one or more CDR or one or more CDR fragments within the heavy chain variable regions or the light chain variable regions of the antibody. In another instance, the idiotopes may be a single amino acid which is located at the variable regions of the antibody, for example, CDR sites.

[0104] In some instances, the idiotope is any single antigenic determinant or epitope within the variable portion of an antibody. In some cases it can overlap the actual antigen-binding site of the antibody, and in some cases it may comprise variable region sequences outside of the antigen-binding site of the antibody. The set of individual idiotopes of an antibody is in some instances referred to as the "idiotype" of such antibody.

[0105] The terms "complementarity determining region," and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework regions" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

[0106] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1): 55-77 ("IMGT" numbering scheme), and Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3): 657-70, ("Aho" numbering scheme).

[0107] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0108] Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located between CDR-L1 and CDR-L2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop. Table 1 CDR Kabat Chothia Contact CDR-L1L24--L34L24--L34L30--L36CDR-L2L50--L56L50--L56L46--L55CDR-L3L89--L97L89--L97L89--L96CDR-H1 (Kabat Numbering 1< )H31--H35BH26--H32..34H30--H35BCDR-H1 (Chothia Numbering 2< )H31--H35H26--H32H3 0--H35CDR-H2H50--H65H52--H56H47--H58CDR-H3H95--H102H95--H102H93--H1011 - Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 2 - Al-Lazikani et al., (1997) JMB 273,927-948

[0109] Thus, unless otherwise specified, a "CDR" or "complementary determining region," or individual specified CDRs (e.g., "CDR-H1, CDR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given V H or V L amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes. In some instances, specified CDR sequences are specified.

[0110] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.

[0111] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (V H and V L , respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a V H or V L domain from an antibody that binds the antigen to screen a library of complementary V L or V H domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0112] Among the disclosed antibodies are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. In particular instances, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0113] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain instances, a single-domain antibody is a human single-domain antibody.

[0114] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some instances, the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

[0115] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework regions (FRs) amino acid residues are derived from human FRs. In some instances, the humanized forms of a non-human antibody, e.g., a murine antibody, are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulin. In certain instances, the humanized antibodies are antibodies from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region (FR) from a human immunoglobulin molecule. In some instances, a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some instances, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. (See, e.g., Queen, U.S. Pat. No. 5,585,089 and Winter, U.S. Pat. No. 5,225,539.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0116] In certain instances, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a heavy chain variable region of the recipient are replaced by residues from a heavy chain variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In some instances, a nucleic acid sequences encoding human variable heavy chains and variable light chains are altered to replace one or more CDR sequences of the human (acceptor) sequence by sequence encoding the respective CDR in the nonhuman antibody sequence(donor sequence). In some instances, the human acceptor sequence may comprise FR derived from different genes. In particular instances, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. In some instances, the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. In some instances, disclosed herein are humanized anti-idiotype antibodies.

[0117] In particular instances, an antibody, e.g., an anti-idiotype antibody, is humanized. In certain instances, the antibody is humanized by any suitable known means. For example, in some instances, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. In particular instances, humanization can be essentially performed by following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), such as by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In certain instances, the humanized antibody is a human antibody in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0118] Sequences encoding full length antibodies can be subsequently obtained by joining the rendered variable heavy and variable light chain sequences to human constant heavy chain and constant light chain regions. Suitable human constant light chain sequences include kappa and lambda constant light chain sequences. Suitable human constant heavy chain sequences include IgG1, IgG2 and sequences encoding IgG1 mutants which have rendered immune-stimulating properties. Such mutants may have a reduced ability to activate complement and / or antibody dependent cellular cytotoxicity and are described in U.S. Pat. No. 5,624,821; WO 99 / 58572, U.S. Pat. No. 6,737,056. A suitable constant heavy chain also includes an IgG 1 comprising the substitutions E233P, L234V, L235A, A327G, A330S, P331S and a deletion of residue 236. In another instance, the full length antibody comprises an IgA, IgD, IgE, IgM, IgY or IgW sequence.

[0119] Suitable human donor sequences can be determined by sequence comparison of the peptide sequences encoded by the mouse donor sequences to a group of human sequences, preferably to sequences encoded by human germ line immunoglobulin genes or mature antibody genes. A human sequence with a high sequence homology, preferably with the highest homology determined may serve as the acceptor sequence to for the humanization process.

[0120] In addition to the exchange of human CDRs for mouse CDRs, further manipulations in the human donor sequence may be carried out to obtain a sequence encoding a humanized antibody with optimized properties (such as affinity of the antigen).

[0121] Furthermore the altered human acceptor antibody variable domain sequences may also be rendered to encode one or more amino acids (according to the Kabat numbering system) of position 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 of the light variable region and 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 of the heavy variable region corresponding to the non-human donor sequence (Carter and Presta, U.S. Pat. No. 6,407,213)

[0122] In particular instances, it is generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in some instances, the humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and imported sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0123] In particular instances, choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody. See, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies. See, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.

[0124] Among the disclosed antibodies are human antibodies. A "human antibody" is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0125] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.

[0126] Among the disclosed antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.A. SJ25C1-derived Antibodies

[0127] In some instances, disclosed are anti-idiotype antibodies specific to a target anti-CD19 antibody that is or is derived from antibody SJ25C1 or an antigen-binding fragment thereof. In some instances, the disclosed antibodies or antigen-binding fragments are specific to an SJ25C1-derived scFv.

[0128] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (V H ) region comprising at least 90% sequence identity to the V H region amino acid sequence set forth in SEQ ID NO: 1, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0129] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region containing a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence set forth in SEQ ID NO: 11 or 84 and / or a CDR-H3 contained within the heavy chain variable (V H ) sequence set forth in SEQ ID NO: 1.

[0130] In some of any such instances, the V H region includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 9, 78, 79, or 80, and / or a CDR-H1 contained within the V H sequence set forth in SEQ ID NO: 1; and / or a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 10, 81, 82, or 83 and / or a CDR-H2 contained within the V H sequence set forth in SEQ ID NO: 1.

[0131] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 9, 78, 79, or 80; the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 10, 81, 82, or 83; and / or the CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or 84. In some instances, disclosed are antibodies or antigen-binding fragments thereof that include a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 9, 78, 79, or 80; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 10, 81, 82, or 83; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 11 or 84.

[0132] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that includes a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3, respectively, comprising the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 1.

[0133] In some of any such instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 1. In some instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 1. In some instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 1.

[0134] In some of any of such instances, the V H region has the sequence of amino acids set forth in SEQ ID NO: 1.

[0135] In some of any such instances, the anti-idiotype antibody or antigen-binding fragment is a heavy chain only, a VH-only, and / or does not include a VL or antigen-binding portion thereof and / or the antigen-binding site of the anti-idiotype antibody or fragment includes residues from the heavy chain only and / or does not include residues from a light chain.

[0136] In some of any such instances, the anti-idiotype antibody or fragment does not contain a light chain variable (V L ) region, does not contain a CDR-L1, CDR-L2, and / or CDR-L3, and / or is a single-domain antibody (sdAb) containing only the V H region. In some instances, the antibody or fragment is a sdAb that only contains a VH region from any as described.

[0137] In some instances of any of the anti-idiotype antibodies or fragments containing any of the above VH region sequences, the anti-idiotype antibody or fragment further comprises a light chain variable (V L ) region. In some such instances, the V L region has at least 90% sequence identity to the V L region amino acid sequence set forth in SEQ ID NO: 5, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in SEQ ID NO: 5.

[0138] In some of any such instances, the V L region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 14 or 87. In some of any such instances, the V L region comprises a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence set forth in SEQ ID NO: 14 or 87.

[0139] In some of any such instances, the V L region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 12 or 85, and / or a CDR-L1 contained within the V L sequence set forth in SEQ ID NO: 5; and / or a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 13 or 86, and / or a CDR-L2 contained within the V L sequence set forth in SEQ ID NO: 5. In some of any such instances, the V L region comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence set forth in SEQ ID NO: 12 or 85, and / or a CDR-L1 contained within the V L sequence set forth in SEQ ID NO: 5; and / or a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence set forth in SEQ ID NO: 13 or 86, and / or a CDR-L2 contained within the V L sequence set forth in SEQ ID NO: 5.

[0140] In some of any such instances, the V L region comprises a CDR-L1 containing the amino acid sequence set forth in SEQ ID NO: 12 or 85; a CDR-L2 containing the amino acid sequence set forth in SEQ ID NO: 13 or 86; and a CDR-L3 containing the amino acid sequence set forth in SEQ ID NO: 14 or 57.

[0141] In some of any such instances, the V L region comprises the CDR-L1, CDR-L2, and CDR-L3, respectively, comprising the amino acid sequences of a CDR-L1, a CDR-L2, and a CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 5.

[0142] In some of any such instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 having at least 90% sequence identity, respectively, to the FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 5. In some instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 5. In some instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 5.

[0143] In some of any such instances, the V L region has the amino acid sequence set forth in SEQ ID NO: 5.

[0144] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the V H region amino acid sequence set forth in SEQ ID NO: 1; and / or comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 5.

[0145] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include the V H and V L regions having amino acid sequences having at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 1 and 5, respectively.

[0146] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include the V H and V L regions having amino acid sequences set forth in SEQ ID NOs: 1 and 5, respectively.

[0147] In some of any such instances, the V H and V L regions include the amino acid sequences of SEQ ID NOs: 1 and 5, respectively.

[0148] In some instances, the anti-idiotype antibody specific to antibody SJ25C1 or an antigen-binding fragment thereof is a single-chain antibody fragment, such as an scFv or diabody. In some instances, the single-chain antibody includes one or more linkers joining two antibody domains or regions, such as a variable heavy chain (V H ) region and a variable light chain (V L ). The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker. Among the linkers are those rich in glycine and serine and / or in some cases threonine. In some instances, the linkers further include charged residues such as lysine and / or glutamate, which can improve solubility. In some instances, the linkers further include one or more proline.

[0149] In some instances, the anti-idiotype antibody is an intact antibody or full-length antibody. In some instances, the anti-ID may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some instances, the constant regions include a light chain constant region (CL) and / or a heavy chain constant region 1 (CH1). In some instances, the anti-ID includes a CH2 and / or CH3 domain, such as an Fc region. In some instances, the Fc region is an Fc region of a human IgG, such as IgG1 or IgG4. In some instances, the anti-idiotype antibody contains the CH domain set forth in SEQ ID NO:2 or a portion thereof or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2 or a portion thereof. In some instances, the anti-idiotype antibody contains the CL domain set forth in SEQ ID NO:6 or a portion thereof or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6 or a portion thereof.

[0150] In some instances, the anti-idiotype antibody specific for SJ25C1 comprises the heavy chain sequence set forth in SEQ ID NO:3 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 and / or comprises the light chain sequence set forth in SEQ ID NO:7 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7. In some instances, the anti-idiotype antibody specific for SJ25C1 comprises the heavy chain sequence set forth in SEQ ID NO:3 and / or the light chain sequence set forth in SEQ ID NO:7. In some instances, the heavy chain and / or light chain of the anti-idiotype antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 8.

[0151] In some instances, the anti-idiotype antibody is an antigen-binding fragment. In some instances, the antigen-binding fragment is selected from the group consisting of fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, a single chain variable fragment (scFv) or a single domain antibody.

[0152] Accordingly, disclosed are single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain fragments, typically comprising linker(s) joining two anti-idiotype antibody domains or regions, such V H and V L domains. The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0153] In some aspects, the linkers rich in glycine and serine (and / or threonine) include at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% such amino acid(s). In some instances, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and / or threonine. In some instances, the linker is comprised substantially entirely of glycine, serine, and / or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGS (3GS; SEQ ID NO: 29) or GGGGS (4GS; SEQ ID NO: 26), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of a sequence set forth in SEQ ID NO: 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 33 (GSTSGSGKPGSGEGSTKG).

[0154] In some instances, the anti-idiotype antibodies include isolated antibodies. In some instances, the anti-ID is humanized, recombinant, and / or monoclonal. In some instances, the anti-ID is human.

[0155] In some instances, the anti-idiotype antibody specific for SJ25C1 is humanized. In particular instances, all or substantially all CDR amino acid residues of the humanized anti-idiotype antibody specific for SJ25C1 are derived from anti SJ25C1non-human CDRs. In some instances, the humanized anti-idiotype antibody specific for SJ25C1 includes at least a portion of an antibody constant region derived from a human antibody.

[0156] In certain instances, the humanized anti-idiotype antibody specific for SJ25C1 includes a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced by residues from a heavy chain variable region of the nonhuman anti-idiotype antibody specific for SJ25C1. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In some instances, the humanized antibody contains FR derived from different genes. In some instances, the humanized anti-idiotype antibody specific for SJ25C1 contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0157] In some instances, the humanized anti-idiotype antibody specific for SJ25C1 contains an altered human acceptor antibody variable domain sequences that have been rendered to encode one or more amino acid residues of position 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 (Kabat) of the light variable region and 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92(Kabat) of the heavy variable region corresponding to the non-human donor sequence.

[0158] In certain instances, the anti-idiotype antibody specific for SJ25C1 is humanized. In particular instances, the humanized anti-idiotype antibody specific for SJ25C1 contains one or more of a CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 region of a non-human anti-idiotype antibody that is specific for SJ25C1. In some instances, some or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 region of contain one or more amino acid modifications. In some instances, the modifications replacing a nonhuman amino acid residue with a human residue. In particular instances, the one or more of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 are inserted into the FR regions of a human antibody. In particular instances, the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody are the CDRs of the VH and VL regions having amino acid sequences set forth in SEQ ID NOs: 1 and 5, respectively. In some instances, all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the anti-idiotype antibody specific for SJ25C1 are inserted into the FRs of the human antibody. In particular instances, the CDR and FR regions are the regions as identified by Kabat, Chothia, AbM, and / or and Contact schemes.

[0159] In particular instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for SJ25C1 are inserted into framework regions of a human antibody. In certain instances, the human antibody is an IgA, IgD, IgE, IgG, and IgM antibody. In particular instances, the human antibody is one of a subclass of human IgA, IgD, IgE, IgG, and IgM, e.g., human IgG1, IgG 2 , IgG 3 , IgG 4 , IgAi, or IgA 2 . In some instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for SJ25C1 are inserted into framework regions of an antigen binding region that is from and / or is derived from a human antibody. In certain instances, the antigen binding fragment is from and / or is derived from a human IgA, IgD, IgE, IgG, and IgM antibody. The subunit structures and three-dimensional configurations of different classes of human immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). In some instances, the human antibody or antigen binding fragment thereof may be part of a larger fusion molecule, formed by covalent or non-covalent association of the human antibody with one or more other proteins or peptides.

[0160] In some instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for SJ25C1 are inserted into framework regions of a human antibody or antigen-binding fragment thereof having all or a portion of an Fc region. In certain instances, the humanized anti-idiotype antibody specific for SJ25C1 contains all or a portion of an Fc region. In some instances, the Fc region has one or more modifications, such as an amino acid modification (e.g. a substitution, insertion, or deletion) at one or more amino acid positions. Such modifications can be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and / or alter effector functions. In some instances, modified Fc regions have altered (e.g., decreased) binding to FcαRs, relative to that of an unmodified Fc region. In certain instances, the humanized anti-idiotype antibody contains all or a portion of a modified Fc region having an altered (e.g., decreased) binding to Fc receptor relative to that of an unmodified Fc region. Non-limiting examples of Fc modifications that alter its binding to the Fc receptors are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; and U.S. Application Nos. US 2010 / 0143254 and 2010 / 0143254.

[0161] In certain instances, a humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 132 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 133. In particular instances, a humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 134 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 135. In some instances, a humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 136 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 137.B. FMC63-derived Antibodies

[0162] In some instances, disclosed are anti-idiotype antibodies specific to a target anti-CD19 antibody that is or is derived from antibody FMC63 or an antigen-binding fragment thereof. In some instances, the disclosed antibodies or antigen-binding fragments are specific to an FMC63-derived scFv.

[0163] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (V H ) region comprising at least 90% sequence identity to the V H region amino acid sequence set forth in SEQ ID NO: 36 or 58, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0164] In some instances, disclosed are antibodies or antigen-binding fragments thereof that include a VH region having a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence of GYX 3 FX 5 X 6 YX 8 MX 10 (SEQ ID NO: 108), wherein X 3 is T or S, X 5 is T or S, X 6 is D or R, X 8 is Y or W, and X 10 is K or N; and / or a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence of WIGX 4 IX 6 PX 8 X 9 X 10 X 11 TX 13 X 14 NQX 17 FKX 20 (SEQ ID NO: 109), wherein X 4 is D or M, X 6 is Nor H, X 8 is N or S, X 9 is N or D, X 10 is G or S, X 11 is G or E, X 13 is D or R, X 14 is Y or L, X 17 is N or K, and X 20 is G or D; and / or a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence of AX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 (SEQ ID NO: 110), wherein X 2 is R or S, X 3 is E or I, X 4 is G or Y, X 5 is N or Y, X 6 is N or E, X 7 is Y or null, X 8 is G or null, X 9 is S or null, X 10 is R or null, X 11 is D or null, X 12 is A or null, X 13 is M or null, X 14 is D or E, and X 15 is Y or A.

[0165] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region containing a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence set forth in SEQ ID NO: 46, 67, 94 or 104 and / or a CDR-H3 contained within the heavy chain variable (V H ) sequence set forth in SEQ ID NO: 36 or 58.

[0166] In some of any such instances, the V H region includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 44, 65, 88, 89, 90, 98, 99, or 100, and / or a CDR-H1 contained within the V H sequence set forth in SEQ ID NO: 36 or 58; and / or a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103, and / or a CDR-H2 contained within the V H sequence set forth in SEQ ID NO: 36 or 58.

[0167] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 44, 65, 88, 89, 90, 98, 99, or 100; the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103; and / or the CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 46, 67, 94 or 104. In some instances, disclosed are antibodies or antigen-binding fragments thereof that include a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 44, 65, 88, 89, 90, 98, 99, or 100; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 46, 67, 94 or 104.

[0168] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that includes a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3, respectively, comprising the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the V H region amino acid sequence set forth in SEQ ID NO: 36 or 58.

[0169] In some instances, the anti-idiotype antibody or antigen-binding fragment thereof includes a CDR-H1 set forth in SEQ ID NOS: 44, 88, 89 or 90; a CDR-H2 set forth in SEQ ID NOS: 45, 91, 92 or 93; and / or a CDR-H3 set forth in SEQ ID NO: 46 or 94. In some instances, the anti-idiotype antibody or antigen-binding fragment thereof includes a CDR-H1 set forth in SEQ ID NO: 65, 98, 99 or 100; a CDR-H2 set forth in SEQ ID NO: 66, 101, 102 or 103; and / or a CDR-H3 set forth in SEQ ID NO: 67 or 104, respectively.

[0170] In some of any such instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 36 or 58. In some instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 36 or 58. In some instances, the V H region contains a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 36 or 58.

[0171] In some of any of such instances, the V H region has the sequence of amino acids set forth in SEQ ID NO: 36 or 58.

[0172] In some of any such instances, the anti-idiotype antibody or antigen-binding fragment is a heavy chain only, a VH-only, and / or does not include a VL or antigen-binding portion thereof and / or the antigen-binding site of the anti-idiotype antibody or fragment includes residues from the heavy chain only and / or does not include residues from a light chain.

[0173] In some of any such instances, the anti-idiotype antibody or fragment does not contain a light chain variable (V L ) region, does not contain a CDR-L1, CDR-L2, and / or CDR-L3, and / or is a single-domain antibody (sdAb) containing only the V H region. In some instances, the antibody or fragment is a sdAb that only contains a VH region from any as described.

[0174] In some instances of any of the anti-idiotype antibodies or fragments containing any of the above VH region sequences, the anti-idiotype antibody or fragment further comprises a light chain variable (V L ) region. In some such instances, the V L region has at least 90% sequence identity to the V L region amino acid sequence set forth in SEQ ID NO: 40 or 62, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V L region amino acid sequence set forth in SEQ ID NO: 40 or 62.

[0175] In some instances, disclosed are antibodies or antigen-binding fragments thereof that include a VH region having a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence of X 1 AX 3 X 4 X 5 X 6 X 7 X 8 YX 10 X 11 WY (SEQ ID NO: 111), wherein X 1 is S or R, X 3 is S or R, X 4 is S or G, X 5 is G or N, X 6 is V or I, X 7 is I or H, X 8 is N or null, X 10 is M or L, and X 11 is Y or A; and / or a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence of X 1 X 2 X 3 YX 5 X 6 X 7 X 8 LAX 11 (SEQ ID NO: 112), wherein X 1 is P or L, X 2 is W or L, X 3 is I or V, X 5 is L or N, X 6 is T or A, X 7 is S or K, X 8 is N or T, and X 11 is S or D; and / or a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence of QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 113), wherein X 2 is Q or H, X 3 is W or F, X 4 is S or W, X 5 is S or W, X 6 is N or T, and X 8 is L or Y.

[0176] In some of any such instances, the V L region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 49, 97, 70, or 107. In some of any such instances, the V L region comprises a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence set forth in SEQ ID NO: 49, 97, 70, or 107.

[0177] In some of any such instances, the V L region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 47, 68, 95, or 105, and / or a CDR-L1 contained within the V L sequence set forth in SEQ ID NO: 40 or 62; and / or a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 48, 69, 96, or 106, and / or a CDR-L2 contained within the V L sequence set forth in SEQ ID NO: 40 or 62. In some of any such instances, the V L region comprises a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence set forth in SEQ ID NO: 47, 68, 95, or 105, and / or a CDR-L1 contained within the V L sequence set forth in SEQ ID NO: 40 or 62; and / or a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence set forth in SEQ ID NO: 48, 69, 96, or 106, and / or a CDR-L2 contained within the V L sequence set forth in SEQ ID NO: 40 or 62.

[0178] In some of any such instances, the V L region comprises a CDR-L1 containing the amino acid sequence set forth in SEQ ID NO: 47, 68, 95, or 105; a CDR-L2 containing the amino acid sequence set forth in SEQ ID NO: 48, 69, 96, or 106; and a CDR-L3 containing the amino acid sequence set forth in SEQ ID NO: 49, 97, 70, or 107.

[0179] In some of any such instances, the V L region comprises the CDR-L1, CDR-L2, and CDR-L3, respectively, comprising the amino acid sequences of a CDR-L1, a CDR-L2, and a CDR-L3 contained within the V L region amino acid sequence set forth in SEQ ID NO: 40 or 62.

[0180] In some instances, the anti-idiotype antibody or antigen-binding fragment thereof includes a CDR-L1 set forth in SEQ ID NOS: 47 or 95; a CDR-L2 set forth in SEQ ID NOS: 48 or 96; and / or a CDR-L3 set forth in SEQ ID NO: 49 or 97. In some instances, the anti-idiotype antibody or antigen-binding fragment thereof includes a CDR-L1 set forth in SEQ ID NO: 68 or 105; a CDR-L2 set forth in SEQ ID NO: 69 or 106; and / or a CDR-L3 set forth in SEQ ID NO: 70 or 107, respectively.

[0181] In some of any such instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 having at least 90% sequence identity, respectively, to the FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 40 or 62. In some instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and / or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and / or FR4 of the amino acid sequence set forth in SEQ ID NO: 40 or 62. In some instances, the V L region comprises a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 40 or 62.

[0182] In some of any such instances, the V L region has the amino acid sequence set forth in SEQ ID NO: 40 or 62.

[0183] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the V H region amino acid sequence set forth in SEQ ID NO: 36 or 58; and / or comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 40 or 62.

[0184] Disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include the V H region having amino acid sequences having at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 36 or 58 and V L region having amino acid sequences having at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 40 or 62.

[0185] In some instances, disclosed are anti-idiotype antibodies or antigen-binding fragments thereof that include the V H region having amino acid sequences set forth in SEQ ID NOs: 36 or 58 and V L region having amino acid sequences set forth in SEQ ID NOs: 40 or 62. In some of instances, the disclosed antibody contains the V H region set forth in SEQ ID NO: 36 and the V L region set forth in SEQ ID NOs:40. In some of instances, the disclosed antibody contains the V H region set forth in SEQ ID NO: 58 and the V L region set forth in SEQ ID NOs: 62.

[0186] In some instances, the anti-idiotype antibody is a single-chain antibody fragment, such as an scFv or diabody. In some instances, the single-chain antibody includes one or more linkers joining two antibody domains or regions, such as a variable heavy chain (V H ) region and a variable light chain (V L ). The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker. Among the linkers are those rich in glycine and serine and / or in some cases threonine. In some instances, the linkers further include charged residues such as lysine and / or glutamate, which can improve solubility. In some instances, the linkers further include one or more proline.

[0187] In some instances, the anti-idiotype antibody is an intact antibody or full-length antibody. In some instances, the anti-ID may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some instances, the constant regions include a light chain constant region (CL) and / or a heavy chain constant region 1 (CH1). In some instances, the anti-ID includes a CH2 and / or CH3 domain, such as an Fc region. In some instances, the Fc region is an Fc region of a human IgG, such as IgG1 or IgG4. In some instances, the anti-idiotype antibody contains the CH domain set forth in SEQ ID NO:37 or 59 or a portion thereof or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:37 or 59 or a portion thereof. In some instances, the anti-idiotype antibody contains the CL domain set forth in SEQ ID NO:41 or 63 or a portion thereof or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:41 or 63 or a portion thereof.

[0188] In some instances, the anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO:38 or 60 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:38 or 60 and / or comprises the light chain sequence set forth in SEQ ID NO:42 or 63 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:42 or 63. In some instances, the anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO:38 and / or the light chain sequence set forth in SEQ ID NO:42. In some instances, the anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO:60 and / or the light chain sequence set forth in SEQ ID NO:63. In some instances, the heavy chain and / or light chain of the anti-idiotype antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence set forth in SEQ ID NO:39, 43, 61 or 64.

[0189] In some instances, the anti-idiotype antibody is an antigen-binding fragment. In some instances, the antigen-binding fragment is selected from the group consisting of fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, a single chain variable fragment (scFv) or a single domain antibody.

[0190] Accordingly, disclosed are single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain fragments, typically comprising linker(s) joining two anti-idiotype antibody domains or regions, such V H and V L domains. The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0191] In some aspects, the linkers rich in glycine and serine (and / or threonine) include at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% such amino acid(s). In some instances, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and / or threonine. In some instances, the linker is comprised substantially entirely of glycine, serine, and / or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGS (3GS; SEQ ID NO: 29) or GGGGS (4GS; SEQ ID NO: 26), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of a sequence set forth in SEQ ID NO: 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 33 (GSTSGSGKPGSGEGSTKG).

[0192] In some instances, the anti-idiotype antibodies include isolated antibodies. In some instances, the anti-ID is humanized, recombinant, and / or monoclonal. In some instances, the anti-ID is human.

[0193] In some instances, the anti-idiotype antibody specific for FMC63 is humanized. In particular instances, all or substantially all CDR amino acid residues of the humanized anti-idiotype antibody specific for FMC63 are derived from non-human, anti-FMC63 CDRs. In some instances, the humanized anti-idiotype antibody specific for FMC63 includes at least a portion of an antibody constant region derived from a human antibody.

[0194] In certain instances, the humanized anti-idiotype antibody specific for FMC63 includes a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced by residues from a heavy chain variable region of the nonhuman anti-idiotype antibody specific for FMC63. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In some instances, the humanized antibody contains FR derived from different genes. In some instances, the humanized anti-idiotype antibody specific for FMC63 contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0195] In some instances, the humanized anti-idiotype antibody specific for FMC63 contains an altered human acceptor antibody variable domain sequences that have been rendered to encode one or more amino acid residues of position 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 (Kabat) of the light variable region and 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92(Kabat) of the heavy variable region corresponding to the non-human donor sequence .

[0196] In certain instances, the anti-idiotype antibody specific for FMC63 is humanized. In particular instances, the humanized anti-idiotype antibody specific for FMC63 contains one or more of a CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for FMC63. In some instances, some or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 region of contain one or more amino acid modifications. In some instances, the modifications replacing a nonhuman amino acid residue with a human residue. In particular instances, the one or more of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 are inserted into human FR regions. In particular instances, the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody are the CDRs of the VH regions having amino acid sequences set forth in SEQ ID NOs: 36 or 58. In certain instances, the CDRs of the nonhuman anti-idiotype VH region are or include a CDR-H1 containing with the amino acid sequence set forth in SEQ ID NO: 44, 65, 88, 89, 90, 98, 99, or 100; the CDR-H2 containing the amino acid sequence set forth in SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103; and / or the CDR-H3 containing the amino acid sequence set forth in SEQ ID NO: 46, 67, 94 or 104. In some instances, the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody are the CDRs of the VL regions having amino acid sequences set forth in SEQ ID NOs: 40 or 62. In some instances, the CDRs of the nonhuman anti-idiotype VL region are or include a CDR-L1 containing the amino acid sequence set forth in SEQ ID NO: 47, 68, 95, or 105; a CDR-L2 containing the amino acid sequence set forth in SEQ ID NO: 48, 69, 96, or 106; and a CDR-L3 containing the amino acid sequence set forth in SEQ ID NO: 49, 97, 70, or 107. In some instances, all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions of the anti-idiotype antibody specific for FMC63 are inserted into the FRs of the human antibody. In particular instances, the CDR and FR regions are the regions as identified by Kabat, Chothia, AbM, and / or and Contact schemes.

[0197] In particular instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for FMC63 are inserted into framework regions of a human antibody. In certain instances, the human antibody is an IgA, IgD, IgE, IgG, and IgM antibody. In particular instances, the human antibody is one of a subclass of human IgA, IgD, IgE, IgG, and IgM, e.g., human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgAi, or IgA 2 . In some instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for FMC63 are inserted into framework regions of an antigen binding region that is from and / or is derived from a human antibody. In certain instances, the antigen binding fragment is from and / or is derived from a human IgA, IgD, IgE, IgG, and IgM antibody. The subunit structures and three-dimensional configurations of different classes of human immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). In some instances, the human antibody or antigen binding fragment thereof may be part of a larger fusion molecule, formed by covalent or non-covalent association of the human antibody with one or more other proteins or peptides.

[0198] In some instances, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the nonhuman anti-idiotype antibody specific for FMC63 are inserted into framework regions of a human antibody or antigen-binding fragment thereof having all or a portion of an Fc region. In certain instances, the humanized anti-idiotype antibody specific for FMC63 contains all or a portion of an Fc region. In some instances, the Fc region has one or more modifications, such as an amino acid modification (e.g. a substitution, insertion, or deletion) at one or more amino acid positions. Such modifications can be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and / or alter effector functions. In some instances, modified Fc regions have altered (e.g., decreased) binding to FcαRs, relative to that of an unmodified Fc region. In certain instances, the humanized anti-idiotype antibody contains all or a portion of a modified Fc region having an altered (e.g., decreased) binding to Fc receptor relative to that of an unmodified Fc region. Non-limiting examples of Fc modifications that alter its binding to the Fc receptors are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; and U.S. Application Nos. US 2010 / 0143254 and 2010 / 0143254.

[0199] In certain instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 138 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 139. In particular instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 140 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 141. In some instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 142 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 143. In certain instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 144 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 145. In particular instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 146 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 147. In some instances, a humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 148 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 149.C. Exemplary Features

[0200] Anti-idiotype antibodies disclosed herein may be identified, screened for, or characterized for their physical / chemical properties and / or biological activities by various known assays. In one aspect, the anti-idiotype antibody is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blotting, and / or flow cytometric assays, including cell-based binding assays, for example, assessing binding of the anti-idiotype antibody (e.g., conjugated to a fluorescent marker or tagged) to a cell presenting the target anti-CD19 antibody moiety, in some cases compared to results using cells that do not express the target anti-CD19 antibody moiety. Binding affinity may be measured as Kd or EC50.

[0201] In some instances of any of the disclosed antibodies, e.g. any of the disclosed antibodies in Section A above, the target anti-CD19 antibody moiety is SJ25C1 or is an antibody derived from SJ25C1.

[0202] In some instances of any of the disclosed antibodies, e.g. any of the disclosed antibodies in Section B above, the target anti-CD19 antibody moiety is FMC63 or is an antibody derived from FMC63.

[0203] Competition assays may be used to identify an antibody that competes with any of the anti-idiotype antibodies described herein. Assays for mapping epitopes bound by the anti-idiotype antibodies and reference antibodies also may be used and are known.

[0204] In some instances, the anti-idiotype antibody does not cross-react with an anti-CD19 antibody moiety different from the target anti-CD19 antibody moiety. In some instances, the target anti-CD19 antibody moiety is derived from the SJ25C1 antibody. In some instances, the target anti-CD19 antibody moiety is derived from the SJ25C1 antibody, and the anti-idiotype antibody does not cross-react with an anti-CD19 antibody moiety derived from the FMC63 antibody, an anti-CD19 antibody comprising a V H comprising the amino acid sequence of SEQ ID NO: 30 and a V L comprising the amino acid sequence of SEQ ID NO: 31.In some instances, the target anti-CD19 antibody moiety is derived from the FMC63 antibody. In some instances, the target anti-CD19 antibody moiety is derived from the FMC63 antibody, and the anti-idiotype antibody does not cross-react with an anti-CD19 antibody moiety derived from the SJ25C1 antibody, an anti-CD19 antibody comprising a V H comprising the amino acid sequence of SEQ ID NO: 23 and a V L comprising the amino acid sequence of SEQ ID NO: 24.

[0205] In some instances, the anti-idiotype antibody specifically binds to a target anti-CD19 antibody moiety that is part of a fusion protein, such as a recombinant receptor. In some instances, the anti-idiotype antibody does not bind to any epitope in the fusion protein outside of the target anti-CD 19 antibody moiety. For example, in some instances, the target anti-CD 19 antibody moiety is, or is part of, the antigen-binding domain of a chimeric antigen receptor (CAR), and the anti-idiotype antibody does not bind any epitope outside of the antigen-binding domain. In some instances, the CAR antigen-binding domain comprises or consists of an scFv.

[0206] In some instances, the anti-idiotype antibody specifically binds to a target anti-CD19 antibody moiety that is an scFv contained in a CAR. In some instances, the anti-idiotype antibody specifically binds to an epitope overlapping one or more complementarity determining regions (CDRs) of the target anti-CD19 scFv. In some instances, the anti-idiotype antibody does not bind any epitopes in the CAR outside of the scFv; in some instances, it does not bind to a reference antibody. In some instances, the reference antibody specifically binds to the same antigen as the target antibody, e.g., to the CD19 and / or comprises one or more variable heavy and / or variable light framework region(s) having at least 90, 95, 96, 97, 98, or 99 % identity to the corresponding framework region(s) of the target antibody (in some aspects, the one or more framework regions comprise an FR1, FR2, FR3, and / or FR4 of the heavy and / or the light chain); and / or contains the same heavy and / or light chain v-gene (or v-gene usage) as the target antibody and / or is derived from the same v-gene sequence as the target antibody. In some aspects, the reference antibody is FMC63. In some aspects, the reference antibody is SJ25C1. In some instances, the CAR comprises a spacer linking the scFv to its transmembrane domain, and the anti-idiotype antibody does not bind any epitope in the spacer. In some instances, the spacer is a sequence derived from CD28, such as an extracellular portion from CD28. In some instances, the spacer comprises the amino acid sequence of SEQ ID NO: 27. In some instances, the anti-idiotype antibody does not bind any epitope in an Fc domain, such as the Fc domain of IgG1. In some instances, the Fc domain is an IgG1 Fc domain lacking the hinge region. In some instances, the Fc domain comprises the amino acid sequence of SEQ ID NO: 32.

[0207] In some instances, the anti-idiotype antibody does not cross-react with a different CAR. In some instances, the anti-idiotype antibody does not cross-react with a different anti-CD19 CAR. In some instances, the anti-idiotype antibody does not cross-react with an anti-CD19 antibody moiety, e.g., of a reference antibody, having one or more different idiotopes compared to the target anti-CD19 scFv. In some instances, the anti-idiotype antibody is specific for a target anti-CD19 scFv of a CAR derived from the SJ25C1 antibody. In some instances, the target anti-CD19 antibody moiety is derived from the SJ25C1 antibody, and the anti-idiotype antibody does not cross-react with a CAR containing an anti-CD19 antibody moiety derived from the FMC63 antibody. In some instances, the anti-idiotype antibody is specific for a target anti-CD19 scFv of a CAR derived from the FMC63 antibody. In some instances, the target anti-CD19 antibody moiety is derived from the FMC63 antibody, and the anti-idiotype antibody does not cross-react with a CAR containing anti-CD19 antibody moiety derived from the SJ25C1 antibody.

[0208] In some instances, the anti-idiotype antibody is an agonist of the CAR. In some instances, the anti-idiotype antibody is an antagonist of the CAR.

[0209] In some instances, the disclosed anti-idiotype antibodies are capable of binding a target anti-CD19 moiety, such as antibody SJ25C1 or FMC63, with at least a certain affinity, as measured by any of a number of known methods. In some instances, the affinity is represented by an equilibrium dissociation constant (K D ); in some instances, the affinity is represented by EC 50 . In certain instances, the binding affinity (EC50) and / or the dissociation constant of the anti-idiotype antibody to the anti-CD19 moiety is at or about or less than at or about 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, such as between at or about 1 nM and at or about 15 nM, e.g., between at or about 5 and at or about 10 nM. In one instance, the extent of binding of an anti-idiotype antibody to a moiety unrelated to the target anti-CD19 moiety is less than, at, or about 10% of the binding of the antibody to the target anti-CD19 moiety as measured, e.g., by a radioimmunoassay (RIA).D. Nucleic Acids

[0210] Also disclosed are nucleic acids encoding the antibodies and / or portions, e.g., chains, thereof. Among the disclosed nucleic acids are those encoding the anti-idiotype antibodies described herein. The nucleic acids may include those encompassing natural and / or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. Exemplary nucleic acids and vectors are those having the sequences set forth as SEQ ID NOs: 15-22, 50-57, 71-77 and CDR-encoding portions thereof, as well as sequences containing at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. The nucleic acid may encode an amino acid sequence comprising the V L and / or an amino acid sequence comprising the V H of the anti-idiotype antibody (e.g., the light and / or heavy chains of the antibody).

[0211] Also disclosed are vectors containing the nucleic acids, host cells containing the vectors, e.g., for producing the antibodies. Also disclosed are methods for producing the antibodies. In a further instance, one or more vectors (e.g., expression vectors) comprising such nucleic acid are disclosed. In a further instance, a host cell comprising such nucleic acid is disclosed. In one such instance, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the V L of the antibody and an amino acid sequence comprising the V H of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the V L of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the V H of the antibody. In some instances, a method of making the anti-idiotype antibody is disclosed, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as disclosed above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).E. Methods of Producing the Antibodies

[0212] Also disclosed are methods of making an anti-idiotype antibody, such as of any of the disclosed instances. In some instances, for recombinant production of the anti-idiotype antibody, nucleic acid encoding an antibody, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0213] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0214] Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6 ®< cells; and NSO cells. In some instances, the antibody heavy chains and / or light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045 A1. In some instances, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and / or light chains. For example, in some instances, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0215] In some instances, the anti-idiotype antibody is produced in a cell-free system. Exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0216] The disclosed instances further include vectors and host cells and other expression systems for expressing and producing the antibodies and other binding proteins, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells such as human cells, as well as cell-free expression systems.

[0217] The anti-idiotype antibodies or antibody moieties can be humanized antibodies or human antibodies. A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some instances, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0218] Among the disclosed anti-idiotype antibodies or antibody moieties are human antibodies. A "human antibody" is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0219] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.F. Immunoconjugates

[0220] In some instances, the anti-idiotype antibody is or is part of an immunoconjugate (anti-idiotype antibody immunoconjugate), in which the anti-idiotype antibody is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic or an imaging agent. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents (e.g., maytansinoids, taxanes, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins. In some instances, the antibody is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0221] Among the anti-idiotype antibody immunoconjugates are antibody-drug conjugates (ADCs), in which an anti-idiotype antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman etal., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

[0222] Also among the anti-idiotype antibody immunoconjugates are those in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0223] Also among the anti-idiotype antibody immunoconjugates are those in which the anti-idiotype antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioactive isotopes include At 211< , I 131< , I 125< , Y 90< , Re 186< , Re 188< , Sm 153< , Bi 212< , P 32< , Pb 212< and radioactive isotopes of Lu.

[0224] Conjugates of an anti-idiotype antibody and cytotoxic agent may be made using any of a number of known protein coupling agents, e.g., linkers, (see Vitetta et al., Science 238: 1098 (1987)), WO94 / 11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell, such as acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0225] Also disclosed are anti-idiotype antibody immunoconjugates comprising an anti-idiotype antibody attached to a label,e.g., a detectable label, which can generate a detectable signal, indirectly or directly. These anti-idiotype antibody immunoconjugates can be used for research or diagnostic applications. The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3< H, 14< C, 32< P, 35< S, 123< I, 125< I, 131< I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase,β-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In some instances, the label is a radioactive atom for scintigraphic studies, for example 99< Tc or 123< I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0226] Examples of detectable labels include but are not limited to radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, coumarin, Alexa488, Oregon green 488, rhodamine green, Alexa 532, Cy3, Bodipy 588 / 586, Alexa586, TAMRA, Rox, Alexa 594, Texas red, Bodipy 630 / 650, Cy5, Alexa647, IR Dye 680, IR Dye 680, IR Dye 700 DX, Cy5.5, Alexa 750, IR Dye 800CW, IR Dye 800, Atto 532, and Atto 465.

[0227] In some instances, the anti-idiotype antibody immunoconjugate is detectable indirectly. For example, a secondary antibody that is specific for the anti-idiotype antibody immunoconjugate and contains a detectable label can be used to detect the anti-idiotype antibody immunoconjugate.Multispecific Antibodies

[0228] In certain instances, the anti-idiotype antibodies are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific. Multispecific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain instances, one of the binding specificities is for an anti-CD19 antibody moiety and the other is for another antigen. In certain instances, bispecific antibodies may bind to two different epitopes of an anti-CD19 antibody moiety. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express an anti-CD19 antibody moiety on their surface, such as anti-CD19 CAR T cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the bispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs.G. Variants

[0229] In certain instances, the anti-idiotype antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and / or mutations, compared to the sequence of an anti-idiotype antibody described herein. Exemplary variants include those designed to improve the binding affinity and / or other biological properties of the anti-idiotype antibody. Amino acid sequence variants of an anti-idiotype antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-idiotype antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the anti-idiotype antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, disclosed that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0230] In certain instances, the anti-idiotype antibodies include one or more amino acid substitutions, e.g., as compared to an anti-idiotype antibody sequence described herein. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an anti-idiotype antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, improved half-life, and / or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some instances, the variant anti-idiotype antibody exhibits retained or improved binding to a target anti-CD19 antibody or fragment thereof. For example, in some instances, the variant anti-idiotype antibody exhibits an increase in binding affinity to the target anti-CD19 antibody of at least about 10% (such as at least about any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000% or more) as compared to the unmodified anti-idiotype antibody.

[0231] In some instances, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is / are substituted. In some instances, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human individual.

[0232] In some instances, alterations are made in CDR "hotspots," residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, with the resulting variant V H or V L being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some instances of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0233] In certain instances, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as disclosed herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain instances of the variant V H and V L sequences disclosed above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0234] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.Modifications

[0235] In certain instances, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and / or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines.

[0236] Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003 / 0157108, US 2004 / 0093621, US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107); WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.); WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0237] Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0238] Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and / or alter effector functions.

[0239] Also among the variants are cysteine engineered antibodies such as "thioMAbs" and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Patent Nos. 7,855,275 and 7,521,541.

[0240] In some instances, the antibodies are modified to contain additional nonproteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.II. METHODS OF IDENTIFYING ANTI-IDIOTYPE ANTIBODIES

[0241] In some instances, there is disclosed a method of identifying an anti-idiotype antibody or antigen-binding fragment that specifically binds to a target antibody or antigen-binding fragment thereof, such as by using hybridoma methods with an immunogen comprising the target antibody or fragment thereof (see for example Kohler and Milstein, Nature, 256: 495 (1975) and Sergeeva et al., Blood, 117(16): 4262-4272). The immunogen may comprise an immunogenicity-enhancing moiety fused to the target antibody or fragment. Such immunogenicity-enhancing moiety may have properties that include, without limitation, increasing solubility and half-life of the immunogen. Exemplary immunogenicity-enhancing moieties include Fc domains or fragments thereof. In some instances, the immunogenicity-enhancing moiety is an Fc domain (such as from IgG1, optionally human). In some instances, the immunogenicity-enhancing moiety is an Fc domain (such as from IgG1, optionally human) lacking all or a portion of the hinge region.

[0242] In some instances, there is disclosed a method of identifying an anti-idiotype antibody or antigen-binding fragment that specifically binds to a target antibody or antigen-binding fragment thereof, comprising: (a) introducing into a subject a soluble immunogen comprising an antigen-binding fragment of the target antibody fused to an immunogenicity-enhancing moiety; and (b) identifying an antibody from the subject that specifically binds to the target antibody or antigen-binding fragment thereof. In some instances, the antigen-binding fragment comprises the variable heavy chain region and / or variable light chain region of the target antibody. In some instances, the antigen-binding fragment is a single chain fragment. In some instances, the antigen-binding fragment is an scFv. In some instances, the antigen-binding fragment is within or included in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR).

[0243] In some instances, the immunogenicity-enhancing moiety is an Fc domain or fragment thereof, which optionally is a human IgG1 Fc. In some instances, the immunogenicity-enhancing moiety is an Fc domain lacking the hinge region. In some instances, the immunogenicity-enhancing moiety comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0244] In some instances, identifying the antibody comprises using hybridoma techniques to identify individual antibody clones produced by the subject and screening the clones for binding to the target antibody or antigen-binding fragment thereof. In some instances, identifying the antibody comprises: (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) screening the hybridomas for production of antibodies that specifically bind the target antibody or the antigen-binding fragment thereof or a chimeric antigen receptor comprising the antigen-binding fragment; and (iii) sequencing an antibody from a hybridoma producing an antibody that specifically binds the target antibody or antigen-binding fragment, thereby identifying the anti-idiotype antibody. In some instances, screening a hybridoma comprises determining the binding affinity of the hybridoma antibody for a target molecule comprising the target antibody or an idiotope of the target antibody, such as an scFv or a CAR or fragment thereof comprising the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions. In some instances, screening a hybridoma further comprises determining the binding affinity of the hybridoma antibody for a non-target molecule that does not comprise an idiotope of the target antibody, such as an Fc or fragment thereof or another antibody or fragment thereof that do not comprise the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions, wherein binding of the hybridoma antibody to the target molecule but not the non-target molecule indicates that the hybridoma antibody specifically binds the target antibody.

[0245] In some of any such instances, the method comprises: (a) introducing into a subject a soluble immunogen comprising an scFv of the target antibody fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that specifically binds to a molecule comprising an idiotope of the target antibody, such as the immunogen. In some instances, the scFv is within or included in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR). In some instances, the Fc domain or fragment thereof is a human IgG1 Fc or fragment thereof. In some instances, the Fc domain or fragment thereof is an Fc domain lacking the hinge region. In some instances, the Fc domain or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 32. In some instances, identifying the antibody comprises using hybridoma techniques to identify individual antibody clones produced by the subject and screening the antibody clones for binding to a molecule comprising an idiotope of the target antibody, such as the immunogen. In some instances, identifying the antibody comprises: (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) screening the hybridomas for production of antibodies that specifically bind a molecule comprising an idiotope of the target antibody, such as the immunogen; and (iii) sequencing an antibody from a hybridoma producing an antibody that specifically binds a molecule comprising an idiotope of the target antibody, such as the immunogen, thereby identifying the anti-idiotype antibody. In some instances, screening a hybridoma comprises determining the binding affinity of the hybridoma antibody for a target molecule comprising an idiotope of the target antibody, such as the immunogen, an scFv, or a CAR or fragment thereof comprising the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions. In some instances, screening a hybridoma further comprises determining the binding affinity of the hybridoma antibody for a non-target molecule that does not comprise an idiotope of the target antibody, such as an Fc or fragment thereof or another antibody or fragment thereof that does not comprise the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions, wherein binding of the hybridoma antibody to the target molecule but not the non-target molecule indicates that the hybridoma antibody specifically binds the target antibody.

[0246] In some of any such instances, the method comprises: (a) introducing into a subject a soluble immunogen comprising an scFv of the target antibody fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising an idiotope of the target antibody, such as the immunogen, an scFv, or a CAR or fragment thereof comprising the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions; and (ii) does not bind to a non-target molecule that does not comprise an idiotope of the target antibody, such as an Fc or fragment thereof or another antibody or fragment thereof that does not comprise the variable heavy chain region and / or variable light chain region of the target antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions. In some instances, the scFv is within or included in the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR). In some instances, the Fc domain or fragment thereof is a human IgG1 Fc or fragment thereof. In some instances, the Fc domain or fragment thereof is an Fc domain lacking the hinge region. In some instances, the Fc domain or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 32. In some instances, identifying the antibody comprises using hybridoma techniques to identify individual antibody clones produced by the subject and screening the antibody clones for binding to the target and non-target molecules. In some instances, identifying the antibody comprises: (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) screening the hybridomas for production of antibodies that bind to the target molecule but not to the non-target molecule; and (iii) sequencing an antibody from a hybridoma producing an antibody that binds to the target molecule but not to the non-target molecule, thereby identifying the anti-idiotype antibody. In some instances, screening a hybridoma comprises determining the binding affinity of the hybridoma antibody for the target molecule and the non-target molecule.

[0247] In some of any such instances, the method comprises: (a) introducing into a subject a soluble immunogen comprising an scFv derived from the FMC63 antibody (such as an scFv comprising the amino acid sequence of SEQ ID NO: 34) fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising an idiotope of the FMC63 antibody, such as the immunogen, an scFv, or a CAR or fragment thereof comprising the variable heavy chain region and / or variable light chain region of the FMC63 antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions; and (ii) does not bind to a non-target molecule that does not comprise an idiotope of the FMC63 antibody, such as an Fc or fragment thereof or another antibody or fragment thereof that does not comprise the variable heavy chain region and / or variable light chain region of the FMC63 antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions. In some instances, the immunogen comprises the amino acid sequence set forth in SEQ ID NO:34 and SEQ ID NO:32, optionally separated by a linker, e.g. set forth in SEQ ID NO:33. In some instances, the immunogen comprises the amino acid sequence of SEQ ID NO: 35. In some instances, the antibody identified in (b) binds to the immunogen and does not bind to either an Fc domain or fragment thereof or a molecule comprising an scFv derived from the SJ25C1 antibody (such as an scFv comprising the amino acid sequence of SEQ ID NO: 28). In some instances, the antibody identified in (b) binds to a target molecule comprising the amino acid sequence of SEQ ID NO: 34 or 35 and does not bind to either a non-target molecule comprising the amino acid sequence of SEQ ID NO: 32 or a non-target molecule comprising the amino acid sequence of SEQ ID NO: 28.

[0248] In some of any such instances, the method comprises: (a) introducing into a subject a soluble immunogen comprising an scFv derived from the SJ25C1 antibody (such as an scFv comprising the amino acid sequence of SEQ ID NO: 28) fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising an idiotope of the SJ25C1 antibody, such as the immunogen, an scFv, or a CAR or fragment thereof comprising the variable heavy chain region and / or variable light chain region of the SJ25C1 antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions; and (ii) does not bind to a non-target molecule that does not comprise an idiotope of the SJ25C1 antibody, such as an Fc or fragment thereof or another antibody or fragment thereof that does not comprise the variable heavy chain region and / or variable light chain region of the SJ25C1 antibody, or portions thereof, such as one or more of the CDRs of the VH and / or VL regions. In some instances, the immunogen comprises the amino acid sequence set forth in SEQ ID NO:28 and SEQ ID NO:32, optionally separated by a linker, e.g. set forth in SEQ ID NO:33. In some instances, the antibody identified in (b) binds to the immunogen and does not bind to either an Fc domain or fragment thereof or a molecule comprising an scFv derived from the FMC63 antibody (such as an scFv comprising the amino acid sequence of SEQ ID NO: 34). In some instances, the antibody identified in (b) binds to a target molecule comprising the amino acid sequence of SEQ ID NO: 28 and does not bind to either a non-target molecule comprising the amino acid sequence of SEQ ID NO: 32 or a non-target molecule comprising the amino acid sequence of SEQ ID NO: 34.

[0249] In some instances, identifying the antibody comprises using hybridoma techniques to identify individual antibody clones produced by the subject and screening the antibody clones for binding to target and non-target molecules. In some instances, identifying the antibody comprises: (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) screening the hybridomas for production of antibodies that bind to the target molecule but not to the non-target molecule; and (iii) sequencing an antibody from a hybridoma producing an antibody that binds to the target molecule but not to the non-target molecule, thereby identifying the anti-idiotype antibody. In some instances, screening a hybridoma comprises determining the binding affinity of the hybridoma antibody for the target molecule and the non-target molecule.

[0250] In an exemplary instance, the identification of anti-idiotype antibodies recognizing the scFv portion of an exemplary anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv derived from SJ25C1 may be generated using an immunogen comprising the sequence set forth as follows: EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDG DTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYW GQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGT NVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFC QQYNRYPYTSGGGTKLEIKR (SEQ ID NO: 28). In some instances, the immunogen may further contain an Fc domain or fragment thereof, e.g. as set forth in SEQ ID NO:32.

[0251] In an exemplary instance, the identification of anti-idiotype antibodies recognizing the scFv portion of an exemplary anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv derived from FMC63 may be generated using an immunogen comprising the sequence set forth as follows:

[0252] In some instances, the immunogen may further contain an Fc domain or fragment thereof, e.g. as set forth in SEQ ID NO:32. In some instances, the identification of anti-idiotype antibodies recognizing the scFv portion of an exemplary anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv derived from FMC63 may be generated using an immunogen comprising the sequence set forth as follows: III. CHIMERIC ANTIGEN RECEPTORS (CARS) AND GENETICALLY ENGINEERED CELLS

[0253] In some instances, the disclosed anti-idiotypic antibodies specifically bind to an antigen-binding portion of a chimeric antigen receptor (CAR), such as an anti-CD 19 CAR containing an antigen-binding portion derived from antibody SJ25C1 or FMC63. In some instances, the disclosed anti-idiotype antibodies bind to such CARs expressed on a cell, such as cells used in connection with adoptive cell therapy. In some instances, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express the recombinant or genetically engineered CAR products of such nucleic acids. In some instances, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and / or persistence in vivo, such as for use in adoptive cell therapy methods. In some instances, the disclosed anti-idiotypic antibodies can be used in methods to modulate one or more of these activities, including to activate, stimulate and / or expand engineered cells expressing the target CAR.

[0254] In some instances, the cells include one or more nucleic acids introduced via genetic engineering in accord with the disclosed methods, and thereby express recombinant or genetically engineered products of such nucleic acids. In some instances, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some instances, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types. In particular instances, the nucleic acids contain a gene that encodes a CAR.

[0255] In some instances, the disclosed methods may be carried out simultaneously, sequentially or concurrently with one or more processing steps for manufacturing or preparing genetically engineered cells. The processing steps of the methods may include any one or more of a number of cell processing steps, alone or in combination. In particular instances, the processing steps include transduction or transfection of the cells with one or more nucleic acids, e.g., a heterologous polynucleotide comprising a gene encoding a recombinant receptor. In certain instances, cells are transduced with viral vector particles containing a retroviral vector, such as one encoding a recombinant product for expression in the cells. In certain instances, the cells are transfected with one or more non-viral nucleic acids, e.g., an episomal plasmid or a transposon. The methods may further and / or alternatively include other processing steps, such as steps for the isolation, separation, selection, washing, suspension, dilution, concentration, and / or formulation of the cells. In some cases, the methods also can include an ex vivo step for cultivation, stimulation or expansion of cells (e.g., stimulation of the cells, for example, to induce their proliferation and / or activation), which, in some cases, can be carried out in accord with the disclosed methods. In some instances, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0256] In some instances, the method includes processing steps carried out in an order in which: cells, e.g., primary cells, are first isolated, such as selected or separated, from a biological sample; selected cells are incubated with viral vector particles for transduction; and transduced cells are formulated in a composition. In some cases, transduced cells are activated, expanded or propagated ex vivo, such as by stimulation in the presence of a stimulation reagent, such as in accord with the disclosed methods. In some instances, the method can include one or more processing steps from among washing, suspending, diluting and / or concentrating cells, which can occur prior to, during, or simultaneous with or subsequent to one or more of the isolation, such as separation or selection, transduction, stimulation, and / or formulation steps.

[0257] In particular instances, the cells to be transfected or transduced are not isolated, selected, or enriched prior to contact with the one or more nucleic acids. In some instances, the cells are not selected prior to contacting the cells with the one or more nucleic acids. In some instances, the cells to be transfected or transduced are not enriched prior to contacting the cells with the one or more nucleic acids.

[0258] In some instances, one or more of the cell processing steps in connection with preparing, processing and / or incubating cells in connection with the disclosed method, including in connection with preparing a composition containing genetically engineered cells, can be carried out in the internal cavity of a centrifugal chamber, such as a substantially rigid chamber that is generally cylindrical in shape and rotatable around an axis of rotation, which can provide certain advantages compared to other available methods. In some instances, all processing steps are carried out in the same centrifugal chamber. In some instances, one or more processing steps are carried out in different centrifugal chambers, such as multiple centrifugal chambers of the same type. Such methods include any of those as described in International Publication Number WO2016 / 073602.

[0259] Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax ®< and Sepax ®< 2 system, including an A-200 / F and A-200 centrifugal chambers and various kits for use with such systems. Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008 / 0171951, and published international patent application, publication no. WO 00 / 38762. Depending on the particular process (e.g., dilution, wash, transduction, formulation), it is within the level of a skilled artisan to choose a particular kit that is appropriate for the process. Exemplary kits for use with such systems include, but are not limited to, single-use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.

[0260] In some instances, the system is included with and / or placed into association with other instrumentation, including instrumentation to operate, automate, control and / or monitor aspects of the various processing steps performed in the system. This instrumentation in some instances is contained within a cabinet. In some instances, the instrumentation includes a cabinet, which includes a housing containing control circuitry, a centrifuge, a cover, motors, pumps, sensors, displays, and a user interface. An exemplary device is described in US Patent No. 6,123,655, US Patent No. 6,733,433 and US 2008 / 0171951.

[0261] In some instances, the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber. In some instances, the containers, such as bags, include one or more containers, such as bags, containing the cells to be transduced or transfected and the vector particles, e.g., viral vector particles or non-viral plasmids, in the same container or separate containers, such as the same bag or separate bags. In some instances, the system further includes one or more containers, such as bags, containing medium, such as diluent and / or wash solution, which is pulled into the chamber and / or other components to dilute, resuspend, and / or wash components and / or compositions during the methods. The containers can be connected at one or more positions in the system, such as at a position corresponding to an input line, diluent line, wash line, waste line and / or output line.

[0262] In some instances, the system, such as a closed system, is sterile. In some instances, all connections of components of the system, such as between tubing line and a container via a connector, are made under sterile conditions. In some instances, connections are made under laminar flow. In some instances, connections are made using a sterile connection device that produces sterile connections, such as sterile welds, between a tubing and a container. In some instances, a sterile connection device effects connection under thermal condition high enough to maintain sterility, such as temperatures of at least 200 °C, such as at least 260 °C or 300 ° C.

[0263] In some instances, the system may be disposable, such as a single-use kit. In some instances, a single-use kit can be utilized in a plurality of cycles of a process or processes, such as at least 2, 3, 4, 5 or more times, for example, in processes that occur in a continuous or a semi-continuous manner. In some instances, the system, such as a single-use kit, is employed for processing of cells from a single patient. In aspects of the methods, the processes need not be performed in the same closed system, such as in the same centrifugal chamber, but can be performed under a different closed system, such as in a different centrifugal chamber; in some instances, such different centrifugal chambers are at the respective points in the methods placed in association with the same system, such as placed in association with the same centrifuge. In some instances, all processing steps are performed in a closed system, in which all or a subset of each one or more processing step is performed in the same or a different centrifugal chamber.A. Target Chimeric Antigen Receptors (CARs)

[0264] In some instances, the disclosed anti-idiotypic antibodies specifically bind the extracellular domain of a target CAR that contains an antigen binding domain of an antibody or antibody fragment that provides specificity for a desired antigen (e.g., tumor antigen) and which is operably linked or connected to an intracellular signaling domain. In some instances, the antigen binding domain includes the antibody SJ25C1 or an antibody fragment of portion derived from SJ25C1. In some instances, the antigen binding domain includes the antibody FMC63 or an antibody fragment of portion derived from FMC63. In some instances, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, providing a primary activation signal. In some instances, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions.

[0265] In some instances, engineered cells, such as T cells, are disclosed that express a CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some instances, the antigen is a polypeptide. In some instances, it is a carbohydrate or other molecule. In some instances, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other instances, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0266] In particular instances, the recombinant receptor, such as a chimeric receptor, contains an intracellular signaling region, which includes a cytoplasmic signaling domain (also interchangeably called an intracellular signaling domain), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, for example, a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g. a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof) that comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0267] In some instances, the chimeric receptor further contains an extracellular ligand-binding domain that specifically binds to a ligand (e.g. antigen). In some instances, the chimeric receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some instances, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some instances, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0268] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, , 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416,and / or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO / 2014055668 A1. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, US 2013 / 0149337, U.S. Patent No.: 7,446,190, U.S. Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, US 2013 / 0149337, U.S. Patent No.: 7,446,190, and U.S. Patent No.: 8,389,282.

[0269] In some instances, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some instances, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0270] In some instances, the antibody or antigen-binding portion thereof is expressed on cells as part of a CAR. Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR. In some instances, the extracellular antigen binding domain specific for an MHC-peptide complex of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some instances, such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a costimulatory receptor.

[0271] In some instances, the recombinant receptor, such as a chimeric receptor (e.g. CAR), includes a ligand-binding domain that binds, such as specifically binds, to an antigen (or a ligand). Among the antigens targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and / or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.

[0272] In some instances, the antigen (or a ligand) is a polypeptide. In some instances, it is a carbohydrate or other molecule. In some instances, the antigen (or a ligand) is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other instances, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0273] In some instances, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.

[0274] In some instances, the antigen (or a ligand) is a tumor antigen or cancer marker. In some instances, the antigen (or a ligand) is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD 171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1 (HLA-AI), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific antigen, an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by and / or characteristic of or specific for HIV, HCV, HBV, HPV, and / or other pathogens and / or oncogenic versions thereof. Antigens targeted by the receptors in some instances include antigens associated with a B cell malignancy, such as any of a number of known B cell markers. In some instances, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0275] In some instances, the antigen is a pathogen-specific antigen. In some instances, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0276] Antigens targeted by the receptors in some instances include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some instances, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In some instances, the antigen is CD19 and is specifically bound by an anti-CD19 antibody, such as SJ25C1 or an antigen-binding fragment derived from SJ25C1 or FMC63 or an antigen-binding fragment derived from FMC63.

[0277] In some instances, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some instances, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab')2, Fv or a single chain Fv fragment (scFv)). In other instances, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another instance, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.

[0278] Among the disclosed antibodies are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular instances, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0279] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0280] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain instances, a single-domain antibody is a human single-domain antibody. In some instances, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.

[0281] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some instances, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some instances, the antibody fragments are scFvs.

[0282] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some instances, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0283] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule (e.g. SJ25C1 or FMC63), generally a variable heavy (VH) chain region and / or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.

[0284] In some instances, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some instances, the antibody or fragment includes an scFv. In some instances, the scFv is derived from SJ25C1 and comprises the sequence of amino acids set forth in SEQ ID NO: 28. In some instances, the scFv is derived from FMC63 and comprises the sequence of amino acids set forth in SEQ ID NO: 34.

[0285] In some instances, the recombinant receptor such as the CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some instances, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some instances, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. In some instances, the spacer has the sequence set forth in SEQ ID NO: 150, and is encoded by the sequence set forth in SEQ ID NO: 151. In some instances, the spacer has the sequence set forth in SEQ ID NO: 152. In some instances, the spacer has the sequence set forth in SEQ ID NO: 153. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19: 3153, international patent application publication number WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US2014 / 0271635.

[0286] In some instances, the constant region or portion is of IgD. In some instances, the spacer has the sequence set forth in SEQ ID NO: 154. In some instances, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5.

[0287] In some instances, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some instances, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some instances, the transmembrane domain is fused to the extracellular domain. In some instances, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. In some instances, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some instances, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0288] In one instance, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0289] The transmembrane domain in some instances is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively the transmembrane domain in some instances is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some instances, the linkage is by linkers, spacers, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.

[0290] In some instances, the extracellular domain and transmembrane domain can be linked directly or indirectly. In some instances, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some instances, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.

[0291] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some instances, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0292] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0293] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some instances, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0294] In some instances, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some instances, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some instances, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

[0295] In some instances, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some instances, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some instances, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement, and / or any derivative or variant of such molecules, and / or any synthetic sequence that has the same functional capability.

[0296] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some instances, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other instances, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0297] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0298] In some instances, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some instances, the CAR includes a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components. In some instances, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0299] In some instances, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some instances, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cells include one or more stimulatory or activating CAR and / or a costimulatory CAR. In some instances, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and / or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0300] In certain instances, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some instances, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.

[0301] In some instances, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[0302] In some instances, the antigen receptor further includes a marker and / or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some instances, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. In instances, the tEGFR contains an amino acid sequence set forth in SEQ ID NO: 155 or 156. In some instances, the tEGFR contains an amino acid sequence with or with about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identify to the sequences set forth in SEQ ID NO: 155 or 156.

[0303] In some instances, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some instances, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as "self' by the immune system of the host into which the cells will be adoptively transferred.

[0304] In some instances, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other instances, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[0305] In some cases, CARs are referred to as first, second, and / or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.

[0306] In some instances, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some instances, the antibody or fragment includes an scFv or a single-domain V H antibody and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some instances, the chimeric antigen receptor includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0307] In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some instances, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some instances, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0308] For example, in some instances, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some instances, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such instances, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.

[0309] In some instances, the transmembrane domain of the receptor, e.g., the CAR is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1), or is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 157 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 157; in some instances, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 158 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0310] In some instances, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0311] In some instances, the intracellular signaling region comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and / or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some instances, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 159 or 160 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 159 or 160. In some instances, the intracellular region comprises an intracellular costimulatory signaling domain of 4-1BB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 161 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 161.

[0312] In some instances, the intracellular signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. In some instances, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 162, 163 or 164 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 162, 163 or 164.

[0313] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO: 150. In other instances, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a C H 2 and / or C H 3 domains. In some instances, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to C H 2 and C H 3 domains, such as set forth in SEQ ID NO: 152. In some instances, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a C H 3 domain only, such as set forth in SEQ ID NO: 153. In some instances, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

[0314] For example, in some instances, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and / or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some instances, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0315] In some instances, nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip element and / or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some instances, T cells expressing an antigen receptor (e.g. CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. U.S. Patent No. 8,802,374). In some instances, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the molecule involved in modulating a metabolic pathway and encoding the recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 131), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 130), Thosea asigna virus (T2A, e.g., SEQ ID NO: 126 or 127), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 128 or 129) as described in U.S. Patent Publication No. 20070116690.

[0316] The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and / or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some instances, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. The receptor may be another receptor such as an immunoinhibitory or costimulatory signal-promoting receptor, such as a CCR or iCAR or non-signaling receptor, e.g., for use in depletion or elimination of cells using the antibodies.B. Genetically Engineered Cells and Methods of Producing Cells

[0317] Among the cells expressing the chimeric antigen receptors are engineered cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into a composition containing the cells, such as by retroviral transduction, transfection, or transformation. Various methods for the introduction of genetically engineered components, e.g., recombinant receptors, e.g., CARs, are well known and may be used. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.1. Vectors and Methods for Genetic Engineering

[0318] Also disclosed are one or more polynucleotides (e.g., nucleic acid molecules) encoding chimeric antigen receptors (CARs), vectors for genetically engineering cells to express such CARs and methods for producing the engineered cells. In some instances, the vector contains the nucleic acid encoding the CAR. In some cases, the vector is a viral vector, such as a retroviral vector, e.g., a lentiviral vector or a gammaretroviral vector.

[0319] In some instances, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some instances, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.

[0320] In some instances, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some instances, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one instance, the gene to be expressed replaces the retroviral gag, pol and / or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, A. D. (1990) Human Gene Therapy 1: 5-14; Scarpa et al. (1991) Virology 180: 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109.

[0321] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0322] In some instances, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some instances, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0323] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Patent No. 7,446,190.

[0324] In some instances, the cells, e.g., T cells, may be transfected either during or after expansion, e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the anti-CD3 / anti-CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in form of a peptide / MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g. natural ligand of a CAR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant regions within the receptor). See, for example, Cheadle et al, "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907: 645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014). In some instances, the cells are stimulated with a disclosed anti-idiotype antibody in accord with the disclosed methods.

[0325] In some cases, a vector may be used that does not require that the cells, e.g., T cells, are activated. In some such instances, the cells may be selected and / or transduced prior to activation. Thus, the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.

[0326] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; genes to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992); see also the publications of PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Patent No. 6,040,177, at columns 14-17.2. Cells and Preparation of Cells for Genetic Engineering

[0327] Disclosed herein are cells, including engineered cells that contain a chimeric antigen receptor (CAR). Also disclosed are population of such cells and compositions containing such cells. Among the compositions are input compositions containing cells in which one or more cells is known or likely or will express a recombinant receptor capable of being recognized or bound by a binding molecule present on one or more particles to which the cells are incubated or contacted. Also among the compositions are compositions produced by the disclosed methods, including output compositions in which is contained stimulated or expanded cells, including compositions enriched for cells containing a recombinant receptor bound or recognized by the binding molecule of the particle, such as in which cells expressing the recombinant receptor, e.g. chimeric receptor, make up at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Thus, disclosed are genetically engineered cells expressing the recombinant receptors e.g., CARs.

[0328] Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also disclosed are methods for engineering, producing or generating such cells, therapeutic methods for administering the cells and compositions to subjects, e.g., patients, and methods for detecting, selecting, isolating or separating such cells.

[0329] In some instances, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some instances, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0330] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some instances, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).

[0331] The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some instances, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some instances, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0332] Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and sub-types thereof, such as stem cell memory T (T SCM ), central memory T (T CM ), effector memory T (T EM ), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0333] In some instances, the cells are natural killer (NK) cells. In some instances, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0334] In some instances, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some instances, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some instances, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0335] In some instances, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some instances, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some instances is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0336] Accordingly, the cells in some instances are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0337] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0338] In some instances, the cells are derived from cell lines, e.g., T cell lines. The cells in some instances are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.

[0339] In some instances, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0340] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets. In some instances, prior to the selection and / or enrichment of cells, the sample or the cells in the sample can be rested or held prior to further processing steps. In some instances, the sample is maintained at or held at a temperature of from or from about 2° C to 8° C for up to 48 hours, such as for up to12 hours, 24 hours or 36 hours. In certain instances, the cells are not selected and / or enriched prior to contacting the cells with the one or more nucleic acids. In some instances, the sample or the cells can be rested or held prior to contacting or incubating the cells with one or more nucleic acids. In certain instances, the sample is maintained at or held at a temperature of from or from about 2° C to 8° C for up to 48 hours, such as for up to 12 hours, 24 hours or 36 hours prior to contacting or incubating the cells with one or more nucleic acids.

[0341] In some instances, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some instances, the cells are washed with phosphate buffered saline (PBS). In some instances, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some instances, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++< / Mg ++< free PBS. In certain instances, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0342] In some instances, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0343] In some instances, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some instances, any known method for separation based on such markers may be used. In some instances, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0344] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0345] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0346] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0347] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 +< , CD62L +< , CCR7 +< , CD27 +< , CD127 +< , CD4 +< , CD8 +< , CD45RA +< , and / or CD45RO +< T cells, are isolated by positive or negative selection techniques.

[0348] For example, CD3 +< , CD28 +< T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander). In particular instances, cells are contacted with anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander) to expand CD3 +< , CD28 +< T cells prior to contacting the cells with the one or more nucleic acids. In certain instances, the cells are not contacted with anti-CD3 / anti-CD28 conjugated magnetic beads prior to contacting the cells with the one or more nucleic acids.

[0349] In some instances, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some instances, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker +< ) at a relatively higher level (marker high< ) on the positively or negatively selected cells, respectively.

[0350] In some instances, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4 +< or CD8 +< selection step is used to separate CD4 +< helper and CD8 +< cytotoxic T cells. Such CD4 +< and CD8 +< populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0351] In some instances, CD8 +< cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some instances, enrichment for central memory T (T CM ) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some instances, combining T CM -enriched CD8 +< T cells and CD4 +< T cells further enhances efficacy.

[0352] In instances, memory T cells are present in both CD62L +< and CD62L -< subsets of CD8 +< peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L -< CD8 +< and / or CD62L +< CD8 +< fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0353] In some instances, the enrichment for central memory T (T CM ) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8 +< population enriched for T CM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (T CM ) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8 +< cell population or subpopulation, also is used to generate the CD4 +< cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0354] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4 +< cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0355] CD4 +< T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4 +< lymphocytes can be obtained by standard methods. In some instances, naive CD4 +< T lymphocytes are CD45RO -< , CD45RA +< , CD62L +< , CD4 +< T cells. In some instances, central memory CD4 +< cells are CD62L +< and CD45RO +< . In some instances, effector CD4 +< cells are CD62L -< and CD45RO -< .

[0356] In one example, to enrich for CD4 +< cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some instances, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some instances, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0357] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0358] In some instances, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0359] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0360] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0361] In certain instances, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain instances, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain instances, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain instances, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0362] In some instances, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some instances, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some instances, the magnetizable particles are biodegradable.

[0363] In some instances, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain instances, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain instances, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0364] In certain instances, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and / or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009 / 072003, or US 20110003380 A1. In one example, the system is a system as described in International Publication Number WO2016 / 073602.

[0365] In some instances, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0366] In some aspects, the separation and / or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[0367] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some instances, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some instances, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some instances, the cell populations for use with the methods described herein are labeled and are retained in the column. In some instances, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[0368] In certain instances, separation and / or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraet al. (2012) Blood.1: 72-82, and Wang et al. (2012) J Immunother. 35(9): 689-701.

[0369] In some instances, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some instances, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain instances, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5): 355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0370] In some instances, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0371] In some instances, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or engineering. In some instances, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some instances, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1: 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0372] In some instances, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. The incubation and / or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some instances, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0373] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0374] In some instances, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some instances, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some instances, such agents and / or ligands may be, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some instances, the stimulating agents include IL-2, IL-15 and / or IL-7. In some aspects, the IL-2 concentration is at least about 10 units / mL.

[0375] In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al.(2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1: 72-82, and / or Wang et al. (2012) J Immunother. 35(9): 689-701.

[0376] In some instances, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some instances, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0377] In some instances, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10: 1.IV. COMPOSITIONS

[0378] Also disclosed are compositions including the binding molecules, such as antibodies, as disclosed herein, including pharmaceutical compositions and formulations. The compositions and formulations generally include one or more optional acceptable carriers or excipients.

[0379] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0380] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0381] In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and / or antibody, and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0382] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0383] In some aspects, the composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0384] Formulations of the antibodies can include lyophilized formulations and aqueous solutions.

[0385] Compositions in some instances are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0386] Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0387] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0388] The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, and the like. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0389] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.V. METHODS AND USES OF THE ANTIBODIES

[0390] In some instances, disclosed herein are methods involving the use of one or more anti-idiotype antibodies. In some aspects, disclosed herein are methods for measuring or detecting a target antibody, such as a CAR or a cell expressing a CAR, and methods for modifying the activity of the target antibody, such as the activity of a CAR or the activity of a cell expressing a CAR. In certain instances, the one or more anti-idiotype antibodies bind, detect, identify, and / or quantify the CAR and / or cells expressing the CAR. In some instances, the methods disclosed herein provide one or more steps of contacting and / or incubating the one or more anti-idiotype antibodies with a cell or a sample containing or thought to be containing cells that express a chimeric antigen receptor (CAR). In some instances, the anti-idiotype antibody is treated, incubated, and / or contacted with the composition or sample under conditions that allow for the formation of a complex between the anti-idiotype antibody and the target antibody, e.g., the CAR. In some aspects, the complex may be utilized for the purposes of detecting, isolating, and / or measuring the CAR. In some instances, the formation of the complex modifies the activity of the target antibody, e.g., the CAR, such as by stimulating receptor signaling activity, or in some instances, antagonizing the activity of the target antibody, e.g., the CAR, by preventing the association of the CAR with an antigen.A. Detection / Isolation Methods

[0391] In some instances, there are disclosed methods involving use of one or more of the anti-idiotype antibodies, and / or molecules (such as conjugates and complexes) containing one or more of such anti-idiotype antibodies, for detecting, binding, and / or isolating an antibody, e.g., a target antibody. In certain instances, the methods provide one or more steps of contacting, incubating, and / or exposing the one or more anti-idiotype antibodies to a sample and / or composition. In some instances, the sample and / or composition has, is likely to have, and / or is suspected of having a target antibody and / or antigen binding fragment thereof that is bound by and / or recognized by the one or more anti-idiotype antibodies. In certain instances, the antibody or antigen binding fragment thereof that is bound by or recognized by the one or more anti-idiotype antibodies contains one or more fusion domains and / or is a fusion protein. In certain instances, the target antibody and / or antigen binding fragment thereof is a CAR. In certain instances, the anti-idiotype antibody, binds to and / or recognizes an anti-CD19 antibody (e.g., antibody SJ25C1 or FMC63), or an antigen-binding fragment thereof, including a chimeric molecule or conjugate including a CAR, containing such anti-CD19 antibody (e.g., antibody fragment).

[0392] The methods in some instances include incubating, treating, and / or contacting a sample and / or a composition containing or suspected of containing the target antibody with the anti-idiotype antibody. In certain instances, the incubating is under conditions permissive for binding of the anti-idiotype antibody to the target antibody present in the composition, for example to form a complex containing the anti-idiotype antibody and the target antibody.

[0393] In some instances, the sample and / or composition contains or is suspected of containing the target antibody, e.g., a CAR. In certain instances, the sample and / or composition contains or is suspected of containing cells that express the target antibody, e.g., a CAR. In certain instances, the sample is a biological sample. In particular instances, the sample is a serum sample or a blood sample. In some instances, the biological sample contains one or more immune cells. In some instances, the biological sample is or is derived from a tissue, such as connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In particular instances, the biological sample is or is derived from heart, vasculature, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, hypothalamus, pituitary gland, pineal gland, thyroid, parathyroid, adrenal gland, kidney, ureter, bladder, urethra, lymphatic system, skin, muscle, brain, spinal cord, nerves, ovaries, uterus, testes, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bone, cartilage, ligaments, or tendons. In particular instances, the biological sample is taken, collected, and / or obtained from a human subject. In certain instances, the sample contains cells that are live and / or intact. In some instances, the sample is or contains a homogenate and / or cells that have been disrupted and / or lysed. In some instances, the biological sample contains proteins and / or antibodies that have been isolated from blood, serum, and / or a tissue.

[0394] In particular instances, the anti-idiotype antibody forms or is capable of forming a complex with a target antibody, e.g., a CAR. In particular instances, the complex is detected, measured, quantified, and / or assessed, for example, to allow for the detection, identification, measurement, and / or quantification of the target antibody, for example in a composition or a sample. In certain instances, the methods include detecting whether a complex is formed 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 instances, the complex contains a detectable label. In particular instances, the anti-idiotype antibody is an immunoconjugate that contains a detectable label. In certain instances, the anti-idiotype antibody contains, is conjugated with, bound to, and / or attached to the detectable label. In some instances, the complex contains an antibody that binds to and / or recognizes the anti-idiotype antibody, e.g., a secondary antibody, that in conjugated with, bound to, and / or attached to a detectable label.

[0395] In some instances, methods for detecting, quantifying, detecting, and / or assessing a target antibody, for example in a sample or composition, includes detecting a complex of the target antibody and the anti-idiotype antibody. In some instances, the complex contains a detectable label. In certain instances, the complex is probed and / or contacted with a detectable label. In some instances, 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.

[0396] In some instances, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell. In particular instances, target antibody, e.g., the CAR is not bound or contained within a cell, for example, in some instances, the target antibody is secreted. In certain instances, the antibody has been detached, removed, and / or lysed from the surface of a cell.

[0397] In some instances, the target antibody or antigen-binding fragment is contained in a chimeric antigen receptor (CAR), such as a CAR expressed on the surface of a cell. In some instances, the cell is a stem cell, e.g., an iPSC, or an immune cell. In some instances, the immune cell is a T cell, e.g., a CD4+ T cell, a CD8+ T cell, naive T (T N ) cell, effector T cell (T EFF ), memory T cell, tumor-infiltrating lymphocyte (TIL), immature T cell, mature T cell, helper T cells, cytotoxic T cell, mucosa-associated invariant T (MAIT) cell, naturally occurring and adaptive regulatory T (Treg) cell, helper T cell, such as a TH1 cell, TH2 cell, TH3 cell, TH17 cell, TH9 cell, TH22 cell, follicular helper T cell, alpha / beta T cell, and / or a delta / gamma T cells. In some instances, the cell is from a tissue, e.g., heart, vasculature, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, hypothalamus, pituitary gland, pineal gland, thyroid, parathyroid, adrenal gland, kidney, ureter, bladder, urethra, lymphatic system, skin, muscle, brain, spinal cord, nerves, ovaries, uterus, testes, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bone, cartilage, ligaments, or tendons.

[0398] In some instances, the target antibody is an anti-CD19 antibody. In some instances, the target antibody is or is derived from antibody SJ25C1 or an antigen-binding fragment thereof. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody is or is derived from antibody FMC63 or an antigen-binding fragment thereof. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0399] In some instances, there is disclosed a method of detecting a target antibody, such as antibody SJ25C1 or FMC63, or an antigen-binding fragment thereof (and / or chimeric molecules comprising such antibody, e.g., antibody fragment, such as a CAR), comprising contacting a composition comprising the target antibody or antigen-binding fragment with an anti-idiotype antibody or antigen-binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment. In some instances, the method further includes detecting whether a complex is formed between the anti-idiotype antibody and the target antibody in the composition, such as detecting the presence or absence or level of such binding. In some instances, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell and the detecting comprises detecting cells bound with the anti-idiotype antibody. In some instances, the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0400] In some instances, there is disclosed a method of isolating a target antibody, such as antibody SJ25C1 or FMC63, or an antigen-binding fragment thereof (and / or chimeric molecules comprising such antibody, e.g., antibody fragment, such as a CAR), comprising contacting a composition and / or a sample containing or suspected of containing the target antibody or antigen-binding fragment with an anti-idiotype antibody or antigen-binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and isolating complexes comprising the anti-idiotype antibody bound to the target antibody or antigen-binding fragment. In certain instances, the target antibody is isolated with an anti-idiotype antibody or antigen binding fragment thereof is an immunoconjugate, such as an immunoconjugate described in Section I-F. In some instances, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell and the isolating comprises isolating cells bound with the anti-idiotype antibody. In some instances, the complexes comprising the anti-idiotype antibody are isolated by affinity-based separation. In some instances, the affinity-based separation is selected from the group consisting of immunoaffinity-based separation, magnetic-based separation, and affinity chromatography. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0401] In some instances, there is disclosed a method of detecting a cell expressing a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen-binding fragment thereof, comprising contacting the cell expressing the CAR with an anti-idiotype antibody or antigen-binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and detecting cells bound with the anti-idiotype antibody. In some instances, the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0402] In certain instances, the methods for detecting a target antibody with an anti-idiotype antibody described herein are used to assess the target antibody in a subject. For example, in some instances, disclosed herein are methods of use for the anti-idiotype antibody for assessing, measuring, and / or quantifying the in vivo pharmacokinetics, expansion, and / or persistence of a CAR expressing cells of a therapeutic cell composition. In some instances, the in vivo pharmacokinetics, expansion, and / or persistence of the cells, and / or changes in cell phenotypes or functional activity of cells, such as CAR expressing cells administered for immunotherapy, e.g. CAR-T cell therapy, in the methods disclosed herein, can be measured with the anti-idiotype antibodies disclosed herein. In some instances, the pharmacokinetics, expansion, and / or persistence of the CAR expressing cells are measured, assessed by detecting the presence and / or amount of cells expressing the CAR in the subject and / or in sample obtained from the subject following the administration of the therapeutic cell composition during and / or after the administration of the therapy with an anti-idiotype antibody disclosed herein.

[0403] In some aspects, the anti-idiotype antibody is used with flow cytometry to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells administered for T cell based therapy) in the blood or serum or organ or tissue sample (e.g., disease site, e.g., tumor sample) of the subject. In some aspects, persistence is quantified as the number of CAR-expressing cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample. In certain aspects, expansion is quantified as the increase in the number of CAR-expressing cells per microliter between samples, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the samples over time. In some instances, the pharmacokinetics, expansion, and / or persistence are measured or assessed by detecting the amount of CAR expressing cells in the subject and / or in samples collected from the subject at multiple time points. In certain instances, the one or more samples are collected, obtained, and / or taken from the subject within 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 6 months, one year, or over one year after the therapeutic cell composition is administered.

[0404] In some instances, there is disclosed a method of selecting cells expressing a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen-binding fragment thereof, comprising contacting a population of cells comprising cells expressing the CAR with an anti-idiotype antibody or antigen-binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and selecting cells bound with the anti-idiotype antibody. In some instances, the cells bound with the anti-idiotype antibody are selected by affinity-based separation. In some instances, the affinity-based separation is selected from the group consisting of immunoaffinity-based separation, flow cytometry, magnetic-based separation, and affinity chromatography. In some instances, the anti-idiotype antibody or antigen-binding fragment thereof or anti-idiotype antibody immunoconjugate is reversibly bound or immobilized to a support or a stationary phase. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0405] In some instances, there is disclosed a method of validating a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen-binding fragment thereof, comprising a) incubating a sample comprising T cells transduced with the CAR with an anti-idiotype antibody or antigen-binding fragment thereof targeting the CAR; b) determining the percent of cells bound with the anti-idiotype antibody or antigen-binding fragment thereof; and c) validating the CAR based on the percent of anti-idiotype antibody-bound T cells. In some instances, the anti-idiotype antibody is labeled, and anti-idiotype antibody-bound T cells are assayed by flow cytometry. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 23 and / or a light chain variable region set forth in SEQ ID NO: 24. In some instances, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 30 and / or a light chain variable region set forth in SEQ ID NO: 31.

[0406] Also disclosed are methods involving use of the disclosed anti-idiotype antibodies, and molecules (such as conjugates and complexes) containing one or more of such anti-idiotype antibodies, for informing treatment decisions in an individual, such as by the detection of CARs recognized by the anti-idiotype antibody, such as CARs comprising a target antibody, such as an anti-CD19 antibody (e.g., antibody SJ25C1 or FMC63), or an antigen-binding fragment thereof. In some instances, the methods are for informing treatment decisions in an individual in association with a therapy comprising administration of CAR T cells, such as anti-CD19 CAR T cells. The methods in some instances include incubating and / or probing a biological sample with the anti-idiotype antibody and / or administering the anti-idiotype antibody to the individual. In certain instances, a biological sample includes a cell or tissue or portion thereof, such as tumor or cancer tissue or biopsy or section thereof. In certain instances, the incubating is under conditions permissive for binding of the anti-idiotype antibody to CARs present in the sample. In some instances, the methods further include detecting whether a complex is formed between the anti-idiotype antibody and CARs in the sample, such as detecting the presence or absence or level of such binding. Such a method may be an in vitro or in vivo method.

[0407] In one instance, an anti-idiotype antibody is used to determine whether adjustment to a CAR T cell therapy in an individual is necessary, e.g. where low levels of the CAR T cells in the individual indicate the need to adjust the therapy. In some instances, the target antibody is an anti-CD19...

Claims

1. An anti-idiotype antibody or antigen-binding fragment thereof comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 89 or 90, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, 92 or 93, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 94; and a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 95, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 96, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 97, wherein: (i) the anti-idiotype antibody or antigen-binding fragment thereof specifically binds to a target antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31; and (ii) the anti-idiotype antibody or antigen-binding fragment thereof is an agonist antibody of a chimeric antigen receptor (CAR) comprising the target antibody or antigen-binding fragment.

2. The anti-idiotype antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises: a light chain variable (VL) region comprising at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 40; and a heavy chain variable (VH) region comprising at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 36.

3. The anti-idiotype antibody or antigen-binding fragment thereof of claim 1 or claim 2, wherein: the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 36; and the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 40.

4. The anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-3, which is humanized.

5. The anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-4, which is an antigen-binding fragment, optionally wherein the antigen-binding fragment is selected from among fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, or a single chain variable fragment (scFv).

6. The anti-idiotype antibody or antigen-binding fragment of any one of claims 1-4, which is a full-length antibody.

7. The anti-idiotype antibody or antigen-binding fragment thereof of any of claims 1-6, wherein the antibody or fragment specifically binds to a single chain variable fragment (scFv) comprised in the extracellular portion of a chimeric antigen receptor, wherein the scFv comprises a heavy chain variable region set forth in SEQ ID NO: 30 and a light chain variable region set forth in SEQ ID NO: 31; and / or comprises the sequence of amino acids set forth in SEQ ID NO: 34.

8. A conjugate, comprising the anti-idiotype antibody or antigen-binding fragment of any one of claims 1-7 and a heterologous molecule or moiety, optionally wherein the heterologous molecule or moiety is: i) a label, optionally wherein the label is selected from a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a streptavidin, a biotin, a luminescent compound or an oligonucleotide; or ii) a protein, peptide, nucleic acid or small molecule, which optionally is or comprises a toxin or a Strep-Tag.

9. A nucleic acid molecule encoding the heavy chain and light chain of the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-7.

10. A vector, comprising the nucleic acid molecule of claim 9.

11. A cell, comprising the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-7 or the nucleic acid molecule of claim 9.

12. A method of producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising expressing the heavy and light chain encoded by the nucleic acid molecule of claim 9 or the vector of claim 10 in a suitable host cell and recovering or isolating the antibody.

13. A composition comprising the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-7, conjugate of claim 8, or the cell of claim 11, optionally further comprising a pharmaceutically acceptable excipient.

14. A method of detecting a target antibody or antigen-binding fragment thereof, comprising: (a) contacting a composition comprising a target antibody or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-7 or the conjugate of claim 8 that specifically binds to the target antibody or antigen-binding fragment thereof, wherein the target antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31; and (b) detecting the anti-idiotype antibody bound to the target antibody or antigen-binding fragment, optionally wherein the target antibody or antigen-binding fragment is expressed on the surface of a cell, wherein the cell expresses on its surface a CAR comprising the target antibody or antigen-binding fragment, and detecting in (b) comprises detecting cells bound with the anti-idiotype antibody.

15. An in vitro or ex vivo method of stimulating cells, comprising incubating an input composition comprising cells expressing a chimeric antigen receptor (CAR) comprising a target antibody or an antigen-binding fragment thereof with the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-7 or the conjugate of claim 8 that specifically binds to the target antibody or antigen-binding fragment thereof, thereby generating an output composition comprising stimulated cells, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable (VH) region sequence set forth in SEQ ID NO: 30 and the light chain variable (VL) region sequence set forth in SEQ ID NO: 31.

16. The method of claim 15, wherein the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, which optionally comprises or is conjugated to a reagent comprising a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof, optionally wherein the reagent comprises a streptavidin mutein; or wherein the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble reagent, which optionally is or comprises a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof, optionally wherein the reagent comprises a streptavidin mutein.

17. The method of claim 15 or claim 16, wherein the incubation is for at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36, 48 hours, 72 hours or 96 hours, and / or wherein the input composition comprises less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% CAR-expressing cells as a percentage of the total cells in the composition.

18. The method of any one of claims 15-17, wherein: the number of CAR-expressing cells in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold or more compared to the number of CAR-expressing cells in the input composition; and / or the percentage of CAR-expressing in the output composition compared to the total cells in the composition is increased by greater than 10 %, 20 %, 40 %, 50 %, 60 %, 70 %, 80 % or more; and / or prior to the introducing and / or incubating the cells are not selected or enriched for CAR-expressing cells.