RU2773355C2 - Anti-idiotypic antibodies and methods related to them - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to an anti-idiotypic antibody or its antigen-binding fragment, its production method, as well as to a conjugate containing it, a composition, and a kit. A nucleic acid molecule encoding a heavy chain and/or a light chain of the above-mentioned antibody or its fragment, as well as a vector and a cell containing it are also disclosed. The invention also relates to a purification method, a detection method, a selection method, a method for the production of a cell composition, as well as to a depletion method and to a stimulation method, providing for the use of the above-mentioned antibody or its fragment.

EFFECT: invention is effective for the treatment of a disease or a disorder, in which CD19 is expressed.

52 cl, 15 dwg, 3 tbl, 11 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims priority to U.S. Provisional Application 62/369008, filed July 29, 2016, entitled "ANTIBODIES AND RELATED METHODS", the contents of which are hereby incorporated by reference in their entirety for all purposes.

Inclusion as a link to a sequence listing

This application is filed with the sequence listing in electronic format. The sequence list is provided as a 735042006540SeqList.TXT file created on July 15, 2017, which is 86050 bytes in size. Information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

The field of technology to which the invention relates

The present disclosure relates in some aspects to anti-idiotypic antibodies that specifically recognize anti-CD19 antibody fragments, in particular anti-CD19 antibody fragments present in recombinant receptors, including chimeric antigen receptors (CARs). The description further relates to the use of anti-idiotypic antibodies to specifically identify or select cells expressing such recombinant receptors, such as anti-CD19 CAR T cells. The description further relates to the use of anti-idiotypic antibodies to specifically activate such cells.

State of the art

Adoptive cell therapy methods are available using engineered cells expressing recombinant receptors such as a chimeric antigen receptor (CAR) containing extracellular antigen-binding domains of antibodies. Various strategies are available to evaluate the activity of such cells in a patient, either in vitro or in vivo . Improved methods are needed to specifically assess the activity of CAR-expressing cells. The present description relates to reagents, compositions and products that meet such needs.

The essence of the invention

Provided herein are agents that specifically bind to antibodies, including antibody fragments such as scFv and chimeric molecules containing them, such as chimeric antigen receptors. Compositions and articles containing such agents are also provided, including those that include a surface to which the agent is associated, such as a hard surface, such as a tablet or microsphere. 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 in therapies involving or suspected to include an antibody or chimeric molecule, such as for example, when detecting, stimulating or using CAR-expressing cells.

In some aspects, the antibody is or includes an anti-idiotypic antibody, or an antigen-binding fragment thereof, that specifically binds to a target antibody that is or contains the variable region(s) of an antibody designated SJ25C1 and/or an antigen-binding fragment thereof. In some embodiments, the agent, e.g., an anti-idiotypic antibody or antigen-binding fragment, contains a light chain variable region (VL) that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity, or 100% identity) with the amino acid sequence of the VL region of SEQ ID NO: 5; and/or a heavy chain variable region (VH) that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity or 100% identity) to the amino acid sequence of the VH region of SEQ ID NO: 1.

Provided herein is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a VL region that is at least 90% sequence identity (and/or at least 95% or 99% sequence identity or 100% sequence identity). ) with the amino acid sequence of the VL region of SEQ ID NO: 5; and/or a VH region that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity or 100% identity) with the amino acid sequence of the VH region of SEQ ID NO: 1.

In some of any such embodiments, the VH region comprises a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 11 or 84, or comprising a CDR-H3 contained in the VH sequence of SEQ ID NO: 1; and/or the VL region contains a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 14 or 87, or containing a CDR-L3 contained in the VL sequence of SEQ ID NO: 5.

In some of any such embodiments, the VH region comprises CDR-H1 and CDR-H2, respectively, comprising the amino acid sequences of CDR-H1 and CDR-H2 contained in the VH region with the amino acid sequence of SEQ ID NO: 1; and/or the VL region contains CDR-L1 and CDR-L2, respectively, containing the amino acid sequences of CDR-L1 and CDR-L2 contained in the VL region with the amino acid sequence of SEQ ID NO: 5.

In some of any such embodiments, the VH region comprises a CDR-H1 of SEQ ID NO: 9, 78, 79, or 80, a CDR-H2 of SEQ ID NO: 10, 81, 82, or 83, and a CDR-H3 of SEQ ID NO: 11 or 84; and/or the VL region contains CDR-L1 with SEQ ID NO: 12 or 85, CDR-L2 with SEQ ID NO: 13 or 86, and CDR-L3 with SEQ ID NO: 14 or 87.

The present description provides an anti-idiotypic antibody or its antigen-binding fragment containing CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence SEQ ID NO: 1; and/or CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 5.

The present description provides an anti-idiotypic antibody or an antigen-binding fragment thereof, comprising a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, 78, 79, or 80, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, 81, 82, or 83, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 11 or 84; and/or a CDR-L1 containing the amino acid sequence of SEQ ID NO: 12 or 85, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 13 or 86, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 14 or 87.

In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 1; and/or the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 1, and the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 5. In some of any such embodiments, the target antibody or antigen binding fragment comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24.

In some embodiments, the agent is an anti-idiotypic antibody, or an antigen-binding fragment thereof, that specifically binds to a target antibody that is or contains the variable region(s) of an antibody designated FMC63, or an antigen-binding fragment thereof, and/or specifically binds to a chimeric molecule containing such an antibody fragment, such as a CAR with a binding domain containing antibody variable regions or portions thereof, derived from FMC63, such as in the form of scFv. In some embodiments, the agent, eg, an anti-idiotypic antibody or antigen-binding fragment, comprises a light chain variable region (VL) that is at least 90% identical to the amino acid sequence of the VL region of SEQ ID NO: 40 or 62; and/or a heavy chain variable region (VH) that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity or 100% identity) to the amino acid sequence of the VH region of SEQ ID NO: 36 or 58. In some embodiments, the antibody or antigen-binding fragment comprises a VL region that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity or 100% identity) with the amino acid sequence of the SEQ VL region. ID NO: 40 or 62; and/or a VH region that is at least 90% sequence identical (and/or at least 95% or 99% sequence identity or 100% identity) with the amino acid sequence of the VH region of SEQ ID NO: 36 or 58.

In some of any such embodiments, the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence GYX ₃ FX ₅ X ₆ YX ₈ MX ₁₀ (SEQ ID NO: 108), where 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; heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence WIGX ₄ IX ₆ PX ₈ X ₉ X ₁₀ X ₁₁ TX ₁₃ X ₁₄ NQX ₁₇ FKX ₂₀ (SEQ ID NO: 109), where X ₄ is D or M, X ₆ is N or 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; heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence 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 ), where 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 none , X ₈ is G or absent, X ₉ is S or absent, X ₁₀ is R or absent, X ₁₁ is D or absent, X ₁₂ is A or absent, X ₁₃ is M or absent, X ₁₄ is D or E and X ₁₅ is Y or A; and/or the VL region contains a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence X ₁ AX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ YX ₁₀ X ₁₁ WY (SEQ ID NO: 111), where 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 absent, X ₁₀ is M or L, and X ₁₁ is Y or A; light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence X ₁ X ₂ X ₃ YX ₅ X ₆ X ₇ X ₈ LAX ₁₁ (SEQ ID NO: 112), where 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; light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence QX ₂ X ₃ X ₄ X ₅ X ₆ PX ₈ T (SEQ ID NO: 113), where 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.

In some embodiments, complementarity determining region 3 (CDR-H3) contains the amino acid sequence of SEQ ID NO: 94 or 104, or contains a CDR-H3 contained in the VH sequence of SEQ ID NO: 36 or 58; and/or light chain complementarity determining region 3 (CDR-L3) contains the amino acid sequence of SEQ ID NO: 97 or 107, or contains a CDR-L3 contained in the VL sequence of SEQ ID NO: 40 or 62.

In some embodiments, the VH region comprises CDR-H1 and CDR-H2, respectively, including the amino acid sequences of CDR-H1 and CDR-H2 contained in the VH region with the amino acid sequence of SEQ ID NO: 36 or 58; and/or the VL region contains CDR-L1 and CDR-L2, respectively, including the amino acid sequences of CDR-L1 and CDR-L2 contained in the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

In some of any such embodiments, the VH region comprises a CDR-H1 of SEQ ID NO: 88, 89, 90, 98, 99, or 100, a CDR-H2 of SEQ ID NO: 91, 92, 93, 101, 102, or 103, and CDR-H3 c SEQ ID NO: 94 or 104; and/or the VL region contains CDR-L1 with SEQ ID NO: 95 or 105, CDR-L2 with SEQ ID NO: 96 or 106, and CDR-L3 with SEQ ID NO: 97 or 107

The present description provides an anti-idiotypic antibody or its antigen-binding fragment containing CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence SEQ ID NO: 36 or 58; and/or CDR-L1, CDR-L2 and CDR-L3, respectively, including the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

The present description provides an anti-idiotypic antibody or an antigen-binding fragment thereof containing a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 88, 89, 90, 98, 99 or 100, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, 92, 93, 101, 102 or 103, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 94 or 104; and/or a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 95 or 105, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 96 or 106, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 97 or 107.

In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 36 or 58; and/or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 40 or 62. In some instances, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 36 or 58 and the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO : 40 or 62.

In some of any such embodiments, the VH region comprises a CDR-H1 of SEQ ID NO: 44, 88, 89, or 90, a CDR-H2 of SEQ ID NO: 45, 91, 92, or 93, and a CDR-H3 of SEQ ID NO: 46 or 94; and/or the VL region comprises a CDR-L1 of SEQ ID NO: 47 or 95, a CDR-L2 of SEQ ID NO: 48 or 96, and a CDR-L3 of SEQ ID NO: 49 or 97. In some of any such embodiments, the VH region contains CDR-H1 with SEQ ID NO: 65, 98, 99 or 100, CDR-H2 with SEQ ID NO: 66, 101, 102 or 103, and CDR-H3 with SEQ ID NO: 67 or 104; and/or the VL region contains CDR-L1 with SEQ ID NO: 68 or 105, CDR-L2 with SEQ ID NO: 69 or 106, and CDR-L3 with SEQ ID NO: 100 or 107.

In some of any such embodiments, the VH region comprises CDR-H1, CDR-H2, and CDR-H3, including the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 36; and/or the VL region contains CDR-L1, CDR-L2 and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 40.

In some of any such embodiments, the VH region comprises CDR-H1, CDR-H2, and CDR-H3, including the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 58; and/or the VL region contains CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 62.

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. In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 58; and/or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 62.

In some of any such embodiments, the target antibody or antigen binding fragment is a single chain fragment. In some aspects, the fragment contains the variable regions of the antibody connected by a flexible linker. In some of any such embodiments, the fragment comprises an scFv.

In some of any such embodiments, the target antibody or antigen binding fragment comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24; and/or is an scFv comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the target antibody or antigen binding fragment comprises the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31; and/or is an scFv containing the amino acid sequence of SEQ ID NO: 34.

In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof specifically binds to the same or overlapping epitope of the target antibody or antigen-binding fragment thereof as the epitope specifically bound to the anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments described herein.

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

In some of any such embodiments, the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from the SJ25C1 antibody contained in the extracellular portion of the chimeric antigen receptor, wherein optionally the scFv derived from the SJ25C1 antibody contains the heavy chain variable region of SEQ ID NO : 23, and/or light chain variable region of SEQ ID NO: 24; and/or contains the amino acid sequence of SEQ ID NO: 28. In some of any such embodiments, the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from the FMC63 antibody contained in the extracellular portion of the chimeric antigen receptor, where optionally the scFv derived from the FMC63 antibody, contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31; and/or contains the amino acid sequence of SEQ ID NO: 34.

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

In some of any such embodiments, the CAR further comprises a transmembrane domain linked to an antigen binding domain via a spacer. In some embodiments, the spacer contains an extracellular portion of CD28, which is not necessarily human CD28. In some aspects, the extracellular portion of CD28 comprises the amino acid sequence of SEQ ID NO: 27. In some of any such embodiments, the transmembrane domain comprises a transmembrane portion of CD28, which is optionally human CD28. In some of any such embodiments, the antibody or fragment thereof does not bind to an epitope in the CAR spacer domain.

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

In some of any such embodiments, the target antibody, or antigen-binding fragment thereof, specifically binds to human CD19. In some of any such embodiments, the anti-idiotypic antibody or fragment thereof does not cross-react with another anti-CD19 antibody that is optionally contained in the extracellular antigen-binding domain of another CAR. In some of any such embodiments, the anti-idiotypic antibody or fragment does not cross-react with another CAR.

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

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

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

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

Some of any such embodiments provide a conjugate comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any of the embodiments described above and a heterologous molecule or fragment. In some embodiments, the heterologous molecule or fragment is a label. In some aspects, the label is selected from a fluorescent dye, fluorescent protein, radioisotope, chromophore, metal ion, gold particle, silver particle, magnetic particle, polypeptide, enzyme, streptavidin, biotin, luminescent compound, or oligonucleotide. In some cases, the heterologous molecule or fragment is a protein, peptide, nucleic acid, or small molecule, which optionally is or contains a toxin, a Strep tag.

In some embodiments, a nucleic acid molecule(s) is provided that encodes the heavy chain and/or light chain of an anti-idiotypic antibody or antigen-binding fragment thereof, according to any of the embodiments described herein. In some aspects, the nucleic acid molecule comprises a nucleotide sequence encoding (i) a heavy chain variable region of SEQ ID NO: 15, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 15; or (iii) a degenerate sequence of (i) or (ii); and/or a nucleotide sequence encoding (iv) a light chain variable region of SEQ ID NO: 19, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 19; or (vi) a degenerate sequence (iv) or (v).

In some of any such embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding (i) a heavy chain of SEQ ID NO: 17, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 17 ; or (iii) a degenerate sequence of (i) or (ii); and/or a nucleotide sequence encoding (iv) a light chain of SEQ ID NO: 21, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 21; or (vi) a degenerate sequence (iv) or (v).

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding (i) a heavy chain variable region of SEQ ID NO: 50 or 71, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 50 or 71; or (iii) a degenerate sequence of (i) or (ii); and/or a nucleotide sequence encoding (iv) a light chain variable region of SEQ ID NO: 54 or 75, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 54 or 75; or (vi) a degenerate sequence (iv) or (v). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding (i) a heavy chain of SEQ ID NO: 52 or 73, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 52, or 73; or (iii) a degenerate sequence of (i) or (ii); and/or a nucleotide sequence encoding (iv) a light chain of SEQ ID NO: 56 or 76, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 56 or 76; or (vi) a degenerate sequence (iv) or (v). In some embodiments, the nucleotide sequence encoding the heavy chain and/or light chain contains a signal sequence.

The present description provides a vector containing a nucleic acid molecule in accordance with any of the embodiments described herein. Also provided herein is a cell comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any of the embodiments described herein, or a nucleic acid molecule according to any of the embodiments described herein.

The present description provides a method for producing an anti-idiotypic antibody or an antigen-binding fragment thereof, comprising the expression of a heavy and/or light chain encoded by a nucleic acid molecule in accordance with any of the embodiments described herein, or a vector in accordance with any of the embodiments. described herein in a suitable host cell, and recovery or isolation of the antibody. In some embodiments, a method for producing an anti-idiotypic antibody or antigen-binding fragment comprises culturing a cell according to any of the embodiments described herein under conditions under which the heavy chain and/or light chain is expressed, and recovering or isolating the antibody. Also provided herein is an anti-idiotypic antibody, or an antigen-binding fragment thereof, produced by a method according to any of the embodiments described herein.

In some embodiments, a composition is provided comprising an anti-idiotype antibody or an antigen-binding fragment thereof according to any of the embodiments described herein, a conjugate according to any of the embodiments described herein, or a cell according to any of embodiments described in this document. In some of any such embodiments, the composition further comprises a pharmaceutically acceptable excipient.

In some embodiments, a kit is provided comprising one or more of an anti-idiotypic antibody or an antigen-binding fragment thereof according to any of the embodiments described herein, a conjugate according to any of the embodiments described herein, a nucleic acid in in accordance with any of the embodiments described herein and, optionally, instructions for use. In some cases, the kit additionally contains a reagent or substrate for immobilizing an anti-idiotypic antibody or its antigen-binding fragment or conjugate, where the specified reagent or substrate is a microsphere, column, microwell, rod, filter, strip, or soluble streptavidin mutein oligomeric reagent.

Detection methods using any of the proposed agents, such as anti-idiotypic antibodies, are also provided. In some embodiments, a method is provided for detecting a target antibody or antigen-binding fragment thereof, such as a CAR containing them, the method comprising (a) contacting a composition containing the target antibody (such as an antibody containing variable regions derived from SJ25C1 antibodies or from an FMC63 antibody or from an antigen-binding fragment of any of such antibodies) with an anti-idiotypic antibody or an antigen-binding fragment thereof; and (b) detecting an anti-idiotypic antibody bound to the target antibody or antigen-binding fragment thereof and/or detecting the presence or absence of the target antibody or agent.

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

In some aspects, the target antibody, or antigen-binding fragment thereof, is associated with or expressed on the cell surface, and the detection in (b) includes detection of cells associated with the anti-idiotypic antibody. In some cases, a cell expresses on its surface a CAR containing the target antibody or its antigen-binding fragment.

In some embodiments, the proposed methods include detecting a CAR containing a target antibody or antigen-binding fragment of any of the embodiments, such as a CAR containing variable domains derived from FMC63 or SJ25C. In some aspects, the methods include (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof, in accordance with any of the described embodiments, or with a conjugate according to any of the described embodiments, which specifically binds to a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof; and (b) detecting cells associated with the anti-idiotypic antibody. In some embodiments, the method includes (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an FMC63 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof, according to any of the described embodiments or with a conjugate according to any of the described embodiments that specifically binds to a target antibody, which is an FMC63 antibody or an antigen-binding fragment thereof; and (b) detecting cells associated with the anti-idiotypic antibody. In some of any such embodiments, the anti-idiotypic antibody, or antigen-binding fragment thereof, is directly or indirectly labeled for detection.

In some embodiments, a method is provided for selecting cells from a cell population, comprising (a) contacting a population of cells expressing a chimeric antigen receptor (CAR) containing a target antibody, or a cell bound to a target antibody, with an anti-idiotypic antibody or antigen-binding antibody thereof. a fragment according to any of the described embodiments or with a conjugate according to any of the described embodiments that specifically binds to a target antibody, which is an SJ25C1 antibody or an antigen-binding fragment thereof, where the target antibody is an SJ25C1 antibody or its antigen-binding fragment; and (b) selecting cells associated with the anti-idiotypic antibody. In some embodiments, the method includes (a) contacting a population of cells expressing a chimeric antigen receptor (CAR) containing the target antibody, or cells associated with the target antibody, with an anti-idiotypic antibody or antigen-binding fragment thereof, in accordance with any of the described embodiments, or with a conjugate according to any of the embodiments described, which specifically binds to a target antibody that is an FMC63 antibody, or an antigen-binding fragment thereof, wherein the target antibody is an FMC63 antibody, or an antigen-binding fragment thereof; and (b) selecting cells associated with the anti-idiotypic antibody.

In some instances, cells bound to the anti-idiotypic antibody are selected by affinity separation. In some aspects, the affinity separation is an immunoaffinity separation. In some of any such embodiments, affinity separation is performed by flow cytometry. In some embodiments, affinity separation is performed by magnetically activated cell sorting. In some aspects, the affinity separation comprises affinity chromatography. In some of any such embodiments, the anti-idiotypic antibody is reversibly bound or immobilized on a support or stationary phase.

Also included are methods of stimulating cells using agents, such as stimulating cells containing a molecule, such as CAR, that is or contains a target antibody recognized by an anti-idiotypic antibody. In some aspects, the methods include incubating an initial composition containing cells expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an SJ25C1 antibody or antigen-binding fragment, with an anti-idiotypic antibody, or antigen-binding fragment thereof, in accordance with any of described embodiments, or with a conjugate according to any of the described embodiments, which specifically binds to the target antibody, which is the SJ25C1 antibody or antigen-binding fragment, thereby generating the final composition containing stimulated cells. In some embodiments, the method includes incubating an initial composition containing cells expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an FMC63 antibody or antigen-binding fragment thereof, with an anti-idiotypic antibody or antigen-binding fragment thereof, in accordance with any from the described embodiments, or with a conjugate according to any of the described embodiments, which specifically binds to the target antibody, which is the FMC63 antibody or antigen-binding fragment, thereby generating the final composition containing stimulated cells.

In some embodiments, the methods result in the proliferation, activation, stimulation, release of cytokines, or other functional outcomes, such as upregulation of an activation marker, or the release or production of cytokines, of cells expressing a chimeric receptor, such as CAR, recognized by an anti-Id antibody. In some aspects, such proliferation or other functional response or findings are induced in such cells to a degree similar to or greater than that induced by incubation of cells with an agent and/or under conditions that stimulate T cell proliferation, such as incubation with microspheres with antibodies against CD3/CD28 and/or cross-linking of antibodies against CD3. In some aspects, the methods do not include cross-linking of the anti-idiotypic antibody. In some aspects of any of the embodiments, the anti-idiotypic agents are capable of inducing the indicated proliferative or functional outcome, or extent thereof, without cross-linking the anti-idiotypic antibody. In some aspects, the anti-idiotypic agents of the present invention are advantageous in their ability to stimulate or induce a specific functional response of T cells or other immune cells expressing the target receptor without the need for anti-Id antibody crosslinking or the use of a secondary agent. In some aspects, the result is achieved using a soluble or tablet-bound form of the anti-idiotypic antibody. In some aspects, the result is achieved using an anti-idity antibody associated with the microsphere.

In some embodiments, a method for preparing a cell composition is provided, comprising (a) introducing into cells a nucleic acid molecule encoding a chimeric antigen receptor (CAR), thereby generating the parent composition; and (b) incubating the parent composition with an anti-idiotypic antibody or an antigen-binding fragment thereof specific for the CAR antigen receptor, thereby obtaining a cell composition.

In some aspects, the CAR contains a target antibody that specifically binds to CD19. In some embodiments, the target antibody is an SJ25C1 antibody or an antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotypic antibody, or antigen-binding fragment thereof, is an anti-idiotypic antibody, or antigen-binding fragment thereof, according to any of the described embodiments, that specifically binds to a target antibody, which is an SJ25C1 antibody or antigen-binding fragment thereof. linking fragment. In some cases, the target antibody is the FMC63 antibody or an antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotypic antibody, or an antigen-binding fragment thereof, specifically binds to a target antibody, which is an FMC63 antibody according to any of the described embodiments, which specifically binds to a target antibody, which is an FMC63 antibody or antigen thereof. -linking fragment.

In some of any such embodiments, the introduction in (a) includes the introduction of the nucleic acid molecule into cells by viral transduction, transposition, electroporation or chemical transfection. In some cases, the introduction in (a) includes the introduction of the nucleic acid molecule into cells by transduction with a retroviral vector containing the nucleic acid molecule, where the viral vector is optionally a retroviral vector or a lentiviral vector. In some aspects, the introduction in (a) includes the introduction of the nucleic acid molecule into cells by transposition with a transposon containing the nucleic acid molecule. In some cases, the introduction in (a) includes the introduction of a nucleic acid molecule into cells by electroporation or transfection of a vector containing a nucleic acid molecule.

In some of any such embodiments, the method further comprises the step of activating the cells prior to step (a). In some aspects, the cell activation step includes contacting the cells with a CD3 agonist and optionally a CD28 agonist. In some cases, the cell activation step includes contacting the cells with a reagent containing anti-CD3 and anti-CD28 agonist antibodies.

In some of any such embodiments, the incubation is carried out under conditions in which the anti-idiotypic antibody, or antigen-binding fragment thereof, binds to the CAR, thereby inducing or modulating a signal in one or more cells of the original composition. In any of such embodiments, the cells comprise T cells. In some cases, T cells contain CD4+ and/or CD8+ T cells.

In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on 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-idiotypic antibody or antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on a soluble reagent, which optionally is or contains a plurality of binding sites capable of reversibly binding to the anti-idiotypic antibody or antigen-binding fragment thereof. In some aspects, the reagent contains a streptavidin mutein.

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.

In some of any such embodiments, the starting 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 number of cells in the composition.

In some of any such embodiments, the number of CAR-expressing cells in the final composition is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the number of CAR-expressing cells in the original composition; and/or the percentage of CAR expression in the final composition compared to the total number of cells in the composition is increased by more than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more.

In some of any such embodiments, cells are not selected or enriched for CAR-expressing cells prior to administration and/or incubation.

In some of any such embodiments, the target antibody or antigen binding fragment comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some of any such embodiments, the target antibody or the antigen-binding fragment contains the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for purifying an antibody or antigen-binding fragment thereof, comprising (a) contacting a composition comprising a target antibody, which is an SJ25C1 antibody or antigen-binding fragment thereof, with an anti-idiotypic antibody or antigen-binding fragment thereof according to any of the described embodiments or with a conjugate according to any of the described embodiments that specifically binds to the target antibody, which is the SJ25C1 antibody or antigen-binding fragment thereof; and (b) isolating complexes containing the anti-idiotypic antibody. In some embodiments, the method includes (a) contacting a composition comprising a target antibody, which is an FMC63 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof, in accordance with any of the described embodiments, or with a conjugate in in accordance with any of the described embodiments that specifically bind to the target antibody, which is the FMC63 antibody or antigen-binding fragment; and (b) isolating complexes containing the anti-idiotypic antibody.

In some embodiments, complexes containing the anti-idiotypic antibody are isolated by affinity separation. In some aspects, the affinity separation is an immunoaffinity separation. In some cases, the affine separation is a separation based on a magnetic field. In some embodiments, affinity separation includes affinity chromatography.

In some embodiments, a method for identifying an anti-idiotypic antibody or antigen-binding fragment thereof is provided, comprising (a) administering to a patient a soluble immunizing reagent comprising an antigen-binding fragment of a target antibody fused to a solubilizing fragment; and (b) identifying a patient antibody that specifically binds to the target antibody or antigen-binding fragment thereof.

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

In some of any such embodiments, the solubilizing fragment is an Fc domain or a fragment thereof, which is optionally a human IgG1 Fc. In some aspects, the solubilizing fragment is an Fc domain lacking a hinge region. In some cases, the solubilizing fragment contains the amino acid sequence of SEQ ID NO: 32.

In some of any such embodiments, identifying the antibody comprises (i) isolating B cells from the patient's spleen and fusing them with immortalized B cells to create hybridomas; (ii) screening hybridomas for the production of antibodies that specifically bind to the target antibody or antigen-binding fragment or chimeric antigen receptor containing the antigen-binding fragment; and (iii) sequencing an antibody from a hybridoma producing an antibody that specifically binds, thereby identifying an anti-idiotypic antibody.

In some of any such embodiments, the target antibody binds to CD19. In some embodiments, the target antibody antigen-binding fragment is derived from the SJ25C1 antibody, wherein the target antibody antigen-binding fragment optionally comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. B some of any such embodiments, the antigen-binding fragment of the target antibody is a single-chain variable fragment (scFv) derived from the antibody SJ25C1, where the scFv optionally contains the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antigen-binding fragment of the target antibody derived from an FMC63 antibody, wherein the antigen-binding fragment of the target antibody optionally comprises the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31. In some embodiments, the antigen-binding fragment of the target antibody is is a single chain variable fragment (scFv) derived from a FMC63 antibodies, where the scFv optionally contains the amino acid sequence of SEQ ID NO: 34.

In some embodiments, a method of cell depletion is provided, comprising administering to a patient a composition comprising an anti-idiotypic antibody, or an antigen-binding fragment thereof, according to any of the described embodiments, or a conjugate, according to any of the described embodiments, that specifically binds to the target antibody. , which is the SJ25C1 antibody, or its antigen-binding fragment, where the patient is injected with a cell expressing a chimeric antigen receptor (CAR), including the target antibody, which is the SJ25C1 antibody, or its antigen-binding fragment. In some embodiments, the method includes administering to a patient a composition comprising an anti-idiotypic antibody, or an antigen-binding fragment thereof, according to any of the described embodiments, or a conjugate, according to any of the described embodiments, that specifically binds to a target antibody, which is an FMC63 antibody. , or with its antigen-binding fragment, where the patient is injected with a cell expressing a chimeric antigen receptor (CAR) containing the target antibody, which is the FMC63 antibody, or its antigen-binding fragment. In some embodiments, the implementation of the depletion occurs through antibody-dependent cell-mediated cytotoxicity (ADCC).

Provided herein is a method for determining the presence or absence of a molecule that binds to a chimeric antigen receptor (CAR), the method comprising (a) contacting the binding reagent with a sample from a patient who has been administered a cell therapy comprising cells engineered with a CAR containing a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, under conditions to form a complex containing a binding reagent and a molecule from the sample that binds to the binding reagent, where the binding reagent contains an extracellular domain from CAR or a part thereof containing the antibody the target or its antigen-binding fragment; and (b) detecting the presence or absence of the complex, thereby determining the presence or absence of a molecule that binds to the CAR. In some embodiments, the method further comprises performing steps (a) and (b) with a positive control sample and optionally determining the presence or absence of the molecule by comparison with a positive control, where the positive control sample contains any of an anti-idiotypic antibody or an antigen-binding fragment thereof described herein, or any of the conjugates described herein, which specifically bind to the target antibody or antigen-binding fragment.

Provided herein is a method for determining the presence or absence of a molecule that binds to a chimeric antigen receptor (CAR), the method comprising (a) contacting the binding reagent with a sample from a patient who has been administered a cell therapy containing cells engineered with a CAR containing a target antibody, which is an FMC63 antibody, or an antigen-binding fragment thereof, under conditions to form a complex comprising a binding reagent and a molecule from the sample that binds to the binding reagent, where the binding reagent contains the CAR extracellular domain or part of the extracellular domain containing the antibody the target or its antigen-binding fragment; and (b) detecting the presence or absence of the complex. In some embodiments, the method further comprises performing steps (a) and (b) with a positive control sample and optionally determining the presence or absence of the molecule by comparison with a positive control, where the positive control sample contains any of the anti-idiotypic antibody or antigen-binding fragment thereof. described herein, or any of the conjugates described herein, which specifically bind to the target antibody or antigen-binding fragment.

In some of any such embodiments, the molecule that binds to the binding reagent is or contains an antibody. In some embodiments, the binding reagent is detectably labeled or capable of generating a detectable signal. In some cases, the binding agent is bound to a solid support or is soluble.

In some of any such embodiments, the complex is detected by immunoassay. In some examples, the immunoassay is an enzyme immunoassay (ELISA), chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR) biosensor (eg, BIAcore), flow cytometry, or Western blotting. In some embodiments, the implementation of the immunoassay includes mesoscale detection. In some cases, the immunoassay is a sandwich assay or a bridge assay.

In some of any such embodiments, the binding reagent is the first binding reagent, and detecting the presence or absence of the complex comprises: (i) contacting the complex formed in step (a) with a second binding reagent, where the second binding reagent (1) contains an extracellular domain a CAR, or portion thereof, comprising the target antibody or antigen-binding fragment thereof, and (2) is detectably labeled or capable of generating a detectable signal; and (ii) assessing the presence or absence of the detected signal. In some aspects, the first binding reagent is associated with a solid support, where the first binding reagent is optionally associated, directly or indirectly, with biotin and/or associated with the solid support through streptavidin; and/or the second binding agent is soluble. In some cases, the extracellular domain of the CAR or part of the first and second binding reagent are the same.

In some of any such embodiments, the detectable label is or comprises a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label; and/or the detected signal is or contains a fluorescent signal, a chemiluminescent signal, an electroluminescent signal, a colorimetric signal, a bioluminescent signal, or a radioactive signal. In some of any such embodiments, the detectable label is or contains a SULFO label.

In some of any such embodiments, the antigen-binding fragment of the target antibody comprises a heavy chain variable region and/or a light chain variable region of the target antibody. In some of any such embodiments, the antigen-binding fragment of the target antibody is a single chain fragment. In some embodiments, the antigen-binding fragment of the target antibody is scFv.

In some of any such embodiments, the sample contains whole blood, serum, or plasma.

Provided herein is an article of manufacture containing any of the anti-idiotypic antibody or antigen-binding fragment thereof described herein, or any of the described conjugates, and instructions for using the anti-idiotypic antibody to detect the SJ25C1 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor containing SJ25C1 antibody or antigen-binding fragment; for selection or enrichment from a cell population of engineered cells expressing a chimeric antigen receptor (CAR) containing the SJ25C1 antibody or antigen-binding fragment; stimulating the original composition containing cells expressing a chimeric antigen receptor containing the SJ25C1 antibody or its antigen-binding fragment.

Provided herein is an article of manufacture comprising any of the anti-idiotypic antibody or antigen-binding fragment thereof described herein, or any of the conjugates described herein, and instructions for using the anti-idiotypic antibody to detect the FMC63 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor containing the FMC63 antibody or antigen-binding fragment thereof; for selection or enrichment from a cell population of engineered cells expressing a chimeric antigen receptor (CAR) containing the FMC63 antibody or an antigen-binding fragment thereof; to stimulate the original composition containing cells expressing a chimeric antigen receptor containing the FMC63 antibody or its antigen-binding fragment.

The present description provides a product containing a binding reagent comprising an extracellular domain of a chimeric antigen receptor (CAR) containing a target antibody that is an FMC63 antibody, or an antigen-binding fragment, and the specified extracellular domain or part thereof contains an antibody or antigen- linking fragment; and containing an anti-idiotypic antibody or antigen-binding fragment described herein, or any of the conjugates described herein. In some embodiments, the implementation of the binding reagent is a first binding reagent, and the product further includes a second binding reagent containing the extracellular domain or part of the CAR.

In some of any such embodiments, the CAR extracellular domain, or portion thereof, of the first and second binding reagents are the same.

In some of any such embodiments, the article of manufacture further includes instructions for using a binding reagent, optionally a first and second binding reagent, to analyze a sample for the presence or absence of a molecule that binds to the binding reagent using an immunoassay, where the immunoassay is optionally a bridge or sandwich -immunoassay, where the sample is optionally taken from a patient who was administered a cell therapy, including cells constructed with a CAR containing a target antibody that is an FMC63 antibody, or an antigen-binding fragment thereof.

The present description provides a product containing a binding reagent comprising an extracellular domain of a chimeric antigen receptor (CAR) containing a target antibody that is an SJ25C1 antibody, or an antigen-binding fragment, and the specified extracellular domain or part thereof contains the antibody or its antigen- linking fragment; and containing an anti-idiotypic antibody or antigen-binding fragment described herein, or any of the conjugates described herein.

In some embodiments, the implementation of the binding reagent is a first binding reagent, and the product further comprises a second binding reagent containing the extracellular domain or part of the CAR. In some aspects, the extracellular domain of the CAR or part of the first and second binding reagent are the same.

In some of any such embodiments, the article of manufacture further comprises instructions for using a binding reagent, optionally a first and second binding reagent, to analyze a sample for the presence or absence of a molecule that binds to the binding reagent using an immunoassay, where the immunoassay is optionally a bridge or sandwich -immunoassay, where the sample is optionally taken from a patient who was administered a cell therapy, including cells constructed with a CAR containing a target antibody that is an SJ25C1 antibody, or an antigen-binding fragment thereof.

In some embodiments, the binding reagent, optionally the first and/or second binding reagent, is detectably labeled or capable of generating a detectable signal. In some cases, one of the first and second binding reagents is attached to a solid support or is capable of attaching to a solid support, and the other of the first and second binding reagents is a detectable label or is capable of generating a detectable signal. In some embodiments, the product further includes a solid support, where optionally one of the first and second binding reagents is directly or indirectly associated with biotin, and the solid support contains a surface coated with streptavidin.

Brief description of the drawings

FIG. 1 shows the results of flow cytometry to assess the functional activity of scFv-specific anti-idiotype antibody clone A-1 (anti-ID A-1) derived from SJ25C1 to stimulate Erk1/2 phosphorylation in Jurkat cells generated with CAR derived from SJ25C1. Activation with anti-CD3 antibody was included as a positive control. Unstimulated or stimulated isotype control cells were included as negative controls.

FIG. 2A-B show the results of a proliferation assay of T cells expressing a CAR containing either a binding domain derived from SJ25C1 (FIG. 2A) or a binding domain derived from FMC63 (FIG. 2B) as assessed by dye dilution using a flow chart. cytometry after stimulation with anti-CD3 (OKT3), anti-ID A-1 or anti-ID B-1 anti-idiotypic antibodies. Unstimulated cells were included as a negative control.

FIG. 2C shows the results of a proliferation assay of CAR mock-transduced and CAR-transduced (dye-labeled) T cells after culturing in the presence of stimulated with a plate-bound anti-idiotypic antibody recognizing the CAR binding domain, as assessed by dye dilution using flow cytometry.

FIG. 3 shows the results of evaluating the expression of two markers of T cell activation, CD69 and CD25, in CD4+ or CD8+ T cells expressing CARs with variable regions derived from SJ25C1, as assessed by flow cytometry after stimulation with anti-CD3 antibodies (OKT3), anti-ID A-1 or anti-ID B-1 associated with the tablet.

FIG. 4 shows the results of evaluating the expression of two markers of T cell activation, CD69 and CD25, in CD4+ or CD8+ T cells expressing CARs with a binding domain having FMC63-derived variable regions, as assessed by flow cytometry after stimulation with coupled with an anti-CD3 (OKT3), anti-ID A-1 or anti-ID B-1 antibody plate, or with a negative control as a non-target anti-idiotypic antibody. Unstimulated cells were included as a negative control.

FIG. 5 shows the results of a bridging ELISA for detecting anti-CAR antibodies using anti-ID B-1 and anti-ID B-2 antibodies recognizing the CAR binding domain over a range of concentrations as positive controls.

FIG. 6 and FIG. 7 shows the fold increase and cumulative cell number of EGFRt+/CD4+ T cells or EGFRt+/CD8+ T cells, respectively, stimulated with the indicated ratio of microspheres coated with anti-idiotypic antibody (anti-ID B-1) or control CD3/CD28 antibody in the presence of or in the absence of cytokines.

FIG. 8 shows the expression level of PD-1 in CD4+ T cells positive for anti-EGFR staining and thus positive for the transduction marker EGFRt after stimulation with the indicated ratio of microspheres coated with anti-idiotypic antibody (anti-ID B-1) or CD3 control antibody. /CD28, in the presence or absence of cytokines, as assessed by flow cytometry on days 3, 7, 10 and 14 of culture.

FIG. 9 demonstrates the viability of CD4+ or CD8+ T cells expressing FMC63 derived CAR as assessed by flow cytometry after stimulation with the indicated ratio of microspheres coated with anti-idiotypic antibody (anti-ID B-1) or control CD3/CD28 antibody, in the presence or absence of cytokines as assessed by flow cytometry on days 3, 7, 10 and 14 of culture.

FIG. 10A shows intracellular staining for the cytokines IL-2, TNFα, and IFNγ for T cells expressing FMC63-derived CAR after stimulation with microspheres coated with an FMC63-derived scFv-specific anti-idiotypic antibody (anti-ID B-1). Results are presented for CD8+ T cells positive or negative for the EGFRt surrogate transduction marker (EGFR+ or EGFRt-).

FIG. 10B shows intracellular staining of cytokines IL-2, TNFα and IFNγ for T cells expressing FMC63 derived CAR after stimulation with antigen expressing K562-CD19 cells. Results are presented for CD8+ T cells positive for anti-EGFR antibody as a surrogate for CAR expression.

FIG. 11 shows the number of population doublings in the sequential stimulation assay over a 14-day culture period of T cells expressing the FMC63-derived scFv-specific CAR after stimulation with the indicated ratio of anti-idiotypic antibody (anti-ID B-1) or control coated microspheres. anti-CD3/CD28 antibody, in the presence or absence of cytokines. Results are presented for CD4+ T cells positive for the surrogate transduction marker EGFRt (EGFRt+/CD4+) or CD8+ T cells positive for EGFRt (EGFRt+/CD8+).

FIG. 12A-12C show results after stimulation of FMC63 derived CD4+ or CD8+ T-CAR expressing cells cultured alone or co-cultured with FMC63 derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated microspheres. The results are presented for two different donors. FIG. 12A depicts the fold expansion of CD4+ T cells or CD8+ T cells in 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 cultures that were positive for EGFRt (EGFRt+/CD4+ or EGFRt+/CD8+). FIG. 12C demonstrates the viability of CD4+ T cells or CD8+ T cells in cultures.

FIG. 13A and 13B show flow cytometry results for T cell surface markers on days 5, 7 and 9 of culture after stimulation of CD4+ or CD8+ T-CAR-expressing FMC63-derived cells cultured alone or co-cultured with FMC63-derived-coated microspheres. scFv-specific anti-idiotypic antibody (anti-ID B-1). FIG. 13A shows surface expression of PD-1 on CD4+ T cells or CD8+ T cells in cultures that were positive for a surrogate EGFRt transduction marker (EGFRt+/CD4+ or EGFRt+/CD8+). FIG. 13B shows surface expression of CD25 on CD4+ T cells or CD8+ T cells in cultures that were positive for an anti-EGFR antibody as a surrogate for CAR expression (EGFRt+/CD4+ or EGFRt+/CD8+).

FIG. 14A shows intracellular levels of the cytokines TNFα, IFNγ, and IL-2 as assessed by flow cytometry of CD4+ or CD8+ T cells present in a thawed composition containing FMC63-derived CAR-expressing T cells that were grown in culture or with CD19-expressing K562 cells or with PMA/ionomycin. Cytokine levels are shown in CD4+ and CD8+ T cells, individually or in co-culture, on thaw day (d=0) or after additional culturing for an additional 9 days in the presence of anti-ID B-1 conjugated microspheres.

FIG. 14B shows the frequency of CD25 or Ki67 positive cells as assessed by flow cytometry of CD4+ or CD8+ T cells present in a thawed formulation containing FMC63 derived CAR expressing T cells that were grown in culture or with K562 cells expressing CD19, or with PMA/ionomycin. Shows the level of markers in CD4+ and CD8+ T cells, individually or in co-culture, on the day of thawing (d=0) or after an additional culturing for an additional 9 days in the presence of microspheres conjugated with anti-ID B-1.

FIG. 15A and 15B show a graph showing the results of cell staining with anti-EGFR antibody or scFv-specific anti-idiotypic antibodies derived from FMC63 (anti-ID B-1 and anti-ID B-2). FIG. 15A shows a graph showing the average fluorescence intensity of cells that have been stained with different antibody concentrations. The cells included a mixture of PBMC and CAR expressing cells. FIG. 15B shows a graph depicting the percentage of CAR-expressing cells in the presence of FMC63-derived scFv-specific anti-idiotypic antibody (anti-ID B-1) detected in cells that were stained with different antibody concentrations. Cells included a mixture of PBMC and CAR expressing cells and PBMC only.

Detailed description of the invention

Provided herein are agents such as anti-idiotypic antibodies and antigen binding fragments (such as single chain fragments including scFv) that specifically recognize anti-CD19 antibody fragments (such as anti-CD19 antibody fragments present in recombinant receptors, including chimeric antigen receptors) . Also provided are uses and methods of use, as well as compositions and articles of manufacture, comprising such agents, including those for specifically identifying, selecting, and/or stimulating and/or activating cells expressing or comprising target antibodies or fragments, such as anti- CD19 CAR T cells. In some embodiments, the provided antibodies can be used to specifically identify and/or select for various anti-CD19 CARs, such as CARs, bound or expressed on a cell surface, and can also be used to specifically activate cells expressing target CARs, such as CARs. -T cells. In some embodiments, antibodies are provided that are specific for anti-CD19 antibodies designated SJ25C1 or FMC63 or antibody fragments derived therefrom, including antibodies and CARs containing variable regions derived from such antibodies and/or an antibody containing an idiotope contained therein. .

In some aspects, the proposed anti-idiotypic antibodies have advantages over conventional reagents for detecting, identifying, manipulating and/or influencing and/or constructing cells that express CAR, and in particular, CAR containing the extracellular domain of an anti-CD19 antibody scFv, or CAR A containing a recognizable idiotype. In some available methods, detection in a sample of the presence or absence or amount of CAR or CAR-expressing cells (and/or stimulation or manipulation of CAR) is performed by assessing the presence or absence or amount of a surrogate molecule, such as that included in a construct encoding a CAR and, thus serving as an indirect or surrogate marker for its expression. In some available methods, detection is performed using a characteristic antibody reagent and/or a reagent that is not specific to the particular CAR being evaluated, for example, 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 that recognize spacer or other domains from the species from which the CAR domain was derived and/or antibodies that recognize specific components used in the target spacer regions, as well as other chimeric receptors. In some available methods developed for detecting the presence or absence of a CAR, the detection is performed using an agent that recognizes the CAR constant region. In some available methods, CAR cells are stimulated using conventional reagents such as anti-CD3/CD28 recognition agents. Some methods use a recombinant CAR ligand (eg, CD19-Fc). Such methods may not be entirely satisfactory in certain contexts and/or have certain limitations. In some cases, CAR ligands such as CD19 may not always be fully effective, for example for use in complex flow cytometry panels. Improved methods and agents are needed, including those providing improved sensitivity and/or selectivity. In the present description proposed options for implementation that meet such needs.

The proposed anti-idiotypic antibodies and antigen-binding fragments, in some embodiments, overcome the problems of low binding affinity associated with target antibody ligands and non-specific binding associated with antibody reagents targeting constant regions of target antibodies, providing the reagent with both high affinity and specificity for its target antibody or its antigen-binding fragment. In some embodiments, the provided antibodies exhibit greater specificity and binding affinity for their target antibodies or antigen-binding fragments, such as an anti-CD19 antibody designated SJ25C1 or FMC63, compared to CD19-Fc and other reagents currently available for detection. or CAR identification.

In addition, in certain embodiments, anti-idiotypic antibodies and antigen-binding fragments that can be selected as agonists or antagonists of chimeric receptors, including their target antibodies or antigen-binding fragments, allow selective detection, isolation, ablation and/or depletion ( for example, killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or stimulating or activating cells using such chimeric receptors bound or expressed on their surface. Provided herein are anti-idiotypic antibody agonists that exhibit activity to stimulate, eg, activate, a CAR containing an extracellular binding domain derived from an anti-CD19 antibody designated SJ25C1 or FMC63. In some aspects, such antibodies can be used in methods for stimulating and expanding specific CAR-expressing cells, including methods for generating and preparing CAR-expressing cells.

Also provided herein are nucleic acids encoding the proposed anti-idiotypic antibodies and fragments, and cells, such as recombinant cells, expressing and intended to produce these anti-idiotypic antibodies and fragments. Also provided are methods for the preparation and use of anti-idiotypic antibodies and fragments thereof, as well as cells expressing or containing anti-idiotypic antibodies and fragments thereof.

All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If the definition given in the present description contradicts or is otherwise inconsistent with the definition set forth in patents, applications, published applications and other publications, which are incorporated here by reference, the definition set forth in the present description shall take precedence over the definition, which is included in this description by reference.

The section headings used in this description are for organizational purposes only and should not be construed as limiting the scope of the description.

I. Anti-idiotypic antibodies

In some aspects, binding molecules, such as anti-idiotypic antibodies or antigen-binding fragments ("anti-IDs"), are provided that specifically recognize a target as an anti-CD19 antibody fragment. In some embodiments, the provided antibodies recognize the target as an anti-CD19 antibody that is SJ25C1 or an antigen-binding fragment thereof, or is an antibody or antigen-binding fragment derived from SJ25C1. In some embodiments, the provided antibodies recognize the target as an anti-CD19 antibody that is FMC63 or an antigen-binding fragment thereof, or is an antibody or antigen-binding fragment derived from FMC63.

SJ25C1 is a mouse IgG1 monoclonal antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody contains the CDRH1, H2 and H3 shown in SEQ ID NOs: 114-116, respectively, and the CDRL1, L2 and L3 sequences shown in SEQ ID NOs: 117-119, respectively. The SJ25C1 antibody contains a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 23 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 24.

In some embodiments, the target antibody is SJ25C1 or an antibody derived from SJ25C1. In some embodiments, an antibody derived from SJ25C1 is an antibody or antigen-binding fragment that contains an SJ25C1 VH and/or VL, an SJ25C1 idiotype, an SJ25C1 paratope, or one or more SJ25C1 complementarity determining regions (CDRs). In some embodiments, the target antibody that is SJ25C1 or an antibody derived from SJ25C1 is an antibody or antigen-binding fragment comprising VH SJ25C1 SEQ ID NO: 23, or a variant thereof having at least 90% identity sequence with SEQ ID NO: 23, and/or VL SJ25C1 SEQ ID NO: 24, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 24. In some embodiments, the implementation of the target antibody, which is SJ25C1, or an antibody derived from SJ25C1, is an antibody or antigen-binding fragment containing VH SJ25C1 SEQ ID NO: 23, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 23, and/or VL SJ25C1 SEQ ID NO: 24, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment thereof comprises VH or VL SJ25C1, representing specified in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In some embodiments, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 23 and/or SEQ ID NO: 24.

In some embodiments, the target antibody that is SJ25C1, or an antibody derived from SJ25C1, is an antibody or antigen-binding fragment containing one or more heavy chain CDRs (CDR-H) of VH SJ25C1 shown in SEQ ID NO: 23 , such as those shown in SEQ ID NOs: 114-116, and/or one or more VL SJ25C1 light chain (CDR-L) CDRs shown in SEQ ID NO: 24, such as those shown in SEQ ID NO: 117- 119. In some embodiments, the antibody or antigen-binding fragment thereof comprises SJ25C1 CDR-H3 (eg SEQ ID NO: 116) and/or SJ25C1 CDR-L3 (eg SEQ ID NO: 119). In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H3 and CDR-L3 SJ25C1 (eg, SEQ ID NOs: 116 and 119, respectively). In some embodiments, the target antibody that is SJ25C1 or an antibody derived from SJ25C1 is an antibody or antigen binding fragment containing one or more of CDR-H1, CDR-H2, and CDR-H3 of SJ25C1 (e.g., those shown in SEQ ID NOs: 114, 115, 116, respectively) and/or one or more of CDR-L1, CDR-L2, and CDR-L3 SJ25C1 (for example, those shown in SEQ ID NOs: 117, 118, 119, respectively). In some embodiments, the target antibody that is SJ25C1, or an antibody derived from SJ25C1, is an antibody or antigen-binding fragment containing SJ25C1 CDR-H1, CDR-H2, and CDR-H3 (e.g., as shown in SEQ ID NO: 114, 115, 116, respectively) and/or CDR-L1, CDR-L2, and CDR-L3 SJ25C1 (eg, those shown in SEQ ID NOs: 117, 118, 119, respectively). In some embodiments, the target antibody that is SJ25C1 or an antibody derived from SJ25C1 is an antibody or antigen binding fragment containing one or more of CDR-H1, CDR-H2, and CDR-H3 of SJ25C1 (e.g., those shown in SEQ ID NOs: 114, 115, 116, respectively) and/or one or more of CDR-L1, CDR-L2, and CDR-L3 SJ25C1 (for example, those shown in SEQ ID NOs: 117, 118, 119, respectively). In some embodiments, the antibody or antigen-binding fragment thereof contains an antigen-binding fragment, such as a fragment (Fab), F(ab') ₂ , Fab', a variable fragment (Fv), or a single chain Fv fragment (scFv). See, for example, Bejcek, BE, et al. (1995). cancer research. 55(11): 2346-2351.

FMC63 is a mouse IgG1 monoclonal antibody raised against JVM3 cells expressing human CD19 (Nicholson et al. (1997). Molecular Immunology. 34 (16-17): 1157-1165). The FMC63 antibody contains CDRH1, H2 and H3 as shown in SEQ ID NOs: 120-122, respectively, and the sequences for CDRL1, L2, and L3 as shown in SEQ ID NOs: 123-125, respectively. The FMC63 antibody contains a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 30 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the target antibody is FMC63 or an antibody derived from FMC63. In some embodiments, an antibody derived from FMC63 is an antibody or antigen-binding fragment that contains an FMC63 VH and/or VL, an FMC63 idiotype, an FMC63 paratope, or one or more FMC63 complementarity determining regions (CDRs). In some embodiments, the target antibody that is FMC63 or an antibody derived from FMC63 is an antibody or antigen-binding fragment comprising VH FMC63 SEQ ID NO: 30, or a variant thereof having at least 90% identity sequences with SEQ ID NO: 30, and/or VL FMC63 SEQ ID NO: 31, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 31. In some embodiments, a target antibody that is FMC63, or an antibody derived from FMC63, is an antibody or antigen-binding fragment containing VH FMC63 SEQ ID NO: 30, or a variant thereof, having at least 90% sequence identity with SEQ ID NO: 30, and/or VL FMC63 SEQ ID NO: 31, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment comprises the VH or VL FMC63 shown in SEQ I D NO: 30 and SEQ ID NO: 31, respectively. In some embodiments, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 30 and/or SEQ ID NO: 31.

In some embodiments, the target antibody that is FMC63, or an antibody derived from FMC63, is an antibody or antigen-binding fragment containing one or more FMC63 heavy chain (CDR-H) VH CDRs shown in SEQ ID NO: 30 , such as those shown in SEQ ID NO: 120-122, and/or one or more VL FMC63 light chain CDRs (CDR-L) shown in SEQ ID NO: 31, such as those shown in SEQ ID NO: 123- 125. In some embodiments, the antibody or antigen binding fragment comprises FMC63 CDR-H3 (eg SEQ ID NO: 122) and/or FMC63 CDR-L3 (eg SEQ ID NO: 125). In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H3 (eg SEQ ID NO: 122) and CDR-L3 FMC63 (eg SEQ ID NO: 125). In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of FMC63 CDR-H1, CDR-H2, and CDR-H3 (represented in SEQ ID NOs: 120, 121, 122, respectively) and/or one or more of CDR-L1, CDR-L2 and CDR-L3 of FMC63 (shown in SEQ ID NOs: 123, 124, 125, respectively). In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 from FMC63 (represented in SEQ ID NOs: 120, 121, 122, respectively) and/or CDR-L1, CDR-L2 and CDR-L3 FMC63 (represented in SEQ ID NOs: 123, 124, 125, respectively). In some embodiments, the antibody or antigen-binding fragment thereof comprises FMC63 CDR-H1, CDR-H2, and CDR-H3 (represented in SEQ ID NOs: 120, 121, 122, respectively) and CDR-L1, CDR-L2, and CDR- L3 from FMC63 (represented in SEQ ID NOs: 123, 124, 125, respectively). In some embodiments, the antibody or antigen-binding fragment thereof comprises an antigen-binding fragment, such as a fragment (Fab), F(ab') ₂ , Fab', a variable fragment (Fv), or a single chain Fv fragment (scFv).

In some embodiments, anti-idiotypic antibodies provided include antibodies that specifically bind to a variable domain (Fv), such as a single chain Fv (scFv) derived from SJ25C1 or FMC63. In some embodiments, anti-idiotypic antibodies specifically bind to a particular Fv epitope or region, and typically the epitope or region contains one or more complementarity determining regions. In some embodiments, anti-idiotypic antibodies specifically bind to an epitope or region overlapping the Fv paratope.

In some embodiments, provided anti-idiotypic antibodies include antibodies that specifically bind to an anti-CD19 antibody fragment derived from SJ25C1 or FMC63 that is contained as part of the extracellular domain of a chimeric antigen receptor (CAR) target. In some embodiments, the CAR target comprises an antigen-binding portion that comprises an SJ25C1 or FMC63 antibody molecule or an antigen-binding fragment, or a portion of an SJ25C1 or FMC63 antibody. In some embodiments, the CAR target comprises an antigen binding domain that is an scFv derived from the VH or VL chains of the SJ25C1 or FMC63 antibodies. In some embodiments, an anti-idiotypic antibody is provided that specifically binds to an anti-CD19 CAR that contains a scFv derived from SJ25C1 or FMC63 antibodies. Exemplary CAR features are described below.

The term "antibody" is used in the present description in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) fragments of antibodies, including fragments of 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 antibody fragments (eg, sdAb, sdFv, nanobody). This 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. -scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" is to be understood to encompass functional fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

The term "anti-idiotypic antibody" refers to an antibody, including antigen-binding fragments thereof, that specifically recognize, specifically target, and/or specifically bind to an antibody idiotope, such as an antigen-binding fragment. Idiotopes of an antibody may include, but are not limited to, residues in one or more complementarity determining regions (CDRs) of an antibody, antibody variable regions, and/or partial or portions of such variable regions and/or such CDRs, and/or any combination of the above. 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 an antibody can be heavy chain variable regions, light chain variable regions, or a combination of heavy chain variable regions and light chain variable regions. Incomplete fragments or portions of heavy chain variable regions and/or light chain variable regions of an antibody may be fragments comprising 2 or more, 5 or more, or 10 or more contiguous amino acids, e.g., from about 2 to about 100, from about 5 to about up to 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids in the heavy chain or light chain variable regions of an antibody; idiotope may include many non-contiguous sections of amino acids. Partial fragments of heavy chain variable regions and light chain variable regions of an antibody may be fragments comprising 2 or more, 5 or more, or 10 or more contiguous amino acids, e.g., from about 2 to about 100, from about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids in the variable regions, and in some embodiments, contain one or more CDRs or CDR fragments. CDR fragments can be 2 or more consecutive or non-sequential, or 5 or more amino acids in a CDR. Therefore, antibody idiotopes can range 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 consecutive amino acids. containing one or more CDRs or one or more CDR fragments in the heavy chain variable regions or the light chain variable regions of an antibody. In another embodiment, idiotopes can be a single amino acid that is located in the variable regions of an antibody, such as CDR sites.

In some embodiments, an idiotope is any single antigenic determinant or epitope in the variable portion of an antibody. In some cases it may overlap the actual antigen-binding site of the antibody, and in some cases it may contain variable region sequences outside the antigen-binding site of the antibody. A set of individual idiotopes of an antibody is, in some embodiments, referred to as the "idiotype" of that antibody.

It is known in the art that the terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to non-contiguous amino acid sequences in the variable regions of an antibody that confer antigen specificity and/or binding affinity. . Typically, 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). It is known in the art that "framework regions" and "FR" refer to the non-CDR portions of the heavy and light chain variable regions. Typically, 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).

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» схема нумерации).The exact boundaries of the amino acid sequence 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

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).

The boundaries of the CDR or FR data may vary depending on the scheme used for identification. For example, the Kabat schema is based on structural alignment and the Chothia schema is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common sequence lengths of antibody regions, where inserts are provided with insertion letters, such as "30a", and deletions found in some antibodies. These two schemes place certain insertions and deletions ("Indels") in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

Table 1 below lists exemplary CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 boundary positions as defined by the Kabat, Chothia and Contact schemes, respectively. For CDR-H1, residue numbering is indicated using the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, FR-L1 is located between CDR-L1 and CDR-L2, and so on. It should be noted that since the Kabat numbering scheme shown places inserts in H35A and H35B, the end of the CDR-H1 loop according to Chothia, when numbered using the Kabat numbering convention shown, varies between H32 and H34 depending on the length of the loop.

Table 1

CDR Kabat Chothia contact CDR-L1 L24--L34 L24--L34 L30--L36 CDR-L2 L50--L56 L50--L56 L46--L55 CDR-L3 L89--L97 L89--L97 L89--L96 CDR-H1
(Kabat Numbering ¹ ) H31--H35B H26--H32..34 H30--H35B CDR-H1
(Chothia Numbering ² ) H31--H35 H26--H32 H30--H35 CDR-H2 H50--H65 H52--H56 H47--H58 CDR-H3 H95--H102 H95--H102 H93--H101

1 - 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

Thus, unless otherwise indicated, the "CDR" or "complementarity determining region" or individual specified CDRs (e.g., CDR-H1, CDR-H2) of a given antibody, or region thereof, such as its variable region, is to be understood to encompass (or defines) a complementarity defining region, as defined by any of the above schemes. For example, if a particular CDR (eg, CDR-H3) is indicated to contain the amino acid sequence of a corresponding CDR in a given VH or VL amino acid sequence, that CDR is understood to have the sequence of the corresponding CDR (eg, CDR-H3) in the variable region as defined any of the above schemes. In some embodiments, specific CDR sequences are defined.

Thus, unless otherwise indicated, "FR" or individual specified FRs (e.g., FR-H1, FR-H2) of a given antibody, or a region thereof, such as its variable region, is to be understood to encompass (or define) a framework region, as defined by any of the above schemes. In some cases, a scheme is provided to identify specific CDRs, FRs, or a plurality of FRs or CDRs, such as CDRs, as determined by the Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR or FR is indicated.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH or VL, respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, 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. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domain from an antibody that binds to the antigen by screening a library of complementary VL or VH domains, respectively See, for example, Portolano et al., J. Immunol 150:880- 887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

Among the proposed antibodies are antibody fragments. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to 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 ; diabody; linear antibodies; single chain antibody molecules (eg scFv); and polyspecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.

Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments containing permutations that do not occur naturally, such as fragments containing two or more antibody or chain regions connected by synthetic linkers, such as peptide linkers, and/or which are not can be obtained by enzymatic cleavage of natural intact antibodies. In some aspects, the antibody fragments are scFv.

A "humanized" antibody is one in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the framework region (FR) amino acid residues are derived from human FRs. In some embodiments, humanized forms of a non-human antibody, such as a mouse antibody, are chimeric antibodies that contain minimal sequences derived from a non-human immunoglobulin. In some embodiments, humanized antibodies are antibodies of a non-human species containing one or more complementarity determining regions (CDRs) from a non-human species and a framework region (FR) from a human immunoglobulin molecule. In some embodiments, a humanized antibody may optionally 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 non-human variant of an antibody that has been humanized, usually to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody. (See, for example, Queen, US Pat. No. 5,585,089 and Winter, US Pat. No. 5,225,539). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced by residues from the heavy chain variable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or a non-human primate having target specificity, affinity and/or ability. In some cases, human immunoglobulin FR residues are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. In some embodiments, nucleic acid sequences encoding human variable heavy chains and human variable light chains are altered to replace one or more CDR sequences of a human (acceptor) sequence with a sequence encoding the corresponding CDR in a non-human antibody sequence (donor sequence). In some embodiments, the implementation of the human acceptor sequence may contain FR derived from different genes. In specific embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of FRs are human immunoglobulin sequences. In some embodiments, the humanized antibody will optionally also include at least a portion of an immunoglobulin (Fc) constant region, typically a human immunoglobulin. For more information, see, for example, 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, for example, 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 pat. No. 9982321 and 7087409 included in the present description by reference. In some embodiments, provided herein are humanized anti-idiotypic antibodies.

In specific embodiments, the antibody, such as an anti-idiotypic antibody, is humanized. In certain embodiments, the antibody is humanized by any suitable known method. For example, in some embodiments, a humanized antibody may have one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from an "import" variable domain. In specific embodiments, humanization may be substantially carried out according to the method of Winter et al. (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988 ) Science 239: 1534-1536), for example, by replacing the hypervariable region sequences with corresponding human antibody sequences. Accordingly, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which a substantially smaller region than the intact human variable domain has been replaced with a corresponding sequence from a non-human species. In some embodiments, the humanized antibody is a human antibody in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

Sequences encoding full-length antibodies can subsequently be obtained by attaching the provided heavy and light chain variable sequences to human heavy chain and light chain constant regions. Suitable human light chain constant sequences include the kappa and lambda light chain constant sequences. Suitable human heavy chain constant sequences include IgG1, IgG2, and sequences encoding IgG1 mutants that have immunostimulatory properties. Such mutants may have reduced ability to activate complement and/or antibody-dependent cellular cytotoxicity and are described in US Patent No. No. 5624821; WO 99/58572, US patent No. 6737056. A suitable heavy chain constant region also includes an IgG1 containing E233P, L234V, L235A, A327G, A330S, P331S substitutions and a deletion of residue 236. In another embodiment, the full-length antibody contains an IgA, IgD, IgE, IgM, IgY, or IgW sequence.

Suitable human donor sequences can be determined by comparing the peptide sequences encoded by the mouse donor sequences with a group of human sequences, preferably those encoded by human germline immunoglobulin genes or mature antibody genes. A human sequence with high sequence homology, preferably the highest defined homology, can serve as an acceptor sequence for the humanization process.

In addition to exchanging human CDRs for mouse CDRs, further manipulations of the human donor sequence can be performed to obtain a sequence encoding a humanized antibody with optimized properties (such as antigen affinity).

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

In specific embodiments, it is generally desirable that antibodies be humanized while retaining high antigen affinity and other beneficial biological properties. To achieve this goal, in some embodiments, humanized antibodies are generated using a parental sequence analysis process and various conceptual humanized products using 3D models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these images allows the analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected and combined from the recipient and imported sequences so that the target characteristic of the antibody, such as increased affinity for the target antigen(s), is achieved. In general, hypervariable region residues are directly and most significantly involved in influencing antigen binding.

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

Among the proposed 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, human cell, or non-human source that utilizes the human antibody repertoire or other sequences encoding human antibodies, including human antibody libraries. The term excludes humanized forms of non-human antibodies containing non-human antigen-binding regions, such as those in which all or substantially all of the CDRs are non-human.

Human antibodies can be generated by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present outside the chromosomes or are randomly integrated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are usually inactivated. Human antibodies can also be obtained from human antibody libraries, including phage display and cell-free libraries containing sequences encoding antibodies derived from the human repertoire.

The proposed antibodies include monoclonal antibodies, including fragments of monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to an antibody derived from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations or occurring during production. preparation of a monoclonal antibody, such variants are usually present in small amounts. Unlike 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 the antigen. The term should not be construed as requiring the production of an antibody by any particular method. A monoclonal antibody can be produced by a variety of methods including, but not limited to, hybridoma production, recombinant DNA techniques, phage display, and other display techniques for producing antibodies.

A. Antibodies derived from SJ25C1

In some embodiments, anti-idiotypic antibodies are provided that are specific for a target anti-CD19 antibody that is or derived from an SJ25C1 antibody or an antigen-binding fragment thereof. In some embodiments, the proposed antibodies or antigen-binding fragments are specific for scFv derived from SJ25C1.

In some embodiments, anti-idiotypic antibodies, or antigen-binding fragments thereof, are provided that comprise a heavy chain variable region (VH) comprising at least 90% identity of the VH region to the amino acid sequence of SEQ ID NO: 1, e.g., at least 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In some embodiments, anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 11 or 84, and /or a CDR-H3 contained in the heavy chain variable sequence (VH) of SEQ ID NO: 1.

In some of any such embodiments, the VH region includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 9, 78, 79, or 80 and/or a CDR-H1 contained in the VH sequence of SEQ ID NO: 1; and/or heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence of SEQ ID NO: 10, 81, 82 or 83 and/or CDR-H2 contained in the VH sequence of SEQ ID NO: 1.

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which include a heavy chain variable region (VH) containing heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, where CDR-H1 contains the amino acid sequence SEQ ID NO: 9, 78, 79 or 80; CDR-H2 contains the amino acid sequence of SEQ ID NO: 10, 81, 82 or 83; and/or CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11 or 84. In some embodiments, antibodies or antigen-binding fragments thereof are provided that include a CDR-H1 having the amino acid sequence of SEQ ID NO: 9, 78, 79, or 80 ; CDR-H2 having the amino acid sequence of SEQ ID NO: 10, 81, 82 or 83; and CDR-H3 having the amino acid sequence of SEQ ID NO: 11 or 84.

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which include heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, respectively, containing the amino acid sequences CDR-H1, CDR-H2 and CDR-H3, contained in the VH region with the amino acid sequence of SEQ ID NO: 1.

In some of any such embodiments, the VH region comprises framework region 1 (FR1), FR2, FR3, and/or FR4 sequences having at least 90% sequence identity, respectively, with FR1, FR2, FR3, and/or FR4, respectively. amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH region comprises framework region 1 (FR1), FR2, FR3, and/or FR4 sequences having at least 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2, FR3 and/or FR4 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH region contains frame region 1 sequences ( FR1), FR2, FR3 and FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively , with FR1, FR2, FR3 and FR4 amino acid sequence of SEQ ID NO: 1.

In some of any such embodiments, the VH region has the amino acid sequence of SEQ ID NO: 1.

In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is only the heavy chain, only VH, and/or does not include VL or the antigen-binding portion thereof, and/or the anti-idiotypic antibody antigen-binding site or fragment includes residues only from the heavy chain and/or do not include residues from the light chain.

In some of any such embodiments, the anti-idiotypic antibody or fragment thereof does not contain a light chain variable region (VL), does not contain CDR-L1, CDR-L2 and/or CDR-L3, and/or is a single domain antibody (sdAb) containing only the VH area. In some embodiments, the antibody or fragment thereof is an sdAb that contains only a VH region of any of those described.

In some embodiments of any of the anti-idiotypic antibodies or fragments thereof comprising any of the above VH region sequences, the anti-idiotypic antibody or fragment thereof further comprises a light chain variable (VL) region. In some such embodiments, the VL region has at least 90% sequence identity with the amino acid sequence of the VL region of SEQ ID NO: 5, e.g., at least 91%, 92%, 93%, 94%, 95% 96%, 97 %, 98% or 99% identity of the VL region with the amino acid sequence of SEQ ID NO: 5.

In some of any such embodiments, the VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 14 or 87. In some of any such embodiments, the VL region comprises a light chain complementarity determining region 3 chain (CDR-L3) having the amino acid sequence of SEQ ID NO: 14 or 87.

In some of any such embodiments, the VL region comprises a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 12 or 85 and/or a CDR-L1 contained in the VL sequence of SEQ ID NO: 5 ; and/or a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 13 or 86 and/or a CDR-L2 contained in the VL sequence of SEQ ID NO: 5. In some of any such embodiments, implementation region VL contains region 1, determining the complementarity of the light chain (CDR-L1), having the amino acid sequence of SEQ ID NO: 12 or 85, and/or the CDR-L1 contained in the VL sequence of SEQ ID NO: 5; and/or light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 13 or 86 and/or a CDR-L2 contained in the VL sequence of SEQ ID NO: 5.

In some of any such embodiments, the VL region comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12 or 85; CDR-L2 containing the amino acid sequence of SEQ ID NO: 13 or 86; and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 14 or 57.

In some of any such embodiments, the VL region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, including the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 contained in the amino acid sequence of the VL region of SEQ ID NO: 5 .

In some of any such embodiments, the VL region comprises a framework region 1 (FR1), FR2, FR3, and/or FR4 that is at least 90% sequence identical, respectively, with FR1, FR2, FR3, and/or FR4 of the amino acid sequence of SEQ. ID NO: 5. In some embodiments, the VL region contains a framework region 1 (FR1), FR2, FR3 and/or FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2, FR3 and/or FR4 of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL region contains the sequence of framework region 1 (FR1), FR2 , FR3 and FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2 , FR3 and FR4 amino acid sequence of SEQ ID NO: 5.

In some of any such embodiments, the VL region has the amino acid sequence of SEQ ID NO: 5.

Proposed anti-idiotypic antibodies or antigen-binding fragments, which contain the amino acid sequence CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 1; and/or contain the amino acid sequences CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) amino acid sequence of SEQ ID NO: 5.

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided which include VH or VL regions having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1 and 5, respectively.

In some embodiments, anti-idiotypic antibodies or antigen-binding fragments thereof are provided that include VH or VL regions having the amino acid sequences shown in SEQ ID NOs: 1 and 5, respectively.

In some of any such embodiments, the VH or VL regions comprise the amino acid sequences of SEQ ID NOs: 1 and 5, respectively.

In some embodiments, the anti-idiotypic antibody specific for the SJ25C1 antibody or antigen-binding fragment thereof is a single chain antibody fragment, such as a scFv or a diabody. In some embodiments, a single chain antibody comprises one or more linkers connecting two antibody domains or regions, such as a heavy chain variable region (VH) and a light chain variable region (VL). The linker is typically a peptide linker, such as a flexible and/or soluble peptide linker. Among the linkers are those that are rich in glycine and serine and/or, in some cases, threonine. In some embodiments, the implementation of the linkers further include charged residues, such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline residues.

In some embodiments, the anti-idiotypic antibody is an intact antibody or a full length antibody. In some embodiments, the anti-ID may comprise at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some embodiments, the constant regions include a light chain constant region (CL) and/or a heavy chain constant region 1 (CH1). In some embodiments, the anti-ID includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is the Fc region of a human IgG, such as IgG1 or IgG4. In some embodiments, the anti-idiotypic antibody comprises a CH domain shown in SEQ ID NO: 2, or a portion thereof, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2 or part thereof. In some embodiments, the anti-idiotypic antibody comprises a CL domain shown in SEQ ID NO: 6, or a portion thereof, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6 or part thereof.

In some embodiments, an anti-idiotypic antibody specific for SJ25C1 contains the heavy chain sequence of 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 with SEQ ID NO: 3, and/or contains a light chain sequence of SEQ ID NO: 7, or a sequence that demonstrates 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 embodiments, the anti-idiotypic antibody specific for SJ25C1 comprises the heavy chain sequence of SEQ ID NO: 3 and/or the light chain sequence of SEQ ID NO: 7. In some embodiments, the heavy chain and/or or the light chain of the anti-idiotypic antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence of SEQ ID NO: 4 or SEQ ID NO: 8.

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

Accordingly, single chain antibody fragments such as scFv and diabodies are provided, in particular human single chain fragments, typically containing a linker(s) connecting two domains or regions of an anti-idiotypic antibody, such as VH or VL domains. The linker is typically a peptide linker, for example a flexible and/or soluble peptide linker such as a glycine and serine rich linker.

In some aspects, glycine and serine (and/or threonine) rich linkers comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they include at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the implementation of the linker consists essentially entirely of glycine, serine and/or threonine. Linkers are typically about 5 to about 50 amino acids in length, typically about 10 to about 30 or about 30, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and in some examples the linkers are 10 to 25 amino acids in length. Typical linkers include linkers having varying numbers of repeats of the sequence GGGS (3GS; SEQ ID NO: 29) or GGGGS (4GS; SEQ ID NO: 26), such as 2, 3, 4 and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of the sequence SEQ ID NO: 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence of SEQ ID NO: 33 (GSTSGSGKPGSGEGSTKG).

In some embodiments, anti-idiotypic antibodies include isolated antibodies. In some embodiments, the anti-ID is humanized, recombinant, and/or monoclonal. In some embodiments, the anti-ID is human.

In some embodiments, the SJ25C1 specific anti-idiotype antibody is humanized. In specific embodiments, all or substantially all of the amino acid residues of the CDRs of the humanized anti-idiotype antibody specific for SJ25C1 are derived from the CDRs of the non-human SJ25C1 antibody. In some embodiments, a humanized anti-idiotype antibody specific for SJ25C1 includes at least a portion of an antibody constant region derived from a human antibody.

In certain embodiments, the humanized anti-idiotypic antibody specific for SJ25C1 comprises a human immunoglobulin (recipient antibody) in which heavy chain variable region residues of the recipient are replaced with heavy chain variable region residues of a non-human SJ25C1 anti-idiotypic antibody. In some cases, human immunoglobulin FR residues are replaced by corresponding non-human residues. In some embodiments, the humanized antibody contains FRs derived from different genes. In some embodiments, the humanized anti-idiotype antibody specific for SJ25C1 comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

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

In certain embodiments, the SJ25C1 specific anti-idiotype antibody is humanized. In specific embodiments, a humanized anti-idiotypic antibody specific for SJ25C1 comprises one or more of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions of a non-human anti-idiotypic antibody that is specific to SJ25C1. In some embodiments, some or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions contain one or more amino acid modifications. In some embodiments, the modifications replace a non-human residue with a human amino acid residue. In specific embodiments, one or more of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 are inserted into the FR regions of a human antibody. In specific embodiments, the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the non-human anti-idiotypic antibody are CDRs of VH or VL regions having the amino acid sequences shown in SEQ ID NO: 1 and 5, respectively. In some embodiments, all CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of an anti-idiotype antibody specific for SJ25C1 are inserted into the FR of a human antibody. In specific embodiments, the CDR and FR regions are regions that are identified by the Kabat, Chothia, AbM and/or Contact schemas.

In specific embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody that is specific for SJ25C1 are inserted into the framework regions of a human antibodies. In some embodiments, the human antibody is an IgA, IgD, IgE, IgG, and IgM antibody. In specific embodiments, the human antibody is one of a subclass of human IgA, IgD, IgE, IgG, and IgM, such as human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody that is specific for SJ25C1 are inserted into the framework regions of the antigen. a binding region that is taken from and/or derived from a human antibody. In some embodiments, the antigen-binding fragment is derived from and/or derived from a human IgA, IgD, IgE, IgG, and IgM antibody. The subunit structures and three-dimensional configurations of the various classes of human immunoglobulins are well known and described generally, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). In some embodiments, the human antibody or antigen-binding fragment thereof may be part of a larger fusion molecule formed by the covalent or non-covalent association of a human antibody with one or more other proteins or peptides.

In some embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody that is specific for SJ25C1 are inserted into human frameworks. an antibody or antigen-binding fragment containing all or part of an Fc region. In certain embodiments, the humanized anti-idiotype antibody specific for SJ25C1 contains all or part of the Fc region. In some embodiments, the Fc region has one or more modifications, such as an amino acid modification (eg, substitution, insertion, or deletion) at one or more amino acid positions. Such modifications may 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 embodiments, the modified Fc regions have altered (eg, reduced) FcαR binding compared to the unmodified Fc region. In some embodiments, the humanized anti-idiotypic antibody comprises all or part of a modified Fc region having altered (eg, reduced) Fc receptor binding compared to that of the unmodified Fc region. Non-limiting examples of Fc modifications that alter its binding to Fc receptors are described, for example, in US Patent Nos. Nos. 7217797 and 7732570; and US Application Nos. US 2010/0143254 and 2010/0143254.

In some embodiments, a humanized anti-idiotypic antibody specific for SJ25C1 has a heavy chain containing the amino acid sequence of SEQ ID NO: 132 and a light chain containing the amino acid sequence of SEQ ID NO: 133. In specific embodiments, a humanized anti-idiotypic antibody specific for SJ25C1, has a heavy chain containing the amino acid sequence of SEQ ID NO: 134 and a light chain containing the amino acid sequence of SEQ ID NO: 135. In some embodiments, a humanized anti-idiotypic antibody specific for SJ25C1 has a heavy chain containing the amino acid sequence of SEQ ID NO: 136 , and a light chain containing the amino acid sequence of SEQ ID NO: 137.

B. Antibodies derived from FMC63

In some embodiments, anti-idiotypic antibodies are provided that are specific for a target anti-CD19 antibody that is or derived from the FMC63 antibody or an antigen-binding fragment thereof. In some embodiments, the proposed antibodies or antigen-binding fragments are specific for scFv derived from FMC63.

In some embodiments, anti-idiotypic antibodies or antigen-binding fragments thereof are provided that include a heavy chain variable region (VH) that is at least 90% identical to the VH region with the amino acid sequence of SEQ ID NO: 36 or 58, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, antibodies or antigen-binding fragments thereof are provided that include a VH region having a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence GYX ₃ FX ₅ X ₆ YX ₈ MX ₁₀ (SEQ ID NO: 108), where 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 heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence WIGX ₄ IX ₆ PX ₈ X ₉ X ₁₀ X ₁₁ TX ₁₃ X ₁₄ NQX ₁₇ FKX ₂₀ (SEQ ID NO: 109), where X ₄ is D or M, X ₆ is N or 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 heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence 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) where 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 absent, X ₈ is G or absent, X ₉ is S or absent, X ₁₀ R or absent, X ₁₁ D or absent, X ₁₂ A or absent, X ₁₃ M or absent, X ₁₄ is D or E, and X ₁₅ is Y or A.

In some embodiments, anti-idiotypic antibodies or antigen-binding fragments thereof are provided that comprise a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NOs: 46, 67, 94 or 104 and/or a CDR-H3 contained in the heavy chain variable sequence (VH) of SEQ ID NO: 36 or 58.

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

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which include a heavy chain variable region (VH) containing heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, where CDR-H1 contains the amino acid sequence SEQ ID NO: 44, 65, 88, 89, 90, 98, 99, or 100; CDR-H2 contains the amino acid sequence of SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103; and/or CDR-H3 contains the amino acid sequence of SEQ ID NO: 46, 67, 94, or 104. In some embodiments, antibodies or antigen-binding fragments thereof are provided that include a CDR-H1 having the amino acid sequence of SEQ ID NO: 44, 65 , 88, 89, 90, 98, 99, or 100; CDR-H2 having the amino acid sequence of SEQ ID NO: 45, 66, 91, 92, 93, 101, 102, or 103; and CDR-H3 having the amino acid sequence of SEQ ID NO: 46, 67, 94, or 104.

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which include heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, respectively, containing the amino acid sequences CDR-H1, CDR-H2 and CDR-H3, contained in the VH region with the amino acid sequence of SEQ ID NO: 36 or 58.

In some embodiments, the anti-idiotypic antibody, or antigen-binding fragment thereof, comprises CDR-H1 c SEQ ID NO: 44, 88, 89, or 90; CDR-H2 c SEQ ID NO: 45, 91, 92 or 93; and/or CDR-H3 of SEQ ID NO: 46 or 94. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises CDR-H1 of SEQ ID NO: 65, 98, 99, or 100; CDR-H2 c SEQ ID NO: 66, 101, 102 or 103; and/or CDR-H3 with SEQ ID NO: 67 or 104, respectively.

In some of any such embodiments, the VH region comprises the sequence of frame region 1 (FR1), FR2, FR3, and/or FR4 having at least 90% sequence identity, respectively, with FR1, FR2, FR3, and/or FR4, respectively. amino acid sequence of SEQ ID NO: 36 or 58. In some embodiments, the VH region comprises a framework region 1 (FR1), FR2, FR3, and/or FR4 sequence having at least 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2, FR3 and/or FR4 of the amino acid sequence of SEQ ID NO: 36 or 58. In some embodiments, the VH region contains the framework sequence region 1 (FR1), FR2, FR3 and FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity , respectively, with FR1, FR2, FR3 and FR4 amino acid sequence of SEQ ID NO: 36 or 58.

In some of these embodiments, the VH region has the amino acid sequence of SEQ ID NO: 36 or 58.

In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is only a heavy chain, only VH, and/or does not include VL or an antigen-binding portion thereof, and/or the anti-idiotypic antibody antigen-binding site or fragment includes residues only from the heavy chain and/or do not include residues from the light chain.

In some of any such embodiments, the anti-idiotypic antibody or fragment thereof does not contain a light chain variable region (VL), does not contain CDR-L1, CDR-L2 and/or CDR-L3, and/or is a single domain antibody (sdAb) containing only the VH area. In some embodiments, the antibody or fragment thereof is an sdAb that contains only a VH region of any of those described.

In some embodiments of any of the anti-idiotypic antibodies or fragments thereof comprising any of the above VH region sequences, the anti-idiotypic antibody or fragment thereof further comprises a light chain variable (VL) region. In some such embodiments, the VL region has at least 90% sequence identity with the amino acid sequence of the VL region of SEQ ID NO: 40 or 62, such as at least 91%, 92%, 93%, 94%, 95% 96% , 97%, 98% or 99% identity of the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

In some embodiments, antibodies or antigen-binding fragments thereof are provided that include a VH region having a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence X ₁ AX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ YX ₁₀ X ₁₁ WY (SEQ ID NO: 111) where 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 absent, X ₁₀ is M or L, and X ₁₁ is Y or A; and/or light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence X ₁ X ₂ X ₃ YX ₅ X ₆ X ₇ X ₈ LAX ₁₁ (SEQ ID NO: 112), where 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 light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence QX ₂ X ₃ X ₄ X ₅ X ₆ PX ₈ T (SEQ ID NO: 113), where 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.

In some of any such embodiments, the VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 49, 97, 70, or 107. In some of any such embodiments, the VL region comprises a region 3 light chain complementarity determining (CDR-L3) having the amino acid sequence of SEQ ID NO: 49, 97, 70, or 107.

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

In some of any such embodiments, the VL region comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 47, 68, 95, or 105; CDR-L2 containing the amino acid sequence of SEQ ID NO: 48, 69, 96, or 106; and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 49, 97, 70, or 107.

In some of any such embodiments, the VL region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, including the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof comprises CDR-L1 c SEQ ID NO: 47 or 95; CDR-L2 c SEQ ID NO: 48 or 96; and/or CDR-L3 of SEQ ID NO: 49 or 97. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof comprises CDR-L1 of SEQ ID NO: 68 or 105; CDR-L2 c SEQ ID NO: 69 or 106; and/or CDR-L3 c SEQ ID NO: 70 or 107, respectively.

In some of any such embodiments, the VL region comprises a framework region 1 (FR1), FR2, FR3, and/or FR4 that is at least 90% sequence identical, respectively, with FR1, FR2, FR3, and/or FR4 of the amino acid sequence of SEQ. ID NO: 40 or 64. In some embodiments, the VL region contains a framework region 1 (FR1), FR2, FR3, and/or FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2, FR3, and/or FR4 of the amino acid sequence of SEQ ID NO: 40 or 62. In some embodiments, the VL region contains the framework sequence 1 ( FR1), FR2, FR3 and FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, with FR1, FR2, FR3 and FR4 the amino acid sequence of SEQ ID NO: 40 or 62.

In some of any such embodiments, the VL region has the amino acid sequence of SEQ ID NO: 40 or 62.

Proposed anti-idiotypic antibodies or antigen-binding fragments, which contain the amino acid sequence CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 36 or 58; and/or contain the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) amino acid sequence of SEQ ID NO: 40 or 62.

Anti-idiotypic antibodies or antigen-binding fragments thereof are provided which include a VH region having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 36 or 58, and a VL region having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 40 or 62.

In some embodiments, anti-idiotypic antibodies or antigen-binding fragments thereof are provided, which include a VH region having the amino acid sequences shown in SEQ ID NO: 36 or 58 and a VL region having the amino acid sequences shown in SEQ ID NO: 40 or 62 In some embodiments, the proposed antibody comprises the VH region of SEQ ID NO: 36, and the VL region of SEQ ID NO: 40. In some embodiments, the proposed antibody comprises the VH region of SEQ ID NO: 58, and the VL region of SEQ ID NO: 62.

In some embodiments, the anti-idiotypic antibody is a single chain antibody fragment, such as a scFv or a diabody. In some embodiments, a single chain antibody comprises one or more linkers connecting two antibody domains or regions, such as a heavy chain variable region (VH) and a light chain variable region (VL). The linker is typically a peptide linker, such as a flexible and/or soluble peptide linker. Among the linkers are those that are rich in glycine and serine and/or, in some cases, threonine. In some embodiments, the implementation of the linkers further include charged residues, such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline residues.

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

In some embodiments, an anti-idiotypic antibody specific for FMC63 comprises a heavy chain sequence of 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 with SEQ ID NO: 38 or 60, and/or contains the light chain sequence of 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 with SEQ ID NO: 42 or 63. In some embodiments, the FMC63-specific anti-idiotype antibody comprises the heavy chain sequence of SEQ ID NO: 38 and/or the light chain sequence of SEQ ID NO: 42. B In some embodiments, an anti-idiotypic antibody specific for FMC63 comprises the heavy chain sequence of SEQ ID NO: 60 and/or the light chain sequence of SEQ ID NO: 63. B in some embodiments, the heavy chain and/or light chain of the anti-idiotypic antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence of SEQ ID NO: 39, 43, 61 or 64.

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

Accordingly, single chain antibody fragments such as scFv and diabodies are provided, in particular human single chain fragments, typically containing a linker(s) connecting two domains or regions of an anti-idiotypic antibody, such as VH or VL domains. The linker is typically a peptide linker, for example a flexible and/or soluble peptide linker such as a glycine and serine rich linker.

In some aspects, glycine and serine (and/or threonine) rich linkers comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they include at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the implementation of the linker consists essentially entirely of glycine, serine and/or threonine. Linkers are typically about 5 to about 50 amino acids in length, typically about 10 to about 30 or about 30, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and in some examples the linkers are 10 to 25 amino acids in length. Typical linkers include linkers having varying numbers of repeats of the sequence GGGS (3GS; SEQ ID NO: 29) or GGGGS (4GS; SEQ ID NO: 26), such as 2, 3, 4 and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of the sequence SEQ ID NO: 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence of SEQ ID NO: 33 (GSTSGSGKPGSGEGSTKG).

In some embodiments, anti-idiotypic antibodies include isolated antibodies. In some embodiments, the anti-ID is humanized, recombinant, and/or monoclonal. In some embodiments, the anti-ID is human.

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

In certain embodiments, the humanized FMC63-specific anti-idiotypic antibody comprises a human immunoglobulin (recipient antibody) in which heavy chain variable region residues of the recipient are replaced with heavy chain variable region residues of a non-human FMC63-specific anti-idiotypic antibody. In some cases, human immunoglobulin FR residues are replaced by corresponding non-human residues. In some embodiments, the humanized antibody contains FRs derived from different genes. In some embodiments, a humanized anti-idiotypic antibody specific for FMC63 comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

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

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

In specific embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody specific for FMC63 is inserted into human antibody framework regions. . In some embodiments, the human antibody is an IgA, IgD, IgE, IgG, and IgM antibody. In specific embodiments, the human antibody is one of a subclass of human IgA, IgD, IgE, IgG, and IgM, such as human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody that is specific for FMC63 are inserted into the framework regions of the antigen. a binding region that is taken from and/or derived from a human antibody. In some embodiments, the antigen-binding fragment is derived from and/or derived from a human IgA, IgD, IgE, IgG, and IgM antibody. The subunit structures and three-dimensional configurations of the various classes of human immunoglobulins are well known and described generally, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (W. B. Saunders, Co., 2000). In some embodiments, the human antibody or antigen-binding fragment thereof may be part of a larger fusion molecule formed by the covalent or non-covalent association of a human antibody with one or more other proteins or peptides.

In some embodiments, one or more or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotypic antibody that is specific for FMC63 are inserted into human frameworks. an antibody or antigen-binding fragment containing all or part of an Fc region. In certain embodiments, the humanized anti-idiotypic antibody specific for FMC63 contains all or part of the Fc region. In some embodiments, the Fc region has one or more modifications, such as an amino acid modification (eg, substitution, insertion, or deletion) at one or more amino acid positions. Such modifications may 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 embodiments, the modified Fc regions have altered (eg, reduced) FcαR binding compared to the unmodified Fc region. In some embodiments, the humanized anti-idiotypic antibody comprises all or part of a modified Fc region having altered (eg, reduced) Fc receptor binding compared to that of the unmodified Fc region. Non-limiting examples of Fc modifications that alter its binding to Fc receptors are described, for example, in US Patent Nos. Nos. 7217797 and 7732570; and US Application Nos. US 2010/0143254 and 2010/0143254.

In some embodiments, a humanized anti-idiotypic antibody specific for FMC63 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 138 and a light chain comprising the amino acid sequence of SEQ ID NO: 139. In specific embodiments, the humanized anti-idiotypic antibody specific for FMC63 has a heavy chain containing the amino acid sequence of SEQ ID NO: 140 and a light chain containing the amino acid sequence of SEQ ID NO: 141. In some embodiments, a humanized FMC63-specific anti-idiotypic antibody has a heavy chain containing the amino acid sequence of SEQ ID NO: 142 , and a light chain containing the amino acid sequence of SEQ ID NO: 143. In some embodiments, the humanized anti-idiotype antibody specific for FMC63 has a heavy chain containing the amino acid sequence of SEQ ID NO: 144 and a light chain containing the amino acid a different sequence of SEQ ID NO: 145. In specific embodiments, a humanized anti-idiotypic antibody specific for FMC63 has a heavy chain containing the amino acid sequence of SEQ ID NO: 146 and a light chain containing the amino acid sequence of SEQ ID NO: 147. In some embodiments, the implementation a humanized anti-idiotypic antibody specific for FMC63 has a heavy chain containing the amino acid sequence of SEQ ID NO: 148 and a light chain containing the amino acid sequence of SEQ ID NO: 149.

C. Typical signs

The anti-idiotypic antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities using various known assays. In one aspect, an anti-idiotypic antibody is tested for its antigen-binding activity, e.g., using known methods such as ELISA, Western blot, and/or flow cytometry, including cell-based binding assays, e.g., to evaluate the binding of an anti-idiotypic antibody (e.g., conjugated to a fluorescent marker or labeled) with a cell presenting the anti-CD19 target antibody fragment, in some cases compared to results using cells that do not express the anti-CD19 target antibody fragment. Binding affinity can be measured as Kd or EC50.

In some embodiments of any of the proposed antibodies, such as any of the proposed antibodies in section A above, the target antibody fragment of an anti-CD19 antibody is SJ25C1, or is an antibody derived from SJ25C1.

In some embodiments of any of the proposed antibodies, such as any of the proposed antibodies in section B above, the target anti-CD19 antibody fragment is FMC63 or is an antibody derived from FMC63.

Competition assays can be used to identify an antibody that competes with any of the anti-idiotypic antibodies described herein. Assays for mapping epitopes associated with anti-idiotypic antibodies and reference antibodies are also known and can be used.

In some embodiments, the anti-idiotypic antibody does not cross-react with an anti-CD19 antibody fragment other than a target anti-CD19 antibody fragment. In some embodiments, the target anti-CD19 antibody fragment is derived from the SJ25C1 antibody. In some embodiments, the CD19 target antibody fragment is derived from the SJ25C1 antibody and the anti-idiotype antibody does not cross-react with the CD19 target antibody fragment derived from the FMC63 antibody, which is an anti-CD19 antibody containing a VH containing the amino acid sequence of SEQ ID NO: 30, and a VL containing the amino acid sequence of SEQ ID NO: 31. In some embodiments, the target anti-CD19 antibody fragment is derived from the FMC63 antibody. In some embodiments, the CD19 target antibody fragment is derived from the FMC63 antibody and the anti-idiotypic antibody does not cross-react with the CD19 target antibody fragment derived from the FMC63 antibody, which is an anti-CD19 antibody containing a VH containing the amino acid sequence of SEQ ID NO: 23, and VL containing the amino acid sequence of SEQ ID NO: 24.

In some embodiments, the anti-idiotype antibody specifically binds to a target anti-CD19 antibody fragment that is part of a fusion protein, such as a recombinant receptor. In some embodiments, the anti-idiotype antibody does not bind to any epitope in the fusion protein outside of the anti-CD19 target antibody fragment. For example, in some embodiments, the target anti-CD19 antibody fragment is all or part of the antigen-binding domain of a chimeric antigen receptor (CAR) and the anti-idiotypic antibody does not bind to any epitope outside the antigen-binding domain. In some embodiments, the implementation of the antigen-binding domain of the CAR contains or consists of scFv.

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

In some embodiments, the anti-idiotypic antibody does not cross-react with another CAR. In some embodiments, the anti-idiotypic antibody does not cross-react with another anti-CD19 CAR. In some embodiments, the anti-idiotypic antibody does not cross-react with an anti-CD19 antibody fragment, for example, with a reference antibody having one or more different idiotopes compared to the target anti-CD19 antibody scFv. In some embodiments, the anti-idiotypic antibody is specific for an anti-CD19 scFv target from a CAR derived from the SJ25C1 antibody. In some embodiments, the target anti-CD19 antibody fragment is derived from the SJ25C1 antibody and the anti-idiotypic antibody does not cross-react with a CAR containing an anti-CD19 antibody fragment derived from the FMC63 antibody. In some embodiments, the anti-idiotypic antibody is specific for an anti-CD19 scFv target from a CAR derived from the FMC63 antibody. In some embodiments, the target anti-CD19 antibody fragment is derived from the FMC63 antibody and the anti-idiotypic antibody does not cross-react with a CAR containing an anti-CD19 antibody fragment derived from the SJ25C1 antibody.

In some embodiments, the anti-idiotype antibody is a CAR agonist. In some embodiments, the anti-idiotype antibody is a CAR antagonist.

In some embodiments, the provided anti-idiotypic antibodies are capable of binding to an anti-CD19 target antibody fragment, such as the SJ25C1 or FMC63 antibody, with at least a certain affinity as measured by any of a number of known methods. In some embodiments, affinity is represented by an equilibrium dissociation constant (KD); in some embodiments, affinity is represented by EC50. In some embodiments, the binding affinity (EC50) and/or dissociation constant of the CD19 anti-idiotype antibody fragment is either about equal to or less than 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 in the range of or about 1 nM to about 15 nM , for example, from about 5 to about 10 nM. In one embodiment, the degree of binding of the anti-idiotypic antibody to a fragment not associated with the target anti-CD19 antibody fragment is less than or about 10% of the binding of the antibody to the target anti-CD19 antibody fragment, as measured, for example, by radioimmunoassay (RIA) .

D. Nucleic acids

Also provided are nucleic acids encoding antibodies and/or parts thereof, such as their chains. Among the proposed nucleic acids are those that encode the anti-idiotypic antibodies described herein. Nucleic acids may include those that include natural and/or non-natural nucleotides and bases, such as those that have backbone chain modifications. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides that includes a nucleic acid molecule or polynucleotide. Examples of nucleic acids and vectors are those having the sequences shown in SEQ ID NOs: 15-22, 50-57, 71-77 and their CDR-coding portions, as well as sequences having with them at least 90 or 91 , 91 or 92, 93, 94, 95, 96, 97, 98 or 99% identity. The nucleic acid may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an anti-idiotypic antibody (eg, antibody light and/or heavy chains).

Also proposed are vectors containing nucleic acids, host cells containing vectors, for example, for the production of antibodies. Methods for producing antibodies are also provided. In a further embodiment, one or more vectors (eg, expression vectors) containing such a nucleic acid are provided. In a further embodiment, a host cell containing such a nucleic acid is provided. In one such embodiment, the host cell contains (e.g., transformed): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing the nucleic acid , which encodes an amino acid sequence containing the VL of the antibody, and a second vector containing a nucleic acid that encodes the amino acid sequence containing the VH of the antibody. In some embodiments, a method for producing an anti-idiotypic antibody is provided, which comprises culturing a host cell containing a nucleic acid encoding an antibody as defined above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or culture medium). host cells).

E. Methods for producing antibodies

Also provided are methods for producing an anti-idiotypic antibody, such as in accordance with any of the proposed embodiments. In some embodiments, for recombinant production of an anti-idiotypic antibody, a nucleic acid encoding an antibody, such as described above, can be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding antibody heavy and light chains).

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts 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 to human cells, resulting in to the formation of an antibody with a partially or fully human glycosylation profile. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

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

In some embodiments, the anti-idiotypic antibody is produced in a cell-free system. Illustrative cell-free systems are described, for example, 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).

Proposed embodiments further include vectors and host cells and other expression systems for the expression and production of 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.

Anti-idiotypic antibodies or antibody fragments can be humanized or human antibodies. A "humanized" antibody is an antibody in which all or substantially all of the amino acid residues of the CDRs are derived from non-human CDRs and all or substantially all of the amino acid residues of the framework FRs are derived from human FRs. The humanized antibody may optionally 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 non-human variant of an antibody that has been humanized, usually to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Among the proposed anti-idiotypic antibodies or antibody fragments are human antibodies. A "human antibody" is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human, human cell, or non-human source that uses the human antibody repertoire or other sequences encoding human antibodies, including human antibody libraries. The term excludes humanized forms of non-human antibodies containing non-human antigen-binding regions, such as those in which all or substantially all of the CDRs are non-human.

Human antibodies can be generated by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present outside the chromosomes or are randomly integrated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are usually inactivated. Human antibodies can also be obtained from human antibody libraries, including phage display and cell-free libraries containing sequences encoding antibodies derived from the human repertoire.

F. Immunoconjugates

In some embodiments, the anti-idiotypic antibody is whole or part of an immunoconjugate (anti-idiotypic antibody immunoconjugate) wherein the anti-idiotypic antibody is conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic or imaging agent. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu radioactive isotopes); chemotherapeutic agents (eg maytansinoids, taxanes, methotrexate, adriamycin, 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 embodiments, 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.

Anti-idiotypic antibody immunoconjugates include antibody-drug conjugates (ADCs) in which the anti-idiotypic antibody is conjugated to one or more drugs, including but not limited to maytansinoid (see U.S. Patent Nos. 52082020, 5416064 and EP 0425235 B1 ); auristatin, such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see US Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see US Pat. 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 US Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tezetaxel and orthotaxel; trichothecene; and CC1065.

Also among the anti-idiotypic 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-chains, non-binding active fragments of diphtheria toxin, exotoxin A-chain (from Pseudomonas aeruginosa), A-chain ricin, 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, restrictin, fenomycin, enomycin and trichothecenes.

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

Anti-idiotypic antibody-cytotoxic agent conjugates can be prepared using any of a number of known protein coupling agents, such as linkers (see Vitetta et al., Science 238: 1098 (1987)), WO 94/11026. The linker may be a "cleavable linker" to facilitate the release of the cytotoxic drug in the cell, such as acid sensitive linkers, peptidase sensitive linkers, light sensitive linkers, dimethyl linkers and disulfide linkers (Chari et al., Cancer Res 52: 127-131 (1992); US patent No. 5208020).

Also provided are anti-idiotypic antibody immunoconjugates comprising an anti-idiotypic antibody fused to a label, such as a detectable label, that can generate a detectable signal, directly or indirectly. These anti-idiotypic antibody immunoconjugates can be used for research or diagnostic purposes. The label is preferably capable of generating, directly or indirectly, a detectable signal. For example, the label may be radiopaque or 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; rendering agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphy, such as ⁹⁹ Tc or ¹²³ I, or a spin label for nuclear magnetic resonance (NMR) (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 can complex with various metal chelating agents and is conjugated to antibodies, for example for PET imaging (WO 2011/056983).

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.

In some embodiments, the anti-idiotypic antibody immunoconjugate is indirectly detected. For example, a secondary antibody that is specific for an anti-idiotypic antibody immunoconjugate and contains a detectable label can be used to detect the anti-idiotypic antibody immunoconjugate.

Multispecific antibodies

In certain embodiments, anti-idiotypic antibodies are multispecific. Among the polyspecific binding molecules are polyspecific antibodies, including, for example, bispecific. Multispecific binding partners, such as antibodies, have binding specificities for at least two different sites, which may be on the same or different antigens. In some embodiments, one of the binding specificities is for an anti-CD19 antibody fragment and the other is for a different antigen. In some embodiments, the bispecific antibodies can bind to two different epitopes on an anti-CD19 antibody fragment. Bispecific antibodies can also be used to localize cytotoxic agents in cells that express an anti-CD19 antibody fragment on their surface, such as anti-CD19 CAR T cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Bispecific antibodies include polyspecific single chain antibodies, such as diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs.

G. Options

In certain embodiments, the anti-idiotypic antibodies comprise one or more amino acid variations, such as substitutions, deletions, insertions, and/or mutations, compared to the anti-idiotypic antibody sequence described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the anti-idiotypic antibody. Variants of the amino acid sequence of an anti-idiotypic antibody can be obtained by introducing appropriate modifications to the nucleotide sequence encoding the anti-idiotypic antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the anti-idiotypic antibody. Any combination of deletion, insertion, and substitution can be made to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

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

In some embodiments, one or more residues in the CDR of the parent antibody (eg, humanized or human antibody) are replaced. In some embodiments, a substitution is performed to convert a sequence or position in a sequence to a germline sequence, such as an antibody sequence found in a germline (e.g., human germline), e.g., to reduce the likelihood of immunogenicity, e.g., after administration to a human.

In some embodiments, changes are made to CDR hotspots, codon-encoded residues that mutate at high rates during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) and/or residues that bind to the antigen, the resulting VH or VL variant being tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any of a variety of methods (eg, reduced precision PCR, strand shuffling, or oligonucleotide directed mutagenesis). Then a secondary library is created. The library is then screened for any antibody variants with the target affinity. Another method of introducing diversity involves CDR-directed approaches, in which several CDR residues (eg, 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, provided such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes (eg, conservative substitutions as provided herein) can be made to the CDR that do not significantly reduce binding affinity. Such changes may, for example, be outside the antigen-binding residues in the CDR. In some embodiments of the VH or VL sequence variants above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

Amino acid sequence insertions include fusions of amino and/or carboxy terminal regions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of end inserts include an antibody with an N-terminal methionine residue. Other insertion options for an antibody molecule include fusion of the N- or C-terminus of the antibody with an enzyme or polypeptide that increases the serum half-life of the antibody.

Modifications

In some embodiments, the antibody is modified to increase or decrease the degree of glycosylation, for example, by removing or inserting one or more glycosylation sites, by changing the amino acid sequence and/or by modifying the attached oligosaccharide(s) with glycosylation sites, for example, using certain cell lines.

Exemplary modifications, variants and cell lines are described, for example, in US patent publications No. 2003/0157108, US 2004/0093621, US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; W02005/035778; 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 Patent Application 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 WO 2003/085107); WO 2003/011878 (Jean-Mairet et al.); US patent No. 6602684 (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.).

Modified antibodies include antibodies having one or more amino acid modifications in the Fc region, such as antibodies having the sequence of a human Fc region or another portion of a constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modification (eg, substitution) at one or more amino acid positions.

Such modifications may be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.

Also included among the variants are cysteine engineered antibodies such as "thioMAb" and other cysteine engineered variants in which one or more antibody residues are replaced with cysteine residues to produce reactive thiol groups at accessible sites, e.g. for use in conjugation agents and linker agents to obtain immunoconjugates. Cysteine-engineered antibodies are described, for example, in US Pat. Nos. 7,855,275 and 7,521,541.

In some embodiments, the antibodies are modified to contain additional non-protein fragments, including water-soluble polymers. Typical polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic copolymer anhydride, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylene polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages 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 an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for modification may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether or not the antibody derivative is to be used in therapy under certain conditions. conditions, etc.

II. Methods for identifying anti-idiotypic antibodies

In some embodiments, a method is provided for identifying an anti-idiotypic antibody, or antigen-binding fragment thereof, that specifically binds to a target antibody or antigen-binding fragment thereof, for example, using hybridoma techniques with an immunogen containing 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 portion fused to the target antibody or fragment. Such an immunogenicity enhancing portion may have properties that include, but are not limited to, increasing the solubility and half-life of the immunogen. Exemplary immunogenicity enhancing moieties include Fc domains or fragments thereof. In some embodiments, the immunogenicity enhancing moiety is an Fc domain (such as IgG1, optionally human). In some embodiments, the immunogenicity enhancing moiety is an Fc domain (eg, from IgG1, optionally human) that lacks all or part of the hinge region.

In some embodiments, a method is provided for identifying an anti-idiotypic antibody or antigen-binding fragment thereof that specifically binds to a target antibody or antigen-binding fragment thereof, comprising: (a) administering to a patient a soluble immunogen comprising a target antigen-binding fragment fused with a part that enhances immunogenicity; and (b) identifying an antibody in the patient that specifically binds to the target antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region of a target antibody. In some embodiments, the antigen binding fragment is a single chain fragment. In some embodiments, the implementation of the antigen-binding fragment is a scFv. In some embodiments, the antigen-binding fragment is within or included in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR).

In some embodiments, the immunogenicity enhancing moiety is an Fc domain or fragment thereof, which is optionally a human IgG1 Fc. In some embodiments, the immunogenicity enhancing moiety is an Fc domain lacking a hinge region. In some embodiments, the immunogenicity enhancing fragment comprises the amino acid sequence of SEQ ID NO: 32.

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

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

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

In some of any such embodiments, the method comprises: (a) administering to the patient a soluble immunogen comprising an scFv derived from an 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 in the patient that (i) binds to a target molecule containing an FMC63 antibody idiotope, such as an immunogen, scFv, or CAR, or a fragment thereof containing the heavy chain variable region and/or light chain variable region of the FMC63 antibody, or parts thereof, such as one or more VH and/or VL CDRs; and (ii) does not bind to a non-target molecule that does not contain an FMC63 antibody idiotope, such as Fc or fragment thereof, or another antibody or fragment thereof that does not contain the heavy chain variable region and/or light chain variable region of the FMC63 antibody or portions thereof, such as one or more VH and/or VL CDRs. In some embodiments, the immunogen comprises the amino acid sequence of SEQ ID NO: 34 and SEQ ID NO: 32, optionally separated by a linker, such as that shown in SEQ ID NO: 33. In some embodiments, the immunogen comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunogen comprises the amino acid sequence of SEQ ID NO: 35. embodiments, the antibody identified in (b) binds to an immunogen and binds neither to an Fc domain or fragment thereof, nor to a molecule containing an scFv derived from an SJ25C1 antibody (such as a scFv containing the amino acid sequence of SEQ ID NO: 28) . In some embodiments, the antibody identified in (b) binds to a target molecule containing the amino acid sequence of SEQ ID NO: 34 or 35 and does not bind to a non-target molecule containing the amino acid sequence of SEQ ID NO: 32, nor with a non-target molecule containing the amino acid sequence of SEQ ID NO: 28.

In some of any such embodiments, the method comprises: (a) administering to the patient a soluble immunogen comprising an scFv derived from an 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 in a patient that (i) binds to a target molecule containing an SJ25C1 antibody idiotope, such as an immunogen, scFv, or CAR, or a fragment thereof comprising a heavy chain variable region and/or a light chain variable region of an SJ25C1 antibody, or parts thereof, such as one or more VH and/or VL CDRs; and (ii) does not bind to a non-target molecule that does not contain an SJ25C1 antibody idiotope, such as Fc or fragment thereof, or another antibody or fragment thereof that does not contain the heavy chain variable region and/or light chain variable region of the SJ25C1 antibody or portions thereof, such as one or more VH and/or VL CDRs. In some embodiments, the immunogen comprises the amino acid sequence of SEQ ID NO: 28 and SEQ ID NO: 32, optionally separated by a linker, such as that shown in SEQ ID NO: 33. In some embodiments, the antibody identified in (b) binds to the immunogen and binds neither to an Fc domain or fragment thereof, nor to a molecule containing an scFv derived from the FMC63 antibody (such as an scFv containing the amino acid sequence of SEQ ID NO: 34). In some embodiments, the antibody identified in (b) binds to a target molecule containing the amino acid sequence of SEQ ID NO: 28 and does not bind to a non-target molecule containing the amino acid sequence of SEQ ID NO: 32 or a non-target molecule containing the amino acid sequence of SEQ ID NO: 34.

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

In a typical embodiment, identification of anti-idiotypic antibodies recognizing the scFv portion of a typical anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv derived from SJ25C1 can be performed using an immunogen containing the sequence shown below:

EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR (SEQ ID NO: 28). In some embodiments, the immunogen may further comprise an Fc domain or fragment thereof, such as those shown in SEQ ID NO: 32.

In a typical embodiment, identification of anti-idiotypic antibodies recognizing the scFv portion of a typical anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv derived from FMC63 can be performed using an immunogen containing the sequence shown below:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 34).

In some embodiments, the immunogen may further comprise an Fc domain or fragment thereof, such as that shown in SEQ ID NO: 32. CD19 derived from FMC63 can be made using an immunogen containing the sequence below:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSpcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgveVHnaktkpreeqynstyrvvsVLtVLhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppVLdsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID NO: 35).

III. Chimeric antigen receptors (CAR) and genetically engineered cells

In some embodiments, the provided anti-idiotypic antibodies specifically bind to an antigen-binding portion of a chimeric antigen receptor (CAR), such as an anti-CD19 CAR containing an antigen-binding portion derived from SJ25C1 or FMC63 antibodies. In some embodiments, the provided anti-idiotypic antibodies bind to such CARs expressed on cells, such as cells used in connection with adoptive cell therapy. In some embodiments, cells include one or more genetically engineered nucleic acids and thereby express recombinant or genetically engineered CAR products of such nucleic acids. In some embodiments, chimeric receptors, when genetically engineered in 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 lifespan, survival and/or resistance in vivo, for example, for use in methods of adoptive cell therapy. In some embodiments, the provided anti-idiotype antibodies can be used in methods to modulate one or more of these activities, including activating, stimulating and/or propagating engineered cells expressing the target CAR.

In some embodiments, cells include one or more nucleic acids introduced through genetic engineering in accordance with the proposed methods, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, that is, not normally present in a cell or sample derived from a cell, such as derived from another organism or cells that, for example, are not normally detectable in the engineered cell and/or organism from which the cell is derived. such a cell. In some embodiments, nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one that contains chimeric combinations of nucleic acids encoding different domains from many different cell types. In specific embodiments, the implementation of the nucleic acids contain a gene that encodes for CAR.

In some embodiments, the implementation of the proposed methods can be performed simultaneously, sequentially or simultaneously with one or more stages of processing to obtain or prepare genetically engineered cells. The processing steps of the methods may include any one or more of a plurality of cell processing steps, alone or in combination. In specific embodiments, the processing steps include transducing or transfecting cells with one or more nucleic acids, such as a heterologous polynucleotide containing a gene encoding a recombinant receptor. In some embodiments, cells are transduced with viral vector particles containing a retroviral vector, such as one that encodes a recombinant product for expression in cells. In some embodiments, cells are transfected with one or more non-viral nucleic acids, such as an episomal plasmid or a transposon. The methods may additionally and/or alternatively include other processing steps such as the steps of isolating, separating, selecting, washing, suspending, diluting, concentrating and/or preparing cells. In some cases, the methods may also include the step of ex vivo culturing, stimulating or expanding cells (eg, stimulating cells, eg, to induce their proliferation and/or activation), which in some cases can be carried out in accordance with the proposed methods. In some embodiments, the methods include isolating cells from a patient, preparing, processing, culturing and/or engineering them, and reintroducing them to the same patient before or after cryopreservation.

In some embodiments, the method includes processing steps in the following order: cells, eg, primary cells, are first isolated, eg, selected or separated from a biological sample; the selected cells are incubated with viral vector particles for transduction; and the transduced cells are included in the composition. In some instances, the transduced cells are activated, expanded, or propagated ex vivo, eg, by stimulation in the presence of a stimulating reagent, eg, according to the methods provided. In some embodiments, the method may include one or more of the processing steps of washing, suspending, diluting and/or concentrating the cells, which may occur before, during, or simultaneously with or after one or more of the isolation steps, such as separation or selection, transduction, stimulation and/or formulation.

In specific embodiments, cells to be transfected or transduced are not isolated, selected, or enriched prior to contact with one or more nucleic acids. In some embodiments, the cells are not selected until the cells are contacted with one or more nucleic acids. In some embodiments, cells to be transfected or transduced are not enriched prior to contacting the cells with one or more nucleic acids.

In some embodiments, one or more of the cell processing steps in connection with obtaining, processing and/or incubating cells in connection with the proposed method, including in connection with obtaining a composition containing genetically engineered cells, can be performed in the interior of a centrifuge chamber. , such as a substantially rigid chamber that is typically cylindrical in shape and can be rotated about an axis of rotation, which may provide certain advantages over other available methods. In some embodiments, all processing steps are carried out in the same centrifuge chamber. In some embodiments, one or more processing steps are performed in different centrifuge chambers, such as multiple centrifuge chambers of the same type. Such methods include any of those described in International Publication No. WO 2016/073602.

Typical centrifuge chambers include those manufactured and sold by Biosafe SA, including those for use with Sepax® and Sepax® 2 systems, including the A-200/F and A-200 centrifuge chambers and various kits for use with such systems. Exemplary chambers, systems, and process equipment and cabinets are described in, for example, US Patent No. 6,125,555, US Patent No. 6,733,433 and Published Application for US Patent Publication No: US 2008/0171951 and Published International Patent Application Publication No. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety. Depending on the particular process (eg, dilution, washing, transduction, formulation), the skilled artisan may select a particular kit that is suitable for the process. Typical kits for use with such systems include, but are not limited to, disposable kits sold by BioSafe SA as products under the names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

In some embodiments, the system is included and/or placed in association with other instrumentation, including instrumentation for controlling, automating, controlling, and/or monitoring aspects of the various processing steps performed on the system. This apparatus, in some embodiments, is contained within a cabinet. In some embodiments, instrumentation includes a cabinet that includes a housing containing control circuitry, a centrifuge, a lid, motors, pumps, sensors, displays, and a user interface. A typical device is described in US Pat. No. 6,125,555, US Pat. No. 6,733,433 and US 2008/0171951.

In some embodiments, the system contains a number of containers, such as bags, tubes, stopcocks, clamps, connectors, and centrifuge chambers. In some embodiments, containers, such as pouches, include one or more containers, such as pouches, containing cells to be transduced or transfected and vector particles, such as viral vector particles or non-viral plasmids, in the same container or in separate containers such as single pack or individual packs. In some embodiments, the system further includes one or more containers, such as pouches, containing a medium such as a diluent and/or wash solution that is drawn into the chamber and/or other components to dilute, resuspend and/or wash the components and/or compositions during ways. The containers may be connected at one or more positions in the system, for example at a position corresponding to an inlet line, a diluent line, a flush line, an outlet line, and/or an outlet line.

In some embodiments, the system, such as a closed system, is sterile. In some embodiments, all connections of the components of the system, for example, between the pipeline and the container through the connector, are performed under sterile conditions. In some embodiments, the connections are made under laminar flow. In some embodiments, connections are made using a sterile connector that creates sterile connections, such as sterile welds, between the tubing and the container. In some embodiments, the implementation of the sterile connector device performs connection under thermal conditions, with a sufficiently high temperature to maintain sterility, such as a temperature of at least 200°C, for example, at least 260°C or 300°C.

In some embodiments, the system may be disposable, such as a disposable kit. In some embodiments, the disposable kit may be used in multiple cycles of the process or processes, such as at least 2, 3, 4, 5, or more times, such as processes that are continuous or semi-continuous. In some embodiments, a system, such as a disposable kit, is used to process cells from a single patient. In aspects of the methods, the processes need not be performed in the same closed system, such as one centrifuge chamber, but may be performed in another closed system, such as another centrifuge chamber; in some embodiments, such different centrifuge chambers are at corresponding points in methods placed in connection with the same system, such as those placed in connection with the same centrifuge. In some embodiments, all processing steps are performed in a closed system, in which all steps or a subset of each of one or more processing steps are performed in the same or different centrifuge chamber.

A. Targeted chimeric antigen receptors (CARs)

In some embodiments, the provided anti-idiotypic antibodies specifically bind to the extracellular domain of the target CAR, which contains an antigen-binding domain of an antibody or antibody fragment that provides specificity for the target antigen (e.g., a tumor antigen) and that is operably linked to or linked to an intracellular signaling domain. In some embodiments, the antigen binding domain comprises an SJ25C1 antibody or an antibody fragment of a portion derived from SJ25C1. In some embodiments, the antigen binding domain comprises an FMC63 antibody or an antibody fragment of a portion derived from FMC63. In some embodiments, an intracellular signaling domain is an activating portion of an intracellular domain, such as a T cell activation domain that provides a primary activation signal. In some embodiments, the implementation of the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to facilitate effector functions.

In some embodiments, engineered cells, such as T cells, are provided that express 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 embodiments, the antigen is a polypeptide. In some embodiments, this is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, such as tumor or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

In specific embodiments, the recombinant receptor, such as a chimeric receptor, comprises an intracellular signaling region that includes a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, e.g. , a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain of the CD3-zeta chain (CD3ζ) zeta chain or a functional variant or signal portion thereof) that contains an immunoreceptor tyrosine activation motif (ITAM).

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

Exemplary antigen receptors, including CARs, and methods for constructing and introducing such receptors into cells include those described, for example, in international patent applications WO 200014257, WO 2013126726, WO 2012/129514, WO 2014031687, WO 2013/166321, WO 2013 /071154, WO 2013/123061 заявке на патент США номер публикации US 2002131960, US 2013287748, US 20130149337, в патентах США №:6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353 and 8479118, and in European patent application number EP 2537416, and/or those described by Sadelain et al., Cancer Discov. April 2013; 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, antigen receptors include CAR as described in US Pat. No. 7,446,190 and receptors as described in International Patent Application Publication No. WO/2014055668 A1. Examples of CARs include those disclosed in any of the above publications such as WO 2014031687, US Patent No. 8339645, US Patent No. 7446179, US 2013/0149337, US Patent No. 7446190, US Patent No. 8389282, 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 WO 2014031687, US Patent No. 8339645, US Patent No. 7446179, US 2013/0149337, US Patent No. 7446190 and US Patent No. 8389282.

In some embodiments, the CAR is constructed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type that is being targeted by adoptive therapy, such as a tumor marker and/or an antigen designed to induce a debilitating response, such as an antigen expressed on cells of normal or healthy type. Thus, a CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigen-binding fragments, a domain or portion, or one or more variable domains of antibodies and/or antibody molecules. In some embodiments, a CAR comprises an antigen-binding portion or portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody (mAb).

In some embodiments, 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 may also be referred to as a TCR-like CAR. In some embodiments, an extracellular antigen-binding domain specific for the MHC-TCR-like CAR peptide complex is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, 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 co-stimulatory receptor.

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

In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, this is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells of the disease or condition, such as tumor or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

In some embodiments, the CAR contains an antibody or antigen-binding fragment (eg, scFv) that specifically recognizes an antigen, such as an intact antigen expressed on a cell surface.

In some embodiments, the antigen (or ligand) is a tumor antigen or tumor marker. In some embodiments, the antigen (or ligand) is an αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, 1B antigen cancer/testis (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, C-C Chemokine motif Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), epidermal growth factor truncated protein (tEGFR), epidermal growth factor receptor type III (EGFR vIII) mutation , epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc-like receptor 5 (FCRL5; also known as the Fc receptor homologue 5 or FCRH5 ), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, fetal acetylcholine r receptor, GD2 ganglioside, O-acetylated GD2 (OGD2), GD3 ganglioside, glycoprotein 100 (gp100), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, antigen, associated with high molecular weight human melanoma (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-receptor 13 alpha 2 (IL-13Ra2), insert kinase domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1CAM), L1-CAM CE7 epitope, leucine-rich repeat-containing family A member (LRRC8A), Lewis Y , melanoma associated antigen (MAGE) -A1, MAGE -A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D ligands (NKG2D) , melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor na, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tyrosine kinase receptor-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), glycoprotein 72 tumor-associated (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific antigen, universal label-related antigen, and/ or biotinylated molecules, and/or molecules expressed and/or characteristic or specific for HIV, HCV, HBV, HPV and/or other pathogens and/or their oncogenic variants. The antigens targeted by the receptors, in some embodiments, include antigens associated with B cell cancer, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

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

The antigens targeted by the receptors, in some embodiments, include antigens associated with B cell cancer, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30. In some embodiments, 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.

In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full length or may be an antigen-binding portion (Fab, F(ab') ₂ , Fv or single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, in particular is selected from, e.g., IgG1, IgG2, IgG3, and IgG4, more specifically , IgG1 (for example, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, in particular kappa.

Among the presented antibodies are fragments of antibodies. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to 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 ; dimers; linear antibodies; heavy chain variable regions (VH), single chain antibody molecules such as scFvs and VH single domain antibodies; and polyspecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH or VL, respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, 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. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domain from an antibody that binds to the antigen by screening a library of complementary VL or VH domains, respectively See, for example, Portolano et al., J. Immunol 150:880 -887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a tumor marker or cell surface antigen of the cell or disease being treated, such as a tumor cell or cancer cell, such as any of the target antigens described in this document or known in the art.

Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments containing permutations that do not occur naturally, such as fragments containing two or more antibody or chain regions connected by synthetic linkers, such as peptide linkers, and/or which are not can be obtained by enzymatic cleavage of natural intact antibodies. In some embodiments, the antibody fragments are scFv.

A "humanized" antibody is an antibody in which all or substantially all of the amino acid residues of the CDRs are derived from non-human CDRs and all or substantially all of the amino acid residues of the framework FRs are derived from human FRs. The humanized antibody may optionally 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 non-human variant of an antibody that has been humanized, typically to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Chimeric receptors, such as CARs, typically include an extracellular antigen-binding domain, such as part of an antibody molecule (e.g., SJ25C1 or FMC63), typically a heavy (VH) chain variable region and/or an antibody light (VL) chain variable region, for example, an scFv antibody fragment.

In some embodiments, the implementation of the chimeric antigen receptor includes an extracellular portion containing the antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling domain. In some embodiments, the implementation of the antibody or its fragment includes scFv. In some embodiments, the scFv is derived from SJ25C1 and contains the amino acid sequence of SEQ ID NO: 28. In some embodiments, the scFv is derived from FMC63 and contains the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the recombinant receptor, such as a CAR, such as an antibody portion thereof, further comprises a spacer, which may be or include at least a portion of an immunoglobulin constant region, or a variant or modified version thereof, such as a hinge region, e.g., a hinge region IgG4 and/or CH1/CL region and/or Fc region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, eg scFv, and the transmembrane domain. The spacer may be of a length that provides increased sensitivity of the cell after antigen binding compared to no spacer. In some examples, the spacer is equal to or about 12 amino acids long, or no more than 12 amino acids long. 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 whole number between the endpoints of any of the listed ranges. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Typical spacers include only an IgG4 hinge, an IgG4 hinge associated with the CH2 and CH3 domains, or an IgG4 hinge associated with the CH3 domain. In some embodiments, the spacer has SEQ ID NO: 150, and is encoded by SEQ ID NO: 151. In some embodiments, the spacer has SEQ ID NO: 152. In some embodiments, the spacer has SEQ ID NO: 153. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19: 3153, in International Patent Application Publication No. WO 2014031687, in US Patent No. 8822647 or in Published Application No. US 2014/0271635.

In some embodiments, the implementation of the constant region or part refers to IgD. In some embodiments, the spacer has the sequence of SEQ ID NO: 154. In some embodiments, the spacer has an amino acid sequence that is 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.

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

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

The transmembrane domain, in some embodiments, is derived from a natural or synthetic source. If the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., include at least the transmembrane region(s)) T-cell receptor alpha, beta or zeta chain, 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 is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain contains 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 the synthetic transmembrane domain. In some embodiments, communication is via linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains the transmembrane portion of CD28.

In some embodiments, the extracellular domain and the transmembrane domain may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any described herein. In some embodiments, the receptor comprises an extracellular portion of the molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28.

Intracellular signaling domains include 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 only one costimulatory receptor. In some embodiments, a short oligo- or polypeptide linker is present, such as a linker 2 to 10 amino acids long, such as a linker containing glycines and serines, such as a glycine-serine doublet, and forms a link between the transmembrane domain and the CAR cytoplasmic signaling domain.

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

A receptor, such as a CAR, typically includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signal sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences include sequences derived from the CD3 zeta chain, FcR-gamma, CD3-gamma, CD3-delta, and CD3-epsilon. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR comprises a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

In some embodiments, the receptor includes an intracellular component of the TCR complex, such as the CD3 TCR chain, which provides T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is associated with one or more cellular signaling modules. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the implementation of the receptor, such as CAR, further includes part 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 formed between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

In some embodiments, upon ligation of a 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 an immune cell, such as a T cell engineered to express CAR. For example, in some contexts, CAR induces T cell function, such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of an intact immunostimulatory chain, for example if it signals an effector function. In some embodiments, the intracellular signaling domain or domains include cytoplasmic T cell receptor (TCR) sequences, and in some aspects also co-receptor sequences, that naturally act in conjunction with such receptors to initiate signal transduction upon interaction with an antigen receptor, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functionality.

In the context of a natural TCR, full activation usually requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating a secondary or costimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating a secondary or costimulatory signal.

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

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

In some embodiments, the activating domain is included in one CAR while the co-stimulatory component is provided by another CAR recognizing a different antigen. In some embodiments, CARs include activating or stimulatory CARs, costimulatory CARs, both of which are expressed in the same cell (see WO 2014/055668). In some aspects, the cells include one or more stimulatory or activating CARs and/or costimulatory CARs. In some embodiments, the cells further comprise inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as CARs that recognize an antigen other than the antigen associated and/or specific for a disease or condition, whereby the activating signal delivered through the disease-targeted CAR is reduced or inhibited by binding the inhibitory CAR to its ligand, for example, to reduce off-target effects.

In some embodiments, the intracellular signaling domain comprises a transmembrane CD28 and a signaling domain associated with an intracellular CD3 domain (eg, CD3 zeta). In some embodiments, the intracellular signaling domain comprises chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains associated with the CD3-zeta intracellular domain.

In some embodiments, the implementation of the CAR includes one or more, for example, two or more co-stimulatory domain and an activation domain, for example, the primary activation domain, in the cytoplasmic part. Typical CARs include the intracellular components of CD3-zeta, CD28 and 4-1BB.

In some embodiments, the antigen receptor further includes a marker and/or CAR expressing cells or other antigen receptor further includes a surrogate marker, such as a cell surface marker, that can be used to confirm transduction or cell design to express the receptor. In some aspects, the marker includes all or part (eg, a truncated form) of CD34, NGFR, or an epidermal growth factor receptor, such as a truncated version of such a cell surface receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. For example, the marker and optionally the linker sequence can be any as described in Published Patent Application No. WO 2014031687. For example, the marker can be a truncated EGFR (tEGFR) which is optionally linked to a linker sequence, such as a cleavable T2A linker sequence. . In embodiments, the tEGFR comprises the amino acid sequence of SEQ ID NO: 155 or 156. In some embodiments, the tEGFR comprises an amino acid sequence having sequence identity equal to or about equal to 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 more than 99% with the sequences shown in SEQ ID NO: 155 or 156.

In some embodiments, the marker is a molecule, such as a cell surface protein, that is not naturally found in T cells or is not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a foreign molecule, such as a foreign protein, ie one that is not recognized as "self" by the host's immune system into which the cells will be adoptively transferred.

In some embodiments, the marker has no therapeutic function and/or no effect other than for use as a marker for genetic engineering, such as the selection of successfully engineered cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise has some intended effect, such as an in vivo ligand for a cell, such as a co-stimulatory or immune control molecule, to enhance and/or attenuate cell responses after adoptive transfer, and encounters with the ligand.

In some cases, CARs refer to first, second and/or third generation CARs. In some aspects, a first generation CAR is one that only provides a CD3 chain-induced signal upon antigen binding; in some aspects, a second generation CAR is one that provides such a signal and a costimulatory signal, for example one that includes 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 various costimulatory receptors.

In some embodiments, the implementation of 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 embodiments, the antibody or fragment thereof comprises an scFv or single domain VH antibody and the intracellular domain comprises ITAM. In some aspects, the intracellular signaling domain includes the CD3-zeta (CD3ζ) zeta chain signal domain. In some embodiments, the implementation of the chimeric antigen receptor includes a transmembrane domain located between the extracellular domain and the intracellular signaling region.

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

For example, in some embodiments, a CAR comprises an antibody, such as an antibody fragment, a transmembrane domain that is or contains a CD28 transmembrane portion or a functional variant thereof, and an intracellular signaling domain comprising a CD28 signal portion or a functional variant thereof and a CD3-zeta signal portion or a functional variant thereof. functional option. For example, in some embodiments, a CAR comprises an antibody, such as an antibody fragment, a transmembrane domain that is or contains a CD28 transmembrane portion or a functional variant thereof, and an intracellular signaling domain comprising a 4-1BB signal portion or a functional variant thereof and a CD3-zeta signal portion. or its functional variant. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, such as an IgG4 hinge, such as a spacer containing only a hinge.

In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (accession number: P10747.1), or is a transmembrane domain that contains the amino acid sequence SEQ ID NO: 157, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 157; in some embodiments, the transmembrane domain-containing portion of the recombinant receptor contains the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In some embodiments, the chimeric antigen receptor contains the intracellular domain of a co-stimulatory T cell molecule. In some aspects, the costimulatory T cell molecule is CD28 or 4-1BB.

In some embodiments, the intracellular signaling region comprises a human CD28 intracellular costimulatory signaling domain, or a functional variant or portion thereof, such as its 41 amino acid domain and/or such domain with an LL-GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence of SEQ ID NO: 159 or 160, or an amino acid sequence that has 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 embodiments, the intracellular region contains the intracellular co-stimulatory signaling domain 4-1BB or a functional variant or portion thereof, such as the human 4-1BB 42 amino acid cytoplasmic domain (accession number Q07011.1), or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 161, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with SEQ ID NO: 161.

In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally stimulating the CD3-zeta signaling domain or a functional variant thereof, such as the human CD3ζ isoform 3 cytoplasmic domain 112 AA (accession number: P20963.2) or the CD3-zeta signaling domain, as described in US Pat. No. 7,446,190 or US Pat. No. 8,911,993. In some embodiments, the intracellular signaling region comprises an amino acid sequence of SEQ ID NO: 162, 163, or 164, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 162, 163 or 164.

In some aspects, the spacer comprises an IgG hinge only, such as an IgG4 or IgG1 hinge only, such as the hinge only spacer shown in SEQ ID NO: 150. with CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge associated with the CH2 and CH3 domains, such as shown in SEQ ID NO: 152. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge associated only with the CH3 domain. , such as those shown in SEQ ID NO: 153. In some embodiments, the spacer is or contains a glycine-serine rich sequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, a CAR includes an antibody, such as an antibody fragment comprising an scFv, 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 a spacer containing An Ig hinge, a transmembrane domain containing all or part of the CD28-derived transmembrane domain, an intracellular signaling domain derived from CD28, and a CD3-zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment thereof such as scFv, a spacer such as any of the spacers containing an Ig hinge, a transmembrane domain derived from CD28, an intracellular signaling domain derived from 4-1BB, and a signaling domain derived from from CD3-zeta.

In some embodiments, the nucleic acid molecules encoding such CAR constructs further comprise a sequence encoding the T2A ribosome skip element and/or a tEGFR sequence, for example, downstream of the sequence encoding the CAR. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be made to express truncated EGFR (EGFRt) as a non-immunogenic selective epitope (e.g., by introducing a construct encoding CAR and EGFRt separated by a T2A ribosome switch for expression of two proteins from the same construct), which can then be used as a marker for the detection of such cells (see, for example, US patent No. 8802374). In some embodiments, a single promoter may direct the expression of an RNA that contains two or three genes (e.g., encoding a molecule involved in the modulation of a metabolic pathway and encoding a recombinant receptor) in a single open reading frame (ORF) separated from each other by sequences encoding a self-cleaving peptide (eg, 2A sequences); or a protease recognition site (eg, furin). Thus, the ORF encodes a single polypeptide, which either during translation (in the case of 2A) or after translation is converted into separate proteins. In some cases, a peptide such as T2A can cause a ribosome to skip (skipping a ribosome) the synthesis of a peptide bond at the C-terminus of element 2A, resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic Vaccines and Ther 2:13 (2004) and deFelipe et al Traffic 5:616-626 (2004)). Many elements of 2A are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, without limitation, 2A sequences from foot-and-mouth disease virus (F2A, e.g. SEQ ID NO: 131), equine rhinitis virus A (E2A, e.g. SEQ ID NO: 130), Thosea asigna virus (T2A, eg SEQ ID NO: 126 or 127) and porcine teshovirus-1 (P2A, eg, SEQ ID NO: 128 or 129) as described in US Patent Publication. No. 20070116690.

Recombinant receptors, such as CARs, expressed by cells administered to a patient typically recognize or bind specifically to a molecule that is expressed, associated, and/or specific to the disease or condition or its cells being treated. When specifically bound to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM transducer signal, into the cell, thereby stimulating an immune response directed at the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by the cell or tissue of the disease or condition, or is associated with the disease or condition. The receptor may be another receptor, such as an immunoinhibitory or co-stimulatory receptor, a stimulatory signal such as CCR or iCAR, or a non-signaling receptor, for example, for use in cell depletion or elimination using antibodies.

B. Genetically engineered cells and methods for producing cells

Among the cells expressing chimeric antigen receptors, there are engineered cells. Genetic engineering typically involves introducing a nucleic acid encoding a recombinant or engineered component into a composition containing cells, for example, by retroviral transduction, transfection, or transformation. Various methods of introducing genetically engineered components, such as recombinant receptors, such as CAR, are well known and can be used. Exemplary methods include methods for transferring receptor-encoding nucleic acids, including via viral, eg, retroviral or lentiviral transfer, transduction, transposons, and electroporation.

1. Vectors and methods of genetic engineering

Also provided are one or more polynucleotides (eg, nucleic acid molecules) encoding chimeric antigen receptors (CARs), vectors for genetically engineered cells to express such CARs, and methods for producing the engineered cells. In some embodiments, the vector contains a nucleic acid encoding a CAR. In some instances, the vector is a viral vector such as a retroviral vector such as a lentiviral vector or a retroviral gamma vector.

In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see, for example, 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.

In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from Moloney mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen necrosis virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any source of avian or mammalian cells. Retroviruses are generally amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the expressed gene replaces the gag, pol and/or env retroviral sequences. A number of exemplary retroviral systems have been described (e.g., US Pat. (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.

Known methods of lentiviral transduction. Typical methods are described, for example, in 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.

In some embodiments, recombinant nucleic acids are transferred to T cells via electroporation (see, for example, Chicaybam et al., (2013) PLoS ONE 8 (3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7 (16 ): 1431-1437). In some embodiments, recombinant nucleic acids are transferred to T cells via transposition (see, for example, 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 for introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; microparticle bombardment of tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International Patent Application Publication No: WO 2014055668 and US Patent No. 7446190.

In some embodiments, cells, eg, T cells, can be transfected either during or after expansion, eg, with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the target receptor gene can be performed, for example, using any suitable retroviral vector. The genetically modified cell population can then be isolated from the original stimulus (eg, an anti-CD3/CD28 antibody stimulus) and subsequently stimulated with a second type of stimulus, eg, via a de novo injected receptor). This second type of stimulus may include an antigenic stimulus in the form of a peptide/MHC molecule, a cognate (cross-linking) ligand of a genetically introduced receptor (eg, a natural CAR ligand), or any ligand (such as an antibody) that binds directly within the receptor scaffold (eg, by recognition of 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 embodiments, the cells are stimulated with the proposed anti-idiotypic antibody in accordance with the proposed methods.

In some cases, a vector may be used that does not require activation of cells, such as T cells. In some such cases, cells may be selected and/or transduced prior to activation. Thus, cells can be constructed before or after cell culture, and in some cases simultaneously or during at least part of the culture process.

Among the additional nucleic acids, for example, are genes for introduction, these are genes that increase the effectiveness of therapy, for example, by stimulating the viability and/or function of the transferred cells; genes for providing a genetic marker for selection and/or evaluation of cells, for example, for evaluation of survival or localization in vivo; genes to improve safety, for example, making the cell susceptible to negative selection in vivo, as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al. Human Human Therapy 3: 319-338 (1992); see also PCT/US 91/08442 and PCT/US 94/05601 Lupton et al. describing the use of bifunctional selectable fusion genes resulting from the fusion of a dominant positive selection marker with a negative selection marker. See, for example, Riddell et al., US Pat. No. 6,040,177, columns 14-17.

2. Cells and preparation of cells for genetic engineering

The present disclosure provides cells, including engineered cells, that contain a chimeric antigen receptor (CAR). Populations of such cells and compositions containing such cells are also provided. Among the compositions are injectable compositions containing cells in which one or more cells are known or likely to express a recombinant receptor capable of being recognized by or bound by a binding molecule present on one or more particles with which the cells are incubated or contacted. Also among the compositions are compositions obtained using the proposed methods, including final compositions that contain stimulated or extended cells, including compositions enriched with cells containing a recombinant receptor bound or recognized by a binding particle molecule, such as cells expressing a recombinant receptor, for example chimeric receptor, is at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more percent of the total number of cells in the composition or cells of a certain type, such like T cells or CD8+ or CD4+ cells. Thus, genetically engineered cells expressing recombinant receptors such as CAR are provided.

Among the compositions are pharmaceutical compositions and formulations for administration, for example for adoptive cell therapy. Also provided are methods for constructing, manufacturing, or generating such cells, therapeutic methods for administering the cells and compositions to individuals, such as patients, and methods for detecting, selecting, isolating, or separating such cells.

In some embodiments, the nucleic acids are heterologous, that is, not normally present in a cell or sample derived from a cell, such as derived from another organism or cells that, for example, are not normally detectable in the engineered cell and/or organism from which the cell is derived. such a cell. In some embodiments, nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one that contains chimeric combinations of nucleic acids encoding different domains from many different cell types.

The cells are typically eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, cells derived from blood, bone marrow, lymph, or lymphoid organs are cells of the immune system, such as cells of innate or adaptive immunity, such as 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).

The cells are typically primary cells, such as cells isolated directly from patient tissues and/or isolated from patient tissues and frozen. In some embodiments, the cells include one or more subgroups of T cells or other cell types, such as entire populations of T cells, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for ability to differentiate, reproduce, recirculate, localize and/or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. For the patient to be treated, the cells may be allogeneic and/or autologous. Methods include predefined 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 embodiments, the methods include isolating cells from a patient, preparing, processing, culturing and/or constructing them, and reintroducing them to the same patient before or after cryopreservation.

Among the subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells are naive T cells (TN), effector T cells (TEFF), memory T cells and their subtypes, such as stem memory T cells. (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells cells, cytotoxic T cells, mucus associated invariant T cells (MAIT), natural and adaptive regulatory T cells (Treg), 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.

In some embodiments, the cells are natural killer (NK). In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

In some embodiments, cells include one or more genetically engineered nucleic acids and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, that is, not normally present in a cell or sample derived from a cell, such as derived from another organism or cells that, for example, are not normally detectable in the engineered cell and/or organism from which the cell is derived. such a cell. In some embodiments, nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one that contains chimeric combinations of nucleic acids encoding different domains from many different cell types.

In some embodiments, obtaining engineered cells includes one or more culturing and/or preparation steps. Cells for administration of a nucleic acid encoding a transgenic receptor, such as a CAR, may be isolated from a sample, such as a biological sample, eg, obtained from or derived from a patient. In some embodiments, the patient from which the cell is isolated is a patient suffering from a disease or condition or in need of cell therapy, or to whom cell therapy will be prescribed. A patient, in some embodiments, is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, such as human primary cells. Samples include tissues, fluids, and other samples taken directly from a patient, as well as samples obtained from one or more processing steps such as separation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source, or a sample that is being 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 from them.

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 leukopheresis product. Sample specimens include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, mucosal-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen , other lymphoid tissues, liver, lung, stomach, intestines, colon, kidneys, pancreas, mammary gland, bones, prostate, cervix, testicles, ovaries, tonsils or other organ and / or cells derived from them. Samples include, in the context of cell therapy, such as adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, such as T cell lines. The cells, in some embodiments, are derived from a xenogeneic source, such as a mouse, a rat, a non-human primate, and a pig.

In some embodiments, cell isolation includes one or more non-affinity based preparation and/or 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 properties such as density, adhesive properties, size, sensitivity, and/or resistance to particular components.

In some examples, cells from the patient's circulating blood are obtained, for example, by apheresis or leukopheresis. Samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and/or platelets, and in some aspects contain cells other than erythrocytes and platelets. In some embodiments, prior to cell selection and/or cell enrichment, the sample or cells in the sample may be quiescent or held prior to further processing steps. In some embodiments, the sample is held or maintained at or about 2 to 8°C for up to 48 hours, such as up to 12 hours, 24 hours, or 36 hours. In certain embodiments, the cells are not selected and/or enriched prior to contacting the cells with one or more nucleic acids. In some embodiments, the sample or cells may be quiescent or held prior to contacting or incubating the cells with one or more nucleic acids. In some embodiments, the sample is held or maintained at or about 2 to 8°C for up to 48 hours, such as up to 12 hours, 24 hours, or 36 hours, before cells are contacted or incubated with one or more nucleic acids.

In some embodiments, blood cells collected from a patient are washed, such as by removing a plasma fraction and placing the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is free of calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, the washing step is performed by a semi-automated "flow through" centrifuge (eg, Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of the blood cell sample are removed and the cells directly resuspended in culture medium.

In some embodiments, the methods include methods for separating cells based on density, such as obtaining leukocytes from peripheral blood by lysing erythrocytes and centrifuging in a Percoll or Ficoll gradient.

In some embodiments, isolation methods include separating different types of cells based on the expression or presence of one or more specific molecules in the cell, such as surface markers, such as surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, isolation in some aspects involves separating cells and cell populations based on cell expression or the level of expression of one or more markers, typically cell surface markers, such as by incubation with an antibody or binding partner that specifically binds to such markers that would normally followed by the step of washing and separating the cells containing the bound antibody or binding partner from those cells that are not bound to the antibody or binding partner.

Such separation steps may be based on positive selection, in which cells containing the bound reagents are retained for later use, and/or negative selection, in which cells not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, negative selection can be particularly useful when there are no antibodies available that specifically identify the cell type in a heterogeneous population, so separation is best done on the basis of markers expressed by cells other than the target population.

Separation should not lead to 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment for cells of a certain type, such as cells expressing a marker, refers to an increase in the number or percentage of such cells, but does not necessarily lead to the complete absence of cells not expressing the marker. Similarly, down-selection, removal or depletion of cells of a certain type, such as marker-expressing cells, refers to a reduction in the number or percentage of such cells, but should not result in the complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, where a positively or negatively selected fraction from one stage 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 the cells with multiple antibodies or binding partners, each specific for the marker targeted by negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells that express one or more surface markers at a high level, or are positive, for example, for CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+ T cells are isolated by positive or negative selection methods.

For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (eg DYNABEADS® M-450 CD3/CD28 T Cell Expander). In specific embodiments, cells are contacted with anti-CD3/CD28 antibody-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T cell expander) to expand CD3+, CD28+ T cells prior to exposing the cells to one or more nucleic acids. . In certain embodiments, cells are not contacted with anti-CD3/CD28 antibody-conjugated magnetic beads prior to contacting the cells with one or more nucleic acids.

In some embodiments, the implementation of the selection is carried out by enriching a certain population of cells by positive selection or depletion of a certain population of cells by negative selection. In some embodiments, positive or negative selection is made by incubating cells with one or more antibodies or other agents that specifically bind to one or more surface markers expressed (marker+) or expressed at a relatively higher level (marker high) at a positive or negatively selected cells, respectively.

In some embodiments, T cells are separated from the PBMC sample by negative selection for markers, such as CD14, expressed on cells other than T cells, such as B cells, monocytes, or other leukocytes. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper cells from CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed at a relatively higher level on one or more T cell subsets of naïve T cells, memory T cells and/or effector T cells. cells.

In some embodiments, CD8+ cells are further enriched or depleted for naïve, 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 embodiments, enrichment in central memory T cells (TCM) is performed to increase efficiency, for example, to improve long-term survival, expansion and/or engraftment after administration, which in some aspects is particularly robust in such subpopulations, See Terakura et al. (2012) Blood.1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

In embodiments, memory T cells are present in both the CD62L+ and CD62L- subgroups of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted in CD62L-CD8+ and/or CD62L+ CD8+ fractions, for example using anti-CD8 and anti-CD62L antibodies.

In some embodiments, central memory (TCM) T cell enrichment is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects it is based on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, the isolation of the CD8+ population enriched in TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells, expressing CD62L. In one aspect, enrichment for central memory T cells (TCM) is performed by starting with a negative fraction of cells selected based on CD4 expression, which is negatively selected based on CD14 and CD45RA expression and positively selected based on CD62L. Such selection is in some aspects performed simultaneously, and in other aspects sequentially, in any order. In some aspects, the same selection step based on CD4 expression used in generating a population or subset of CD8+ cells is also used to generate a population or subset of CD4+ cells such that both positive and negative fractions from a CD4 based separation are retained and used. in subsequent steps of the methods, optionally after one or more additional positive or negative selection steps.

In a specific example, a PBMC sample or other leukocyte sample is subjected to a selection of CD4+ cells where both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19 and positive selection based on the characteristics of central memory T cell markers such as CD62L or CCR7, where positive and negative selection is carried out in any order.

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

In one example, for enrichment of CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is coupled to a solid support or matrix, such as a magnetic microsphere or paramagnetic microsphere, to allow cell separation for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation methods (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).

In some aspects, the sample or cell composition to be separated is incubated with a small magnetizable or magnetically sensitive material, such as magnetically sensitive particles or microparticles, such as paramagnetic microspheres (eg, such as Dynalbeads microspheres or MACS microspheres). The magnetically sensitive material, such as a particle, is typically associated directly or indirectly with a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell, cells, or cell population that is desired to be separated, such as that is desired negatively or positively select.

In some embodiments, the implementation of the magnetic particle or microsphere contains a magnetically sensitive material associated with a particular binding element, such as an antibody or other binding partner. There are many known magnetically sensitive materials used in magnetic separation techniques. Suitable magnetic particles include those described in Molday, US Patent 4452773 and in European patent EP 452342 B, which are incorporated herein by reference. Other examples are colloidal sized particles such as those described in Owen US Pat. 4795698 and Liberti et al., Pat. 5200084 are other examples.

Incubation is usually carried out under conditions under which antibodies or binding partners or molecules, such as secondary antibodies or other reagents that specifically bind to such antibodies or binding partners that are attached to the magnetic particle or microsphere, specifically bind to cell surface molecules, if they are present on the cells in the sample.

In some aspects, the sample is placed in a magnetic field, and those cells to which magnetically sensitive or magnetizable particles are attached will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are kept; cells that are not attracted (unlabeled cells) are kept for negative selection. In some aspects, the 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 subjected to additional separation steps.

In some embodiments, the magnetically sensitive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells by coating with primary antibodies specific for one or more markers. In some embodiments, cells, rather than microspheres, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (eg, streptavidin) are added. In some embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically sensitive particles are left attached to the cells, which are then to be incubated, cultured and/or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, magnetizable or magnetically sensitive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetically activated cell sorting systems (MACS) are capable of selecting cells with magnetized particles attached to them with a high degree of purity. In some embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted following the application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached particles are eluted. Then, after this first elution step is completed, the samples that were magnetically trapped and not eluted are released in some way so that they can be eluted and recovered. In some embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

In some embodiments, isolation or separation is carried out using a system, device, or apparatus that performs one or more of the method steps of isolation, preparation, separation, processing, incubation, culture, and/or formulation. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user manipulation, and/or contamination. In one example, the system is the system described in international patent application, publication number WO 2009/072003 or US 20110003380 A1. In one example, the system is the system described in International Publication No. WO 2016/073602.

In some embodiments, the system or device performs one or more, for example, all of the steps of isolation, processing, design, and formulation in an integrated or stand-alone system and/or in an automated or programmable manner. In some aspects, the system or device includes a computer and/or a computer program in communication with the system or device that allows a user to program, control, evaluate, and/or customize various aspects of the processing, isolation, construction, and formulation steps.

In some aspects, the separation and/or other steps are carried out using the CliniMACS system (Miltenyi Biotec), for example, to automatically separate cells at the clinical scale level in a closed and sterile system. Components may include an embedded microcomputer, a magnetic separation unit, a peristaltic pump and various pinch valves. The built-in computer in some aspects controls all the components of the instrument and instructs the system to carry out repetitive procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a selection column holder. The peristaltic pump controls the flow rate throughout the pipeline and, together with the pinch valves, provides a controlled buffer flow through the system and a constant cell suspension.

The CliniMACS system, in some aspects, uses antibody-bound magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a 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 guard column and separation column, and is for single use only. After starting the separation program, the system automatically applies the cell sample to the separation column. Labeled cells are retained on the column while unlabeled cells are removed through a series of washing steps. In some embodiments, cell populations for use with the methods described herein are unlabeled and are not stored on the column. In some embodiments, cell populations for use with the methods described herein are labeled and stored in a column. In some embodiments, cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field and collected in a cell collection bag.

In some embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system is in some aspects equipped with a cell processing unit that allows automatic washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an integrated camera and image recognition software that determines the optimal cell fractionation end point by identifying macroscopic layers of the original cell product. For example, peripheral blood is automatically separated into layers of erythrocytes, leukocytes and plasma. The CliniMACS Prodigy system can also include an integrated cell culture chamber that performs cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Source ports can allow for sterile removal and replenishment of media, and cells can be monitored with an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35 (9): 651-660, Terakura et al. (2012) Blood.1: 72-82, and Wang et al. (2012) J Immunother. 35(9): 689-701.

In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a liquid stream. In some embodiments, the cell population described herein is harvested and enriched (or depleted) by preparative scale sorting (FACS). In certain embodiments, the cell population described herein is harvested and enriched (or depleted) by using microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, for example, 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 well-defined subsets of T cells to be identified. with a high degree of purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on the binding of antibodies or other binding partners specific for one or more cell surface markers is carried out in a liquid stream, such as by fluorescence-activated cell sorting (FACS), including a preparative scale (FACS) and/or MEMS chips. systems (MEMS), for example, in combination with a flow cytometry detection system. Such methods allow positive and negative selection based on several markers at the same time.

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

In some embodiments, cells are incubated and/or cultured prior to or in connection with genetic engineering. Incubation steps may include culturing, cultivating, stimulating, activating and/or expanding. The incubation and/or construction may be carried out in a culture vessel such as a block, chamber, well, column, tube, tube set, valve, vial, culture dish, bag or other cell culture or culture container. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to cause cells to proliferate, proliferate, activate and/or survive in a population, mimic antigen exposure, and/or prime cells for genetic engineering, such as introducing a recombinant antigen receptor.

Conditions may include one or more of the following: specific environments, temperature, oxygen content, carbon dioxide content, time, agents such as 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 cells.

In some embodiments, the implementation of the stimulating conditions or agents include one or more agents, such as a ligand, that is capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for TCR, such as anti-CD3 antibodies. In some embodiments, stimulatory conditions include one or more agents, such as a ligand, that is capable of stimulating a co-stimulatory receptor, such as an anti-CD28 antibody. In some embodiments, such agents and/or ligands may be associated with a solid support, such as a microsphere, and/or with one or more cytokines. Optionally, the expansion method may further comprise the step of adding the anti-CD3 antibody and/or the anti-CD28 antibody to the culture medium (eg, at a concentration of at least about 0.5 ng/mL). In some embodiments, stimulatory agents include IL-2, IL-15, and/or IL-7. In some aspects, the concentration of IL-2 is at least about 10 units/ml.

In some aspects, the incubation is carried out in accordance with techniques such as those described in US patent No. 6040177, according 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.

In some embodiments, T cells are expanded by adding feeder cells to the starter culture composition, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the resulting cell population contains at least about 5, 1020, or 40 or more PBMC feeder cells for each T-lymphocyte in the initial population to be expanded); and by incubating the culture (eg, for a period of time sufficient to build up the number of T cells). In some aspects, the non-dividing feeder cells may comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of T cell populations.

In some embodiments, stimulation conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees, and typically at or about 37 degrees Celsius. . Optionally, the incubation may further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCLs can be irradiated with gamma rays ranging from about 6000 to 10000 rads. LCL feeder cells, in some aspects, are present in any suitable amount, such as at a ratio of LCL feeder cells to original T lymphocytes of at least about 10:1.

IV. Compositions

Also provided are compositions comprising binding molecules such as antibodies as provided herein, including pharmaceutical compositions and formulations. Compositions and formulations typically include one or more optional acceptable carriers or excipients.

The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the patient to whom the composition will be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the patient. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule and/or antibody, and/or route of administration. Accordingly, there are many suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, 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 from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic 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 counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

Buffer 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 buffer agents is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for the preparation of administered pharmaceutical compositions are known. Typical methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

In some aspects, the composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, 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 from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in 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 counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

Antibody formulations may include lyophilized formulations and aqueous solutions.

Compositions in some embodiments are provided as sterile liquid preparations, eg, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer periods of contact with particular tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent such as a mixture with a suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose or the like. The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosifying agents, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the intended formulation. Standard techniques may in some aspects be taken into account for the preparation of suitable formulations.

Various additives may be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of an injectable pharmaceutical form can be achieved by the use of absorption delaying agents such as aluminum monostearate and gelatin.

The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting, dispersing, or emulsifying agents (eg, methylcellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, coloring agents, and the like. Standard techniques may in some aspects be taken into account for the preparation of suitable formulations.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

V. Methods and uses of antibodies

In some embodiments, provided herein are methods comprising the use of one or more anti-idiotypic antibodies. In some aspects, provided 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 a target antibody, such as the activity of a CAR, or the activity of a cell expressing the CAR. In some embodiments, one or more anti-idiotypic antibodies bind, detect, identify, and/or quantify CARs and/or CAR-expressing cells. In some embodiments, the methods provided herein comprise one or more steps of contacting and/or incubating one or more anti-idiotypic antibodies with a cell or sample containing or suspected of containing cells that express a chimeric antigen receptor (CAR). In some embodiments, the anti-idiotypic antibody is treated, incubated, and/or contacted with the composition or sample under conditions that allow the formation of a complex between the anti-idiotypic antibody and a target antibody, such as CAR. In some aspects, the complex may be used for the purpose of detecting, isolating and/or measuring CAR. In some embodiments, the formation of the complex modifies the activity of the target antibody, e.g., CAR, e.g., by stimulating receptor signaling activity, or in some embodiments, by antagonizing the activity of the target antibody, e.g., CAR, by preventing association of the CAR with the antigen.

A. Detection/Isolation Methods

In some embodiments, methods are provided that include using one or more anti-idiotypic antibodies and/or molecules (such as conjugates and complexes) containing one or more such anti-idiotypic antibodies to detect, bind, and/or isolate an antibody, such as a target antibody. In some embodiments, the methods provide for one or more steps of contacting, incubating, and/or displaying one or more anti-idiotypic antibodies with a sample and/or composition. In some embodiments, the sample and/or composition is likely to contain and/or is suspected to contain a target antibody and/or antigen-binding fragment thereof that binds to and/or is recognized by one or more anti-idiotypic antibodies. In some embodiments, an antibody or antigen-binding fragment thereof that is bound to or recognized by one or more anti-idiotypic antibodies contains one or more fusion domains and/or is a fusion protein. In some embodiments, the target antibody and/or antigen-binding fragment thereof is a CAR. In certain embodiments, the anti-idiotypic antibody binds to and/or recognizes an anti-CD19 antibody (e.g., an SJ25C1 or FMC63 antibody) or an antigen-binding fragment thereof, including a chimeric molecule or conjugate comprising a CAR containing such an anti-CD19 antibody (e.g., an antibody fragment).

Methods in some embodiments include incubating, processing and/or contacting a sample and/or composition containing or suspected to contain a target antibody with an anti-idiotypic antibody. In some embodiments, the incubation is performed under conditions that allow the anti-idiotypic antibody to bind to the target antibody present in the composition, for example, to form a complex containing the anti-idiotypic antibody and the target antibody.

In some embodiments, the sample and/or composition contains or is suspected of containing a target antibody, such as CAR. In some embodiments, the sample and/or composition contains or is suspected of containing cells that express the target antibody, such as CAR. In certain embodiments, the sample is a biological sample. In specific embodiments, the sample is a serum sample or a blood sample. In some embodiments, the biological sample contains one or more immune cells. In some embodiments, the biological sample is or is derived from a tissue, such as connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In specific embodiments, the biological sample is or is derived from the heart, vasculature, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, hypothalamus, pituitary, pineal, thyroid, parathyroid glands, adrenals, kidneys, ureters, bladder, urethra, lymphatic system, skin, muscles, brain, spinal cord, nerves, ovaries, uterus, testicles, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bones , cartilage, ligaments or tendons. In specific embodiments, a biological sample is collected, collected and/or obtained from a human. In certain embodiments, the sample contains cells that are alive and/or intact. In some embodiments, the sample is or contains a homogenate and/or cells that have been disrupted and/or lysed. In some embodiments, the biological sample contains proteins and/or antibodies that have been isolated from blood, serum, and/or tissue.

In specific embodiments, the anti-idiotypic antibody forms or is capable of complexing with a target antibody, such as CAR. In specific embodiments, the complex is detected, measured, quantified, and/or evaluated, eg, to allow detection, identification, measurement, and/or quantification of the target antibody, eg, in a composition or sample. In some embodiments, the methods include detecting whether a complex is formed between an anti-idiotypic antibody and a target antibody in a sample and/or detecting the presence or absence or level of such binding. In some embodiments, the complex contains a detectable label. In specific embodiments, the anti-idiotypic antibody is an immunoconjugate that contains a detectable label. In certain embodiments, the anti-idiotypic antibody comprises, conjugates, binds to, and/or attaches to a detectable label. In some embodiments, the complex contains an antibody that binds to and/or recognizes an anti-idiotypic antibody, eg, a secondary antibody that is conjugated, linked, and/or fused to a detectable label.

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

In some embodiments, the target antibody or antigen-binding fragment is associated with a cell or expressed on the cell surface. In specific embodiments, the implementation of the target antibody, for example, CAR, is not associated with or contained in the cell, for example, in some embodiments, the implementation of the target antibody is secreted. In some embodiments, the implementation of the antibody was separated, removed and/or lysed from the surface of the cell.

In some embodiments, the target antibody, or antigen-binding fragment thereof, is contained in a chimeric antigen receptor (CAR), such as a CAR expressed on a cell surface. In some embodiments, the cell is a stem cell, such as an iPSC or an immune cell. In some embodiments, the immune cell is a T cell, e.g., CD4+ T cell, CD8+ T cell, naive T (TN) cell, effector T cell (TEFF), memory T cell, tumor infiltrating lymphocyte (TIL) , immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal associated invariant T cells (MAIT), natural and adaptive regulatory T cells (Treg), 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/or delta/gamma T cells. In some embodiments, the cell is derived from tissue, e.g., heart, vascular network, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, hypothalamus, pituitary, pineal, thyroid, parathyroid glands, adrenal glands, kidneys, ureter, bladder, urethra, lymphatic system, skin, muscle, brain, spinal cord, nerves, ovaries, uterus, testes, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, bones , cartilage, ligament or tendon.

In some embodiments, the target antibody is an anti-CD19 antibody. In some embodiments, the target antibody is or is derived from an SJ25C1 antibody or antigen-binding fragment thereof. In some embodiments, the target antibody, or antigen-binding fragment thereof, comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody is or is derived from an antibody FMC63 or its antigen-binding fragment. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for detecting a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof (and/or chimeric molecules containing such an antibody, such as an antibody fragment, such as CAR), comprising contacting a composition containing the antibody -target or antigen-binding fragment thereof, with anti-idiotypic antibody or antigen-binding fragment or anti-idiotypic antibody immunoconjugate described herein, and detection of anti-idiotypic antibody associated with the target antibody or antigen-binding fragment. In some embodiments, the method further comprises detecting whether a complex is formed between the anti-idiotypic antibody and the target antibody in the composition, such as detecting the presence or absence or level of such binding. In some embodiments, the target antibody or antigen-binding fragment is associated with a cell or expressed on the surface of the cell, and the detection includes detection of cells associated with the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method is provided for isolating a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof (and/or chimeric molecules containing such an antibody, such as an antibody fragment, such as CAR), comprising contacting the composition and/or a sample containing a target antibody or antigen-binding fragment thereof with an anti-idiotypic antibody or an antigen-binding fragment thereof or an anti-idiotypic antibody immunoconjugate described herein, and isolating complexes containing an anti-idiotypic antibody bound to a target antibody or antigen-binding fragment thereof fragment. In some embodiments, the target antibody is isolated using an anti-idiotypic antibody or an antigen-binding fragment thereof, such as an immunoconjugate, such as the immunoconjugate described in section I-F. In some embodiments, the target antibody, or antigen-binding fragment thereof, is associated with or expressed on the surface of a cell, and isolation comprises isolation of cells associated with the anti-idiotypic antibody. In some embodiments, complexes containing the anti-idiotypic antibody are isolated by affinity separation. In some embodiments, the affinity separation is selected from the group consisting of immunoaffinity separation, magnetic separation, and affinity chromatography. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for detecting a cell expressing a CAR containing a target antibody, such as an SJ25C1 or FMC63 antibody, or antigen-binding fragments thereof, comprising contacting a cell expressing a CAR with an anti-idiotypic antibody or antigen-binding fragment thereof, or an anti-idiotypic immunoconjugate. antibodies described herein, and detection of cells associated with an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In certain embodiments, the methods for detecting a target antibody with an anti-idiotypic antibody described herein are used to evaluate the target antibody in a patient. For example, in some embodiments, provided herein are methods of using an anti-idiotypic antibody to assess, measure, and/or quantify the in vivo pharmacokinetics, expansion, and/or persistence of CAR-expressing cells of a therapeutic cell composition. In some embodiments, in vivo pharmacokinetics, expansion and/or persistence of cells, and/or changes in cellular phenotypes or functional activity of cells, such as CAR-expressing cells, administered for immunotherapy, such as CAR-T cell therapy, in the methods provided in herein can be measured using the anti-idiotypic antibodies provided herein. In some embodiments, the pharmacokinetics, expansion, and/or persistence of CAR-expressing cells is measured by determining the presence and/or number of CAR-expressing cells in a patient and/or in a patient sample following administration of a therapeutic cell composition during and/or after administration of therapy using the anti-idiotypic antibody provided herein.

In some aspects, an anti-idiotypic antibody is used with flow cytometry to evaluate the number of cells expressing the recombinant receptor (e.g., CAR-expressing cells administered for T-cell therapy) in blood, serum, organ, or tissue (e.g., at a disease site, e.g. in a tumor sample) of the patient. In some aspects, persistence is quantified as the number of CAR-expressing cells per microliter of sample, such as blood or serum, or the total number of peripheral blood mononuclear cells (PBMCs) or leukocytes or T cells per microliter of sample. In certain aspects, expansion is quantified as the increase in CAR-expressing cells per microliter between samples, e.g., blood or serum, or in total peripheral blood mononuclear cells (PBMCs) or leukocytes or T cells per microliter of samples over time. In some embodiments, pharmacokinetics, expansion, and/or persistence is measured or assessed by determining the number of CAR-expressing cells in a patient and/or in samples collected from a patient at multiple time points. In certain embodiments, one or more samples are taken, received, and/or collected from a patient over a time period of 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 more than one year after injection therapeutic cell composition.

In some embodiments, a method is provided for selecting CAR-expressing cells containing a target antibody, such as an SJ25C1 or FMC63 antibody, or antigen-binding fragments thereof, comprising contacting a population of cells containing CAR-expressing cells with an anti-idiotypic antibody or antigen-binding fragment thereof. , or the anti-idiotypic antibody immunoconjugate described herein, and selecting cells associated with the anti-idiotypic antibody. In some embodiments, cells bound to the anti-idiotypic antibody are selected by affinity separation. In some embodiments, the affinity separation is selected from the group consisting of immunoaffinity separation, flow cytometry, magnetic separation, and affinity chromatography. In some embodiments, the anti-idiotypic antibody, or an antigen-binding fragment or anti-idiotypic antibody immunoconjugate thereof, is reversibly linked or immobilized on a support or stationary phase. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method is provided for confirming a CAR containing a target antibody, such as an SJ25C1 or FMC63 antibody, or antigen-binding fragments thereof, comprising a) incubating a sample containing CAR-transduced T cells with an anti-idiotypic antibody, or antigen-binding fragment thereof. targeting CAR; b) determining the percentage of cells associated with antiidiotypic antibody or antigen-binding fragment; and c) confirmation of CAR based on the percentage of T cells bound to the anti-idity antibody. In some embodiments, the anti-idiotypic antibody is labeled and anti-idiotypic antibody bound T cells are analyzed by flow cytometry. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

Methods are also provided, including the use of the proposed anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies to inform a person about treatment decisions, such as detection of CARs recognized by an anti-idiotypic antibody, such as CARs containing a target antibody, such as an anti-CD19 antibody (eg, an SJ25C1 or FMC63 antibody), or an antigen-binding fragment thereof. In some embodiments, the methods are designed to inform an individual's treatment decisions in conjunction with a therapy involving the administration of CAR T cells, such as anti-CD19 CAR T cells. Methods in some embodiments include incubating and/or testing a biological sample with an anti-idiotypic antibody and/or administering an anti-idiotypic antibody to an individual. In certain embodiments, the biological sample includes a cell or tissue, or a portion thereof, such as a tumor or cancer tissue, or a biopsy, or a portion thereof. In some embodiments, the incubation is carried out under conditions that allow the anti-idiotypic antibody to bind to CARs present in the sample. In some embodiments, the methods further comprise detecting whether a complex is formed between the anti-idiotypic antibody and CAR 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.

In one embodiment, an anti-idiotypic antibody is used to determine whether an individual needs to adapt to CAR T cell therapy, such as when low levels of CAR T cells in the individual indicate a need for adjustment of therapy. In some embodiments, the target antibody is an anti-CD19 antibody. In some embodiments, the target antibody is or is derived from an SJ25C1 antibody or antigen-binding fragment thereof. In some embodiments, the target antibody, or antigen-binding fragment thereof, comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody is or is derived from an antibody FMC63 or its antigen-binding fragment. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for evaluating a CAR T cell therapy in an individual, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising incubating a sample from the individual with an anti-idiotypic antibody or antigen thereof. binding fragment targeting CARs, and determining the number of T cells associated with the anti-idiotypic antibody, and determining the potential therapeutic benefit of therapy based on the number of T cells associated with the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody is labeled and anti-idiotypic antibody bound T cells are analyzed by flow cytometry. In some embodiments, the sample is a blood-derived sample, or is or is derived from an apheresis or leukopheresis product. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method for evaluating CAR T cell therapy in an individual is provided. In some aspects, the CAR contains a target antibody, such as an antibody that is or derived from SJ25C1 or FMC63, such as an antigen-binding fragment of a full-length SJ25C1 or FMC63. In some embodiments, the method comprises administering to the individual an anti-idiotypic antibody, or an antigen-binding fragment thereof, targeted to CARs (eg, targeted to an antibody, eg, a fragment of a CAR antibody). In some aspects, the introduction is carried out after the start of the first dose of therapy. The method may include determining the presence of an anti-idiotypic antibody in one or more tissues/organs of an individual. In some aspects, the method includes determining the potential therapeutic benefit of therapy based on the presence of an anti-idiotypic antibody in at least one of one or more tissues/organs. In some embodiments, the anti-idiotypic antibody is labeled and the presence of the anti-idiotypic antibody in an individual is determined by imaging it in the individual to detect the label. In some embodiments, determining the presence of an anti-idiotypic antibody in one or more tissues/organs in an individual includes or is performed by determining the level of anti-idiotypic antibody or its binding in one or more tissues/organs.

In some embodiments, the method further comprises administering the anti-idiotypic antibody to the subject upon initiation of a second or subsequent dose of therapy and/or determining the presence of the anti-idiotypic antibody in one or more tissues/organs of the subject. In some aspects, the method further comprises determining a potential therapeutic benefit of therapy based on a difference in anti-idiotypic antibody level in at least one of one or more tissues/organs in the subject between the first and second administration of the anti-idiotypic antibody. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method for adjusting a CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof. In some aspects, the method includes incubating or contacting a sample of an individual with an anti-idiotypic antibody or an antigen-binding fragment thereof that targets CAR. In some aspects, the method includes determining the number of T cells associated with an anti-idiotypic antibody. In some aspects, the method includes adjusting therapy based on the number of T cells associated with the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody is labeled directly or indirectly. In some aspects of such embodiments, anti-idiotypic antibody bound T cells are imaged in vivo or ex vivo, such as in some cases by flow cytometric analysis of a patient sample administered with CAR-T cells and anti-idiotypic antibody. In some embodiments, the sample is a blood-derived sample and/or is or is derived from an apheresis or leukopheresis product. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method for adjusting CAR T cell therapy in an individual is provided. In some aspects, such a method is carried out for CAR-T cell therapy, where the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, including an SJ25C1 or FMC63 antigen-binding fragment. The method, in some embodiments, comprises administering to an individual an anti-idiotypic antibody, or an antigen-binding fragment thereof, that targets or binds to a CAR upon initiation of a first dose of therapy, and determining the presence, absence, or level of the anti-idiotypic antibody in one or more tissues/organs in the individual . In some aspects, the method includes adjusting therapy based on the presence, absence, or level of an anti-idiotypic antibody in at least one of one or more tissues/organs. In some embodiments, the anti-idiotypic antibody is labeled directly or indirectly, and in some such embodiments, the presence of the anti-idiotypic antibody in an individual is determined by imaging it in the individual to detect a label. In some embodiments, determining the presence of an anti-idiotypic antibody in one or more tissues/organs in an individual includes determining the level of the anti-idiotypic antibody or antigen-binding fragment thereof in one or more tissues/organs. In some embodiments, the method further comprises administering an anti-idiotypic antibody to an individual after initiation of a second or subsequent dose of administration of therapy and, in some aspects, determining the presence, absence, or level of an anti-idiotypic antibody in one or more tissues/organs in the individual and/or adjusting therapy based on observations such as as, for example, based on the difference in the level of anti-idiotypic antibody in at least one or more of the tissues/organs in the individual between the first and second administration of anti-idiotypic antibodies. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some aspects, therapy is adjusted (i) if the number of T-cell therapy cells detected in the blood or other biological sample after they were detected are not detected or the number decreases, while decreasing optionally compared to the previous time point after introduction of T-cell therapy; (ii) the number of T-cell therapy cells detected in the blood or other biological sample is reduced by exactly or more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more the peak or maximum number of T-cell therapy cells detected in the blood or biological sample of the patient after the start of the administration of T-cell therapy, optionally the first, second or subsequent dose; (iii) at the time when the peak or maximum level of T cell therapy cells is detected in the patient's blood, the number of T cells or cells derived from T cells detected in the patient's blood is less than 10%, less than 5 %, less than 1% or less than 0.1% of the total number of peripheral blood mononuclear cells (RVMS) in the patient's blood; and/or (iv) if the number of CD3+ or CD8+ cell therapy cells detected in the blood is less than 20 cells per µl, 15 cells per µl, 10 cells per µl, less than 5 cells per µl, or less than 1 cell per µl. In some embodiments, therapy is adjusted by administering one or more additional doses of CAR-T cell therapy, administering an increased dose of CAR-T cell therapy, administering an alternative CAR-T cell therapy specific for the same or a different antigen, administering one or more immunomodulatory agents or other agents to stimulate or increase the expansion or persistence of CAR-T cells.

Various methods known in the art can be used to detect specific antibody-antigen binding. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator fragment or group of tags can be attached to anti-idiotypic antibodies and chosen to meet the needs of various applications of the method, which are often determined by the availability of assay equipment and compatible immunoassay procedures. Examples of labels include radionuclides (e.g. ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H or ³² P and/or chromium ( ⁵¹ Cr), cobalt ( ⁵⁷ Co), fluorine ( ¹⁸ F), gadolinium ( ¹⁵³ Gd, ¹⁵⁹ Gd) , germanium ( ⁶⁸ Ge), holmium ( ¹⁶⁶ Ho), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, ¹¹¹ In), iodine ( ¹²⁵ I, ¹²³ I, ¹²¹ I), lanthanum ( ¹⁴⁰ La), lutetium ( ¹⁷⁷ Lu ), manganese ( ⁵⁴ Mn), molybdenum ( ⁹⁹ Mo), palladium ( ¹⁰³ Pd), phosphorus ( ³² P), praseodymium ( ¹⁴² Pr), promethium ( ¹⁴⁹ Pm), rhenium ( ¹⁸⁶ Re, ¹⁸⁸ Re), rhodium ( ¹⁰⁵ Rh), ruthenium ( ⁹⁷ Ru), samarium ( ¹⁵³ Sm), scandium ( ⁴⁷ Sc), selenium ( ⁷⁵ Se), ( ⁸⁵ Sr), sulfur ( ³⁵ S), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Tl), tin ( ¹¹³ Sn, ¹¹⁷ Sn), tritium ( ³ H), xenon ( ¹³³ Xe), ytterbium ( ¹⁶⁹ Yb, ¹⁷⁵ Yb), yttrium ( ⁹⁰ Y)), enzymes (e.g. alkaline phosphatase, horseradish peroxidase, luciferase or β -galactosidase), fluorescent moieties or proteins (eg, fluorescein, rhodamine, phycoerythrin, GFP, or BFP) or luminescent moieties (eg, Qdot™ nanoparticles available from Quantum Dot Corporation, Palo Alt Oh California). Various general techniques are known to be used in performing the various immunoassays noted above.

In some embodiments, the anti-idiotypic antibodies do not need to be labeled, and their presence can be detected using a labeled antibody that binds to any anti-idiotypic antibody.

The anti-idiotypic antibodies provided herein can be used in any known assay method such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Anti-idiotypic antibodies can also be used for in vivo diagnostic assays such as in vivo imaging. Typically, the anti-idiotypic antibody is labeled with a radionuclide (such as ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, or ³ H) so that the cells or tissue of interest can be localized in vivo upon administration to an individual.

In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized or associated with a solid support, where one or more target cells containing CAR-T cells are contacted with the solid support. In some embodiments, the solid support is a microsphere. In some embodiments, the solid support is the surface of a well or plate, such as a cell culture plate. In some embodiments, the implementation of the solid support is a resin or matrix present or contained in the chromatographic column, for example, to allow chromatographic isolation or selection of CAR+ T cells. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment is reversibly linked or capable of reversibly binding to a solid support. In some embodiments, the solid support is an affinity chromatography matrix containing one or more binding sites capable of binding, eg, reversibly binding, to a binding partner present in the anti-idiotypic antibody. In one exemplary embodiment, the anti-idiotypic antibody comprises a streptavidin-binding peptide or other streptavidin-binding fragment capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a solid support, and which, in some cases, can dissociate in the presence of a competitive substance such as biotin . Examples of such systems include those described in U.S. Published Application No. US 20150024411.

In some aspects, the anti-ID antibodies provided herein can be expressed on the surface of a cell. In some aspects, an anti-ID antibody expressed by a cell can be used to induce or stimulate a CAR expressing cell, such as part of a system for selective growth of CAR cells. In some aspects, the anti-ID antibody, or antigen-binding fragment thereof, is expressed on an artificial antigen-presenting cell (aAPC). Anti-ID expressing AAPCs can be used as reagents to stimulate or expand CAR-T cell populations.

Methods for obtaining or generating aAPC are known, see, for example, US patent. 6225042, 6355479, 6362001, 6790662; 7754482; US Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO 2007/103009. Various aAPCs are known in the art, see, for example, US Pat. No. 8,722,400, Published Application No. US 2014/0212446; Butler and Hirano (2014) Immunol Rev., 257(1):10. 1111/imr.12129; Suhoshki et al. (2007) Mol. Ther., 15: 981-988).

aAPCs include features of naturally occurring APCs, including expression of the MHC molecule, stimulatory and costimulatory molecule(s), Fc receptor, adhesion molecule(s), and/or the ability to produce or secrete cytokines (eg, IL-2). Typically, aAPC is a cell line that lacks the expression of one or more of the above and is generated by introducing (e.g., by transfection or transduction) one or more of the missing elements needed to stimulate the cell, e.g., a CAR T cell.

In some embodiments, cells selected to become aAPCs have deficiencies in intracellular antigen processing, intracellular peptide transport, and/or intracellular class I or class II MHC molecular peptide loading. In some aspects, such cells also lack the ability to express a class I or class II MHC molecule and/or molecules involved in or associated with antigen processing. Exemplary aAPCs are or are derived from a cell line deficient in an antigen processing associated transporter (TAP), such as an insect cell line. An exemplary cell line is a Drosophila cell line, such as the Schneider 2 cell line (see, for example, Schneider, J. Embryol. Exp. Morph. 1972 Vol 27, pp. 353-365). Exemplary methods for obtaining, growing, and culturing Schneider 2 cells are provided in US Patent Nos. 6225042, 6355479 and 6362001.

In some embodiments, the cell is a K652 cell or a cell derived from K562. In some embodiments, the cell is a cell line available from ATCC No. CCL-243.

In some aspects, the aAPC is further engineered to express an additional molecule to enhance, potentiate, or increase stimulation of a CAR-expressing T cell. In some embodiments, the additional molecule is an immunostimulatory ligand, a co-stimulatory ligand, a cytokine, or an adhesion molecule. In some embodiments, the costimulatory ligand specifically binds to at least one costimulatory molecule present on the T cell. In some embodiments, the aAPC is generated, e.g., by transduction or transfection to express one or more co-stimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL , OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, ILT3, ILT4, 3/TR6 or B7-H3 ligand or antibody that specifically binds with CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor or CD83 ligand), cell adhesion molecules ( e.g. ICAM-1 or LFA-3) and/or a cytokine (e.g. IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon -alpha (IFNα), interferon-beta (IFNβ), interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), tumor necrosis factor beta (TNFβ), granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (GCSF)). In some cases, aAPC does not normally express the MHC molecule, but may be engineered to express the MHC molecule or, in some cases, is or may be induced to express the MHC molecule, for example, by stimulation with cytokines. In some cases, aAPCs may also be loaded with a stimulatory or co-stimulatory ligand, which may include, for example, an anti-CD3 antibody, an anti-CD28 antibody, or an anti-CD2 antibody. In some embodiments, the aAPC expresses a molecule capable of mediating a primary signal in a cell, such as a signal mediated through a T cell receptor/CD3 complex on a T cell. In some embodiments, the aAPCs contain a stimulatory ligand that specifically binds to the TCR/CD3 complex such that primary signaling occurs.

In some embodiments, because the anti-ID is capable of signaling through the CAR, the aAPC does not express a stimulatory ligand that specifically binds to the TCR/CD3 complex. In some embodiments, the aAPC does not express a costimulatory molecule.

In some embodiments, the anti-ID is expressed as a single chain fragment (scFv) for cell surface expression. The nucleic acid encoding the scFV may be fused to DNA sequences encoding the transmembrane domain. Preferably used transmembrane regions include those derived from the T cell receptor alpha, beta or zeta chain transmembrane region(s), CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33. , CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Exemplary transmembrane domains include those derived from CD8 or CD28.

B. Use in cell stimulation

In some embodiments, the provided anti-idiotypic antibodies, or antigen-binding fragments thereof, are agonists and/or exhibit specific activity to stimulate cells expressing the target antibody, including conjugates or chimeric receptors containing it, such as an anti-CD19 antibody (e.g., an SJ25C1 antibody or FMC63) or an antigen-binding fragment thereof. In some embodiments, methods are provided comprising using the provided anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies to stimulate or activate CAR-expressing cells or expressing another chimeric receptor, such as T cells. In some aspects, the CAR or other receptor comprises a target antibody, such as an anti-CD19 antibody (eg, an SJ25C1 or FMC63 antibody), or an antigen-binding fragment thereof.

In some embodiments, the methods may be used in connection with methods for producing genetically engineered T cells, such as methods for expanding genetically engineered T cells or other cells into which a nucleic acid molecule encoding a chimeric receptor, such as a CAR containing an antibody- target, for example, by transfection, transduction, or non-viral nucleic acid transfer, such as the transposon approach. In some aspects, the target antibody is an anti-CD19 antibody (eg, an SJ25C1 or FMC63 antibody) or an antigen-binding fragment thereof. In specific embodiments, the target antibody is or contains a CAR, such as an anti-CD19 CAR. In specific embodiments, the anti-CD19 CAR comprises a scFv that is taken from and/or derived from an anti-CD19 antibody, such as an SJ25C1 or FMC63 antibody.

Methods in some embodiments include incubating a sample containing CAR-transduced T cells with an anti-idiotypic antibody. In some embodiments, the methods further comprise detecting whether CAR-T cells are activated or stimulated, for example, by assessing viability, proliferation, and/or expression of activation markers in CAR-T cells. In some embodiments, the target antibody is an anti-CD19 antibody. In some embodiments, the target antibody is or is derived from SJ25C1 or FMC63 antibodies or antigen-binding fragments thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, a method for stimulating cells is provided, comprising incubating an initial composition containing CAR-expressing cells containing a target antibody, such as an SJ25C1 or FMC63 antibody, or antigen-binding fragment thereof, with an anti-idiotypic antibody, or antigen-binding fragment thereof, as described herein, thereby generating the final composition containing stimulated cells. In some embodiments, the incubation is carried out under conditions in which the anti-idiotypic antibody, or antigen-binding fragment thereof, binds to the CAR, thereby inducing or modulating a signal in one or more cells in the original composition. In some embodiments, the cells include T cells. In some embodiments, the T cells include CD4+ and/or CD8+ T cells.

In some embodiments, provided herein is a method of promoting or expanding cells that express a CAR by incubating an initial composition containing CAR expressing cells with an anti-ID antibody that binds to and/or recognizes a CAR. In some embodiments, binding between the anti-ID antibody and the CAR induces expansion of the CAR-expressing cells, thereby producing a final composition containing expanded cells.

In some embodiments, the anti-idiotypic antibody is contacted or incubated with an initial composition of one or more cells to generate the final composition. In certain embodiments, the original cells and/or the original composition is a composition and/or a plurality of cells that are or are to be processed, incubated, or contacted under conditions that will result in one or more changes in at least a portion of the cells of the original composition, so thereby transforming the original composition into the final composition. In some embodiments, the cells administered are an immune cell composition, such as a T cell composition, that contains CAR-expressing cells. In specific embodiments, at least a portion of the cells in the original composition is activated, expanded and/or enriched in the generated final composition by using the proposed methods.

In certain embodiments, the implementation of the anti-idiotypic antibody contributes to the expansion or enrichment of the cells of the original composition, expressing CAR. In some embodiments, the initial composition contains eukaryotic cells, such as mammalian cells. In certain embodiments, the initial composition contains human cells. In some embodiments, the initial composition contains cells that are derived from blood, bone marrow, lymph, or lymphoid organs. In specific embodiments, the initial composition contains cells of the immune system, ie. cells of innate or acquired immunity, for example, myeloid or lymphoid cells, including lymphocytes, usually T cells and/or NK cells. In some embodiments, the initial composition contains stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In specific embodiments, the initial composition contains CD3+ cells. In certain embodiments, the initial composition contains CD4+ cells. In some embodiments, the initial composition contains CD8+ cells. In some embodiments, the initial composition is a composition of CD4+ cells. In specific embodiments, the initial composition is a composition of CD8+ cells.

In some embodiments, the methods and agents are capable of stimulating T cells deficient or downregulated by one or more natural signaling molecules, such as one or more co-stimulatory receptors, or antigen receptors, or cytokine receptors, but the cells still express a chimeric receptor, e.g. , CAR recognized by the anti-Id antibody. In some embodiments, the cells of the parent composition are low or negative for surface expression of CD28 or another costimulatory molecule or other signaling molecule. Thus, in some embodiments, the provided agents and methods have certain advantages over certain other activating or stimulatory agents or methods that may require or depend on the surface expression of CD28 or other endogenous signaling molecule to provide the desired signal and/or the full extent of such signal, for example, to provide a costimulatory signal and/or to achieve full activation. In some embodiments, the proposed agents and methods are advantageous in such respects over anti-CD3/CD28 reagents (eg, microspheres); in some aspects, the proposed anti-ID antibodies have the advantage that they are able to stimulate or achieve the desired effect, such as activation or proliferation of cells that are low or negative for CD28 or another natural signaling molecule. In some aspects, signaling through the CAR via stimulation with an anti-ID antibody results in primary and secondary (costimulatory) signaling through the CAR using only one reagent. In some embodiments, the initial composition contains CD3+ cells that low-express CD28 or another endogenous signaling molecule. In some embodiments, the initial composition contains CD3+ cells that are CD28 negative or are negative for another endogenous signaling molecule. In some embodiments, the anti-ID antibody stimulates the activation and/or expansion of cells that express low levels of CD28 or cells that are CD28 negative. In some embodiments, cells are contacted with an anti-idiotypic antibody or antigen-binding fragment that is immobilized on or associated with a solid support. In some embodiments, the solid support is a microsphere. In some embodiments, the solid support is the surface of a well or plate, such as a cell culture plate. In some examples, the anti-ID antibody is soluble. In certain embodiments, the cells are not contacted with anti-CD3/CD28 antibody-conjugated reagents prior to contacting the cells with the anti-idiotypic antibody or antigen-binding fragment.

In some embodiments, an anti-idiotypic antibody is applied to, coupled to, or incubated with an initial composition of cells that have been transduced or transfected with a nucleotide encoding a CAR. In specific embodiments, the implementation of the incubation, processing and/or contacting of the original cells with anti-idiotypic antibody leads to the expansion and/or enrichment of cells expressing CAR. In specific embodiments, incubation, treatment, and/or contact of parent cells with anti-idiotypic antibody does not result in expansion and/or enrichment of cells that do not express CAR. In specific embodiments, incubation, treatment, and/or contact of the original cells with anti-idiotypic antibody results in expansion and/or enrichment of cells that do not express CAR, which is at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% less than expansion and/or enrichment cells that express CAR. In some embodiments, the anti-idiotypic antibodies provided herein are used to expand CAR-expressing cells of a parent composition that has experienced poor transduction and/or transfection efficiency and/or that contains low numbers of CAR-expressing cells. In certain embodiments, the anti-idiotypic antibody promotes selective expansion and/or enrichment of CAR-expressing cells.

Some embodiments suggest that an anti-idiotypic antibody is more effective at expanding and/or enriching cells of an initial composition with low transduction or transfection efficiency and/or that has few CAR-expressing cells than expanding and/or enriching cells by polyclonal stimulation, e.g. , stimulation with an anti-CD3 antibody and/or an anti-CD28 antibody. In specific embodiments, polyclonal stimulation results in the expansion of cells that express and cells that do not express CAR in the original composition, and therefore, in some embodiments, may not be able to enrich CAR-expressing cells, especially when the original the composition contains a low amount of CAR-expressing cells. In contrast, in some embodiments, incubation with an anti-idiotypic antibody results in selective expansion of CAR-expressing cells, and therefore, in certain embodiments, results in selective expansion and/or enrichment of CAR-expressing cells. In some embodiments, incubation, contact, and/or treatment of parent cells with an anti-idiotypic antibody results in greater enrichment and/or expansion of CAR-expressing cells than polyclonal stimulation.

In specific embodiments, the anti-idiotypic antibody is incubated, applied to, and/or linked to parent cells that have been transfected and/or transduced with fewer viral particles, viral vector particle copy ratios to cells, and/or infectious units (IUs) than the parent cells, which are expanded and/or enriched by polyclonal stimulation. For example, in some embodiments, the initial composition that is incubated with the anti-idiotypic antibody is generated from cells that have been transduced using IE per cell that is or is at least 0.5, 1, 2, 3, 4, 5, 10 , 15, 20, 30, 40, 50 or 60 IU less per cell than the original composition, which is increased and/or enriched with polyclonal stimulation. In some embodiments, the initial composition that is incubated with the anti-idiotypic antibody is generated from cells that have been transduced with a viral vector particle titer of at least 1×10 ⁵ IU/ml, 5×10 ⁵ IU/ml, 1×10 ⁶ IU/ml, 5×10 ⁶ IU/ml, 6×10 ⁶ IU/ml, 7×10 ⁶ IU/ml, 8×10 ⁶ IU/ml, 9×10 ⁶ IU/ml or 1×10 ⁷ IE/ml is less than the original composition, which is increased and/or enriched using polyclonal stimulation.

In specific embodiments, cell transduction with a high IE/per cell will result in high transduction efficiency, but in some embodiments, may also result in transfected cells with high vector copy number (VCN), which may present safety risks and may not be appropriate. regulatory requirements. In specific embodiments, reducing the IE/per cell to which the cells are transduced will decrease the efficiency of transduction but will decrease the VCN. In specific embodiments, an increase in IE/per cell by which cells are transduced will increase transduction efficiency but also increase VCN.

In some embodiments, the initial composition comprises a population of cells that have been transduced or transfected, or cells that are derived from cells that have been transduced or transfected with one or more nucleic acids encoding a CAR that binds to or is recognized by an anti-idiotypic antibody. In some embodiments, the starting composition contains less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40% , less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of cells that are CAR-expressing cells. In specific embodiments, cells of the original composition were transfected or transduced as described in section III. In some embodiments, the parent cells comprise a population of cells that have been transduced or transfected with one or more nucleic acids encoding an anti-CD19 CAR, such as an anti-CD19 CAR that contains an scFv taken from and/or derived from an anti-CD19 antibody, such as an SJ25C1 or FMC63 antibody, or a population of cells derived from them.

In specific embodiments, incubation, contacting or treatment of cells from the original composition with an anti-idiotypic antibody is carried out under conditions for stimulation, expansion and / or activation of cells, and the conditions may include one or more of the following: specific environments, temperature, oxygen content, dioxide content carbon, time, agents, such as 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 cells.

In some embodiments, cells of the original composition are transfected or transduced with a single nucleic acid containing a gene encoding CAR, and the cells are bound, incubated, or treated with an anti-idiotypic antibody that binds to or recognizes the recombinant receptor. In some embodiments, the cells of the original composition are treated, incubated, or coupled with an anti-idiotypic antibody after the cells are transduced or transfected with a CAR-encoding nucleic acid. In specific embodiments, the cells of the original composition are treated, incubated, or contacted with the anti-idiotypic antibody immediately, for about 1 minute, for about 5 minutes, for about 30 minutes, for about 1 hour, for about 2 hours, in for about 4 hours for about 6 hours for about 8 hours for about 12 hours for about 24 hours for about 2 days for about 3 days for about 4 days for about 5 days, for about 6 days, for about 1 week, for about 2 weeks, for about 3 weeks, for about 4 weeks, for about 5 weeks, or for about 6 weeks after the original cells the compositions were transduced or transfected.

In some embodiments, the cells of the original composition are treated, incubated, and/or contacted with a soluble anti-idiotypic antibody, an antibody that does not cross-link, and/or an antibody that is not bound or attached to a solid support.

In some embodiments, the methods result in the proliferation, activation, stimulation, release of cytokines, or other functional outcomes, such as upregulation of an activation marker or the release or production of cytokines, of cells expressing a chimeric receptor, such as CAR, recognized by an anti-Id antibody. In some aspects, such proliferation or other functional response or outcome is induced in such cells to a degree similar to or greater than that induced by incubation of cells with an agent and/or under conditions that stimulate T cell proliferation, such as in the presence of microspheres with anti-CD3/CD28 antibody and/or by cross-linking an anti-CD3 antibody. In some aspects, the methods do not include cross-linking of the anti-idiotypic antibody. In some aspects of any of the embodiments, the anti-idiotypic agents are capable of inducing the specified proliferative or functional outcome, or the extent thereof, without anti-idiotypic antibody crosslinking. In some aspects, the anti-idiotypic agents of the present disclosure are advantageous in their ability to stimulate or induce a specific functional response of T cells or other immune cells expressing the target receptor without the need for anti-Id antibody crosslinking or the use of a secondary agent. In some aspects, the result is achieved using a soluble or tablet-bound form of the anti-idiotypic antibody. In some aspects, the result is achieved using an anti-idity antibody associated with the microsphere.

In specific embodiments, the cells of the original composition are treated, incubated and/or brought into contact at a concentration of from 10 pg/ml to 100 μg/ml, from 1 pg/ml to 1 ng/ml, from 1 ng/ml to 1 μg/ml from 100 ng/ml to 1.0 µg/ml, from 1 ng/ml to 100 ng/ml, from 10 ng/ml to 1.0 µg/ml, from 100 ng/ml to 10 pg/ml, from 250 ng/ml and 10 µg/ml, 250 to 1 ng/ml, 1 to 10 µg/ml, 250 to 2.5 µg/ml, or 1 to 10 µg/ml.

In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on 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-idiotypic antibody or antigen-binding fragment thereof. In some embodiments, the solid support is the surface of the plate or well. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized in a soluble reagent, which optionally is or contains a plurality of binding sites capable of reversibly binding to the anti-idiotypic antibody or antigen-binding fragment thereof. In some embodiments, the reagent contains a streptavidin mutein. In one exemplary embodiment, the anti-idiotypic antibody comprises a streptavidin-binding peptide or other streptavidin-binding fragment capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a soluble reagent, and which, in some cases, can dissociate in the presence of a competitive agent such as biotin . Examples of such systems include those described in PCT Published Application No. WO 2015/158868.

In specific embodiments, the cells of the original composition are treated, incubated, and/or contacted with an anti-idiotypic antibody that is attached, bound, coated, and/or conjugated to a solid surface or carrier, such as a plate or well. In some embodiments, the anti-idiotypic antibody has been attached, bound, coated and/or conjugated to a solid surface or support by incubating the solid surface or support with a certain concentration of anti-idiotypic antibody. In specific embodiments, the solid surface or support is incubated with an anti-idiotype antibody at a concentration of 10 ng/mL to 100 μg/mL, 100 ng/mL to 1.0 μg/mL, 250 ng/mL to 10 μg/mL , 250 ng/mL to 1 µg/mL, 1 µg/mL to 10 µg/mL, 250 ng to 2.5 µg/mL, or 1 µg/mL to 10 µg/mL. In some embodiments, the solid surface or support is incubated with an anti-idiotypic antibody at a concentration of 250 ng/mL to 10 μg/mL. In some embodiments, the solid surface or support is incubated with an anti-idiotypic antibody at or about 0.25 µg/mL, 0.5 µg/mL, 1 µg/mL, 1.25 µg/mL, 2 µg/mL, 2.5 µg/ml, 5 µg/ml or 10 µg/ml.

In some embodiments, the incubation lasts at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours. . In some embodiments, the starting 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 number of cells in the composition. In some embodiments, the number of CAR-expressing cells in the final composition is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to with the number of CAR-expressing cells in the original composition; and/or the percentage of CAR-expressing cells in the final composition compared to the total number of cells in the composition is increased by more than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more. In some embodiments, the cells are not selected or enriched for CAR-expressing cells prior to incubation. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31.

In some embodiments, the anti-idiotypic antibody is contacted or incubated with cells from the original composition, e.g., containing CAR-expressing cells, for some time to grow one or more cells of the original composition, for example, to grow cells of the original composition that express the recombinant receptor. In specific embodiments, cells from the original composition are contacted, incubated, or treated with an anti-idiotypic antibody for at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, or for at least about 4 weeks. In specific embodiments, cells from the original composition are contacted, incubated, or treated with anti-idiotypic antibody for less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than than about 6 days or less than about 12 days. In some embodiments, cells from the original composition are contacted, incubated, or treated with anti-idiotypic antibody for about 1 day to about 14 days, about 3 days to 7 days, or 4 days to 6 days.

In specific embodiments, cells from the original composition, for example, containing cells that express CAR, are incubated, bound, or treated with anti-idiotypic antibody at temperatures above room temperature to grow cells of the original composition that express the recombinant receptor. In some embodiments, the processing, incubation, or contacting is carried out at a temperature greater than about 25°C, such as typically greater than or greater than about 32°C, 35°C, or 37°C. In some embodiments, the processing, contacting or incubation is carried out at a temperature equal to or equal to about 37°C ± 2°C, such as at a temperature equal to or equal to about 37°C.

In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, about 100%, or 100% of the cells in the final composition express CAR.

In specific embodiments, the number of cells that express CAR in the final composition that has been incubated, treated and/or contacted with the anti-idiotypic antibody is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% , at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 95%, at least 100%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or at least 100 times more than the number of cells that express CAR in the original composition.

In specific embodiments, the percentage of cells that express CAR in the final composition that has been incubated, treated and/or contacted with the anti-idiotypic antibody is at least 1%, at least 5%, at least 10%, by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100% at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or at least 100 times more, how the number of cells that express CAR in the original composition.

In some embodiments, the number of cells that express CAR in the final composition that has been incubated, treated and/or contacted with an anti-idiotypic antibody is at least 1%, at least 5%, at least 10%, by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100% , at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or at least 100 times more than the number of cells in the final composition that received polyclonal stimulation and that were incubated with antibodies against CD3 and CD28.

In some embodiments, the percentage of cells that express CAR in the final composition that has been incubated, treated, and/or contacted with the anti-idiotype antibody is at least 1%, at least 5%, at least 10%, by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100% , at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or at least 100 times more than the number The number of cells in the final composition that received polyclonal stimulation, for example, that were incubated with antibodies against CD3 and CD28.

In some embodiments, cells that express CAR in the final composition that has been incubated, treated and/or contacted with an anti-idiotypic antibody contain at least 1%, at least 5%, at least 10%, with at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least least 99% lower VCN than the cells of the final composition, which received polyclonal stimulation, for example, they were incubated with antibodies against CD3 and CD28. In some embodiments, the mean VCN of CAR-expressing cells in output is no more than or about 10, 5, 4, 2.5, 1.5, or 1.

In some embodiments, such methods may be used as part of production, analysis, and/or quality control methods, for example, in connection with the development of cell therapy with cells expressing recombinant polypeptides containing an antibody or fragment thereof recognized by an anti-idiotypic antibody, such as CAR- T cells, for testing purposes, including testing the expression and/or activity of the engineered receptor, for example, in cells engineered for use in therapy in an individual. In some embodiments, cell compositions can be tested at any stage during the generation of CAR-expressing T cells. In specific embodiments, a cell sample may be collected from the cell composition at any stage of the process and stored, for example, by cryo-freezing and/or cryopreservation for later testing and/or analysis. Test compositions may be pharmaceutical compositions, for example, including those containing cells and a pharmaceutically acceptable recipient and/or cryopreservative.

In some embodiments, the anti-idiotypic antibody stimulates cells expressing the target antibody, eg, CAR, in vivo. Specific embodiments suggest that CAR-T cell therapy is effective in the treatment of cancer and other diseases and disorders. However, in some contexts, the available approaches to CAR-T cell therapy may not always be completely satisfactory. For example, in some embodiments, the exposure and persistence of CAR-expressing cells in a patient decreases or decreases over time. However, observations indicate that in some cases, increased exposure of CAR-expressing cells may improve the efficacy and therapeutic outcomes of CAR-T cell therapy. Thus, in some embodiments, an anti-idiotypic antibody is administered to enhance, increase and/or increase the persistence and/or expansion of cells expressing CAR.

In certain embodiments, the anti-idiotypic antibody is administered to a patient, such a patient who has previously received a therapeutic cell composition containing CAR-expressing cells. In some embodiments, administration of an anti-idiotypic antibody to a patient promotes re-expansion of CAR-expressing cells in the patient, which in some cases may reach or exceed an initial peak level of expansion prior to administration of the anti-idiotypic antibody. In some embodiments, an anti-idiotypic antibody is administered to modulate the expansion and/or persistence of CAR-expressing cells at times when levels of CAR-expressing cells are reduced or not detected. In some embodiments, the CAR-expressing cells that are re-expanded with the anti-idiotypic antibody exhibit increased activity in the patient to whom they are administered, for example, compared to that prior to administration of the anti-idiotypic antibody.

In some embodiments, administration of an anti-idiotypic antibody increases or enhances the persistence of CAR-expressing cells in a patient. In some embodiments, CAR-expressing cells are detected in the patient after or at least 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 2 months, 3 months , 4 months, 5 months, 6 months, or more than 6 months after anti-idiotypic antibody administration. In some aspects, increased exposure of a patient to cells includes expansion and/or increased expansion of cells.

In some embodiments, CAR-expressing cells are expanded in a patient following administration of an anti-idiotypic antibody. In specific embodiments, administration of the anti-idiotypic antibody results in a maximum concentration in the blood, serum, or other body fluid, organ, or tissue of the patient that is at least 100, 500, 1000, 1500, 2000, 5000, 10,000, or 15,000 copies of the nucleic acid coding for CAR per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 CAR-expressing cells per microliter. In some embodiments, CAR-expressing cells are detected at at least 10%, 20%, 30%, 40%, 50%, or 60% of the total amount of PBMC in the patient's blood and/or at that level for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks after anti-idiotype antibody administration, or within 1, 2, 3, 4, or 5 or more years after administration of anti-idiotypic antibodies. In some aspects, administration of an anti-idiotypic antibody results in at least 2, at least 4, at least 10, or at least 20-fold increase in the number of copies of a nucleic acid encoding a recombinant receptor, e.g., CAR, per microgram of DNA, e.g. , in serum, plasma, blood, or tissue, such as in a patient's tumor sample. In specific embodiments, administration of the anti-idiotypic antibody results in an increase of at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold increase in the number of circulating CAR-expressing cells in the patient.

In some aspects, at least about 1×10 ² , at least about 1×10 ³ , at least about 1×10 ⁴ , at least about 1×10 ⁵ , or at least about 1×10 ⁶ , or at least about 5×10 ⁶ , or at least about 1×10 ⁷ , or at least about 5×10 ⁷ , or at least about 1×10 ⁸ CAR-expressing cells and/or at least 10 , 25, 50, 100, 200, 300, 400 or 500 or 1000 CAR-expressing cells per microliter e.g. at least 10 per microliter detected or present in the patient or in fluid, plasma, serum, tissue or compartment thereof, for example in the blood, for example, in peripheral blood, or at the site of the disease after administration of an anti-idiotypic antibody. In some embodiments, such a number or concentration of cells is detected in a patient for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years after administration of the anti-idiotypic antibody.

Various delivery systems are known and can be used to administer an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody is administered by encapsulation and/or attachment to liposomes, microparticles, and microcapsules. Methods for administering the anti-idiotypic antibody include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-idiotypic antibody may be administered by any convenient route, such as infusion, bolus injection, absorption through epithelial or mucosal (eg, oral, rectal, and intestinal mucosa, etc.), and may be administered together with other biologically active substances. The introduction can be systemic or local. Pulmonary administration can also be used, for example, using an inhaler or nebulizer and a composition with an aerosol agent. In some embodiments, the anti-idiotypic antibody is delivered in a vesicle, in particular in a liposome (Langer, 1990, Science 249: 1527-1533), for example in a cationic liposome (WO 98140052).

In some embodiments, a method for preparing a cell composition is provided, comprising introducing into cells a nucleic acid molecule encoding a CAR, thereby generating an initial composition, and incubating the initial composition with an anti-idiotypic antibody or an antigen-binding fragment thereof specific for the CAR antigen-binding domain, obtaining thereby cellular composition. In some embodiments, the CAR includes a target antibody, or antigen-binding fragment thereof, that specifically binds to CD19. In some embodiments, the target antibody is an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is an anti-idiotypic antibody or antigen-binding fragment thereof as described herein. In some embodiments, the anti-idiotypic antibody, or antigen-binding fragment thereof, is a CAR agonist. In some embodiments, administration includes introducing the nucleic acid molecule into cells by viral transduction, transposition, electroporation, or chemical transfection. In some embodiments, administration comprises introducing the nucleic acid molecule into cells by transduction with a retroviral vector containing the nucleic acid molecule, by transduction with a lentiviral vector containing the nucleic acid molecule, by transposition with a transposon containing the nucleic acid molecule, or by electroporation or transfection of a vector containing nucleic acid molecule.

In some embodiments, the method further comprises the step of stimulating or activating cells prior to administering a nucleic acid molecule encoding a CAR. In some embodiments, activating cells includes contacting cells with a CD3 agonist and optionally a CD28 agonist. In some embodiments, cell activation comprises contacting cells with a reagent containing anti-CD3 and anti-CD28 agonist antibodies. In some such embodiments, during at least a portion of contact with an anti-CD3/CD28 antibody and/or during at least a portion of administration of a nucleic acid encoding a CAR, the method comprises incubating or contacting cells with an anti-idiotypic antibody or antigen thereof. linking fragment. In some embodiments, the incubation is carried out under conditions in which the anti-idiotypic antibody, or antigen-binding fragment thereof, binds to the CAR, thereby inducing or modulating a signal in one or more cells in the original composition. In some embodiments, the implementation of the cells contain T cells.

In some such embodiments, the T cells comprise CD4+ and/or CD8+ T cells. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on 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-idiotypic antibody or antigen-binding fragment thereof. In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized in a soluble reagent, which optionally is or contains a plurality of binding sites capable of reversibly binding to the anti-idiotypic antibody or antigen-binding fragment thereof. In some embodiments, the reagent contains a streptavidin mutein. In one exemplary embodiment, the anti-idiotypic antibody comprises a streptavidin-binding peptide or other streptavidin-binding fragment capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a soluble reagent, and which, in some cases, can dissociate in the presence of a competitive agent such as biotin . Examples of such systems include those described in PCT Published Application No. WO 2015/158868. In some embodiments, the incubation lasts at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours. In some embodiments, the starting 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 number of cells in the composition. In some embodiments, the number of CAR-expressing cells in the final composition is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to with the number of CAR-expressing cells in the original composition; and/or the percentage of CAR-expressing cells in the final composition compared to the total number of cells in the composition is increased by more than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more. In some embodiments, the cells are not selected or enriched for CAR-expressing cells prior to incubation. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for monitoring the activity of a CAR containing a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, comprising the steps of incubating a sample containing CAR-transduced T cells with an agonistic anti-idiotypic antibody or its an antigen-binding moiety that targets or binds to CARs; and/or detecting the presence, absence, or amount of CAR T cell activation, stimulation, and/or expansion, thereby controlling CAR T cell activity. In some embodiments, such methods may be used to validate CARs, in which case the method may include: c) validating CARs based on the level of activation, stimulation, and/or expansion of CAR-T cells.

In some embodiments, activation, promotion and/or expansion of CAR-T cells is assessed by determining the viability, proliferation and/or expression of T cell activation markers in CAR-T cells after a period of incubation with an anti-idiotypic antibody. In some embodiments, the viability of CAR T cells is assessed by calculating the percentage of live relative to total CAR transduced T cells after incubation with anti-idiotypic antibody. In some embodiments, proliferation of CAR T cells is assessed by diluting the dye used to stain CAR T cells prior to incubation with anti-idiotypic antibody. In some embodiments, expression of T cell activation markers is assessed by flow cytometry staining for antibodies recognizing T cell activation markers. In some embodiments, T cell activation markers are selected from the group consisting of CD25, CD26, CD27, CD28, CD30, CD69, CD71, CD134, CD137, and CD154. In some embodiments, the incubation period is from about 1 to about 10 days (eg, about any range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including any ranges in between). In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method for monitoring a preparation of CAR-T cells is provided, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising a) incubating a portion of the preparation with an agonistic anti-idiotypic antibody or antigen thereof. a binding fragment that targets or binds to the CAR; and b) determining the presence, absence or amount of activation, stimulation and/or expansion of CAR T cells. In some embodiments, the implementation of CAR-T cells may be cells produced or manufactured under specific conditions suitable for testing. In some embodiments, monitoring is performed in connection with a release assay, such as cell validation prior to administration to a patient. In some aspects, the method further comprises validating the drug based on the level of CAR T cell activation. In some embodiments, the activation of CAR-T cells in the preparation is assessed by determining the viability, proliferation and/or expression of T cell activation markers in CAR-T cells after a period of incubation with an anti-idiotypic antibody. In some embodiments, viability of CAR T cells is assessed by calculating the percentage of live relative to total CAR transduced T cells after incubation with anti-idiotypic antibody. In some embodiments, proliferation of CAR-T cells is assessed by diluting the dye used to stain CAR-T cells prior to incubation with anti-idiotypic antibody. In some embodiments, expression of T cell activation markers is assessed by flow cytometry staining for antibodies recognizing T cell activation markers. In some embodiments, T cell activation markers are selected from the group consisting of CD25, CD26, CD27, CD28, CD30, CD69, CD71, CD134, CD137, and CD154. In some embodiments, the incubation period is from about 1 to about 10 days (for example, about any of the ranges of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including any ranges between these values ). In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

C. Use in cell inactivation/depletion

In some embodiments, the provided anti-idiotypic antibodies or antigen-binding fragments thereof are antagonists and/or exhibit specific activity for inhibition, ablation, and/or depletion (e.g., killing via antibody-dependent cell-mediated cytotoxicity, ADCC) of cells expressing antibody- a target such as an anti-CD19 antibody (eg SJ25C1 or FMC63 antibody) or an antigen-binding fragment thereof. Also provided are methods comprising using the proposed anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies to inactivate, ablate and/or deplete CAR-T cells, wherein the CAR comprises a target antibody such as an anti-CD19 antibody (eg, an SJ25C1 or FMC63 antibody), or an antigen-binding fragment thereof.

Methods in some embodiments include treating, contacting and/or incubating a composition and/or sample containing CAR transduced T cells with an anti-idiotypic antibody. In some embodiments, the methods further comprise detecting whether CAR-T cells are inactivated, for example, by evaluating viability, proliferation, and/or expression of activation markers in CAR-T cells. In some embodiments, the implementation of the methods associated with therapy, including the introduction of CAR-T cells. The methods, in some embodiments, include administering an anti-idiotypic antibody to an individual. In one embodiment, an anti-idiotypic antibody or conjugate is used to remove and/or deplete (eg, kill) CAR-T cells in an individual. In some embodiments, the target antibody is an anti-CD19 antibody. In some embodiments, the target antibody is or is derived from an SJ25C1 or FMC63 antibody or antigen-binding fragments thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, an anti-idiotypic antibody is administered to deplete, reduce, and/or reduce the number of CAR-expressing cells in a patient. In specific embodiments, administration of an anti-idiotype antibody depletes, reduces, and/or reduces the number of CAR-expressing cells, e.g., circulating CAR-T cells, by at least 25%, at least 50%, at least 60%, at least at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100%, or about 100% . In some embodiments, the depletion, decrease, and/or decrease is related to the number of CAR-expressing cells in a patient prior to administration of the anti-idiotypic antibody. In specific embodiments, the depletion, decrease, and/or decrease is related to the number of CAR-expressing cells in a patient who is not receiving anti-idiotypic antibody. In some embodiments, CAR-expressing cells are not detected in a patient after administration of an anti-idiotypic antibody. In specific embodiments, the anti-idiotypic antibody is a human or humanized antibody.

In some embodiments, a method is provided for inactivating CAR-T cells, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising incubating a sample containing CAR-T cells with an antagonistic an anti-idiotypic antibody or antigen-binding fragment thereof that targets CAR, thereby inactivating CAR-T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to attenuate the activation of CAR-T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to substantially inactivate CAR-T cells in the sample. In some embodiments, incubation with an anti-idiotypic antibody results in ablation and/or depletion of CAR-T cells in the sample. In some embodiments, the anti-idiotypic antibody is used in an amount sufficient to result in clearance of CAR-T cells in the sample. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, an anti-idiotypic antibody is administered to deplete, decrease and/or decrease the activity of CARs and/or CAR-expressing cells in a patient. In specific embodiments, administration of an anti-idiotype antibody reduces and/or reduces the stimulation and/or activation of a CAR and/or a CAR-expressing cell by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100%, or about 100%. In some embodiments, the decrease and/or decrease is associated with the stimulation and/or activity of CAR-expressing cells and/or CAR in the patient prior to administration of the anti-idiotypic antibody. In specific embodiments, the decrease and/or decrease is associated with the stimulation and/or activity of CAR and/or CAR-expressing cells in a patient who is not administered anti-idiotypic antibody. In some embodiments, activity and/or stimulation refers to one or more aspects of CAR or CAR-T cell receptor activity, and may be measured by any suitable known methods, including any of the methods provided herein. In some embodiments, activity and/or stimulation of CAR and/or CAR-expressing cells in a patient is not detected in the patient after administration of the anti-idiotypic antibody. In specific embodiments, the anti-idiotypic antibody is a human or humanized antibody.

In some embodiments, an anti-idiotypic antibody is administered to prevent, reduce, and/or reduce the binding and/or ability of CAR and/or CAR-expressing cells to bind to an antigen. In specific embodiments, administration of an anti-idiotypic antibody reduces and/or reduces antigen binding of a CAR and/or a CAR expressing cell by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100%, or about 100%. In some embodiments, the reduction and/or reduction is related to antigen binding and/or the ability of CARs and/or CAR-expressing cells to bind to an antigen in a patient prior to administration of the anti-idiotypic antibody. In specific embodiments, the decrease and/or reduction is related to antigen binding and/or antigen binding ability of CAR and/or CAR expressing cells in a patient who has not received an anti-idiotypic antibody. In specific embodiments, the anti-idiotypic antibody is a human or humanized antibody.

In some embodiments, a method is provided to ablate and/or deplete (e.g., kill) CAR-T cells, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising incubating a sample containing CAR-T cells, with an anti-idiotypic antibody or antigen-binding fragment thereof targeted to CAR, thereby removing and/or depleting CAR-T cells in the sample. In some embodiments, the implementation of the ablation and/or depletion occurs through antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the anti-idiotype antibody is used in an amount sufficient to result in ablation and/or depletion of substantially all CAR-T cells in the sample. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for regulating a CAR-T cell therapy in an individual, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising administering to the individual an antagonistic anti-idiotypic antibody, or an antigen-binding fragment thereof. fragment targeting CAR, thereby inactivating CAR T cells. In some embodiments, the anti-idiotypic antibody is administered in an amount sufficient to attenuate CAR-T cell activation in the subject. In some embodiments, the anti-idiotypic antibody is administered in an amount sufficient to substantially inactivate CAR-T cells in the subject. In some embodiments, administration of an anti-idiotypic antibody results in ablation and/or depletion of CAR-T cells in an individual. In some embodiments, the anti-idiotypic antibody is administered in an amount sufficient to result in clearance of CAR-T cells in the subject. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

In some embodiments, a method is provided for regulating CAR-T cell therapy in an individual, wherein the CAR comprises a target antibody, such as an SJ25C1 or FMC63 antibody, or an antigen-binding fragment thereof, the method comprising administering to the individual an anti-idiotypic antibody immunoconjugate targeting CAR, wherein the anti-idiotypic antibody immunoconjugate contains a cytotoxic agent. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to attenuate CAR-T cell therapy in the subject. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to substantially terminate CAR-T cell therapy in the subject. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to result in clearance of CAR-T cells in the subject. In some embodiments, the cytotoxic agent is selected from the group consisting of 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. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24. In some embodiments, the target antibody or antigen-binding fragment thereof contain the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31.

D. Use in Binding Assay or Method

Provided herein are methods for assessing the presence or absence of a molecule in a sample that binds to a chimeric antigen receptor (CAR), such as the extracellular domain of CAR, or a portion thereof containing an antigen binding domain. In some embodiments, the methods can be used to evaluate the presence or absence of a humoral or antibody response from a patient to an administered cell therapy comprising a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor comprises a target antibody, which is an FMC63 antibody or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises a target antibody, which is an SJ25C1 antibody or an antigen-binding fragment thereof. In some embodiments, an anti-idiotypic antibody or an antigen-binding fragment thereof specific for the extracellular domain of CAR, such as any of those described herein, can be used as a positive method control.

In specific embodiments, the method comprises contacting a sample with an anti-idiotypic antibody or an antigen-binding fragment thereof specific for the CAR extracellular domain at an anti-idiotypic antibody concentration of 10 ng/mL to 100 μg/mL, from 100 ng/mL. and 1.0 µg/mL, 250 ng/mL to 10 µg/mL, 250 ng/mL to 1 µg/mL, 1 µg/mL to 10 µg/mL, 250 ng to 2.5 µg/mL ml or from 1 µg/ml and 10 µg/ml. In some embodiments, the anti-idiotype antibody concentration is between 250 ng/mL and 10 μg/mL. In some embodiments, the anti-idiotype antibody concentration is about 0.1 µg/mL, 0.25 µg/mL, 0.5 µg/mL, 1.0 µg/mL, 1.25 µg/mL, 2 µg/mL, 2 .5 µg/ml or 5 µg/ml.

In some aspects, adoptive cell therapy may be associated with the development of a patient's immune response to the injected cells and/or construct. For example, in some cases, the effect of the chimeric receptor may be limited by the host's immune response against the recombinant receptors expressed by the introduced cells, which may prematurely kill the cells. It has been observed that even in some patients with B-cell malignancies, who are often immunocompromised, an immune response specific to the receptor regions expressed by the cells administered in adoptive cell therapy can be detected. For example, patients, such as humans, who are injected with cells genetically engineered using CARs may develop a specific immune response to the immunogenic region of the chimeric region, including regions that may contain non-human sequences (e.g., murine scFvs), and/or to a region containing a junction between two domains or parts of a chimeric receptor, such as the transmembrane and costimulatory domain of CAR.

In some embodiments, methods are provided that include contacting or incubating a binding reagent with a patient sample that has been administered a cell therapy comprising cells engineered with a chimeric antigen receptor, wherein the binding reagent is a protein that includes the extracellular domain of CAR or a portion thereof containing an antibody. -target or its antigen-binding fragment. In some embodiments, the methods further comprise detecting whether a binding reagent is complexed with a molecule, e.g., a binding molecule, such as an antibody present in the sample, and/or detecting the presence or absence or level of such binding. In some embodiments, contacting or incubation is carried out under conditions that allow binding of the binding reagent to a molecule present in the patient sample. In certain aspects, the method can be further carried out on a positive control sample containing an anti-idiotypic antibody or a CAR-specific antigen-binding fragment thereof, such as any of those described herein. In some embodiments, determining the presence, absence, or level of binding of a molecule to a binding reagent may include comparing binding or detecting binding or detecting a positive control sample with a binding reagent.

In some embodiments, the cell therapy is or contains genetically engineered cells expressing an anti-CD19 CAR, comprising a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, where the binding reagent contains an extracellular domain of the CAR, or a portion thereof, containing the antibody SJ25C1 or its antigen-binding fragment. In some embodiments, the positive control comprises an anti-iditopic antibody as described in subsection I.A.

In some embodiments, the cell therapy is or contains genetically engineered cells expressing an anti-CD19 CAR, comprising a target antibody, which is the FMC63 antibody, or an antigen-binding fragment thereof, where the binding reagent contains the extracellular domain of the CAR, or a portion thereof, containing the antibody FMC63 or its antigen-binding fragment. In some embodiments, the positive control comprises an anti-iditopic antibody as described in subsection I.B.

In some embodiments, the methods include detecting whether a binding reagent is complexed with a molecule, e.g., a binding molecule, such as an antibody present in a sample, and/or detecting the presence or absence or level of such binding. In some embodiments, contacting or incubation is carried out under conditions that allow binding of the binding reagent to a molecule present in the patient sample. In some aspects, the complex is detected using an immunoassay, optionally a sandwich or bridge assay. For example, the immunoassay is an enzyme immunoassay (ELISA), chemiluminescent, electrochemiluminescent, surface plasmon resonance (SPR) biosensor (eg, BIAcore), flow cytometry, or Western blot analysis. In some embodiments, the immunoassay is or includes mesoscale detection.

In some aspects, the immunoassay is a sandwich assay or a bridge assay. In a sandwich or bridging assay, the binding reagent is the first binding reagent, and detecting the presence or absence of a molecule or a complex comprising the molecule comprises contacting the complex formed between the first binding reagent and the molecule with a second binding reagent, wherein the second binding reagent is an agent capable of binding to the same or similar molecule as the first binding agent. In some embodiments, the implementation of the second binding reagent contains the extracellular domain of CAR or part thereof. In some aspects, the extracellular domain of the CAR, or a portion thereof, of the first binding agent and the second binding agent are the same or substantially the same.

In some embodiments, the binding reagent, such as the first and/or the second binding reagent, is detectably labeled or capable of generating a detectable signal. A binding reagent, such as a first and/or a second binding reagent, is directly or indirectly associated with a detectable label. In some embodiments, the detectable label is or includes a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label. In some embodiments, a binding reagent, such as a first and/or a second binding reagent, is directly or indirectly linked to a SULFO tag. In some embodiments, at least one of the first and second binding reagents is detectably labeled or capable of generating a detectable signal, and the other of the first and second binding reagents is attached or immobilized to a solid support. In some aspects, the first binding agent is affixed to or immobilized on a solid support, or may be affixed to or immobilized on a solid support. Methods for directly or indirectly attaching a binding agent to a solid support are well known in the art. Attachment methods typically involve non-specific adsorption of the binding agent to a solid support, or covalent attachment of the binding agent, usually via a free amino group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Attachment methods also include indirectly attaching a binding reagent to a solid support, such as by applying a capture reagent such as streptavidin to the solid support and adding affinity-labeled binding reagents, such as biotin labeled reagents, to the solid support so that the interaction between the affinity tag ( eg biotin) and a capture reagent (eg streptavidin) binds the binding reagent to the solid support. In some embodiments, the implementation of the first binding reagent is directly or indirectly associated with biotin. In some examples, the first soluble reagent is associated with a solid support coated with streptavidin. In some embodiments, the second binding reagent is directly or indirectly linked to a detectable label, optionally a SULFO label.

In specific embodiments, the sample is contacted with a first binding reagent that is attached, bound, coated, and/or conjugated to a solid surface or support, such as a plate or well. In some embodiments, the first binding reagent is attached, bonded, coated, and/or conjugated to a solid surface, or by indirectly attaching the binding reagent to a solid support, such as by applying a capture reagent, such as streptavidin, to the solid support and adding to a solid support of affinity-labeled binding reagents, such as biotin-labeled reagents, such that the interaction between the affinity tag (eg, biotin) and the capture reagent (eg, streptavidin) binds the binding reagent to the solid support. In some embodiments, the sample is contacted with a second binding agent that is directly or indirectly associated with the SULFO tag. In specific embodiments, the first and/or second binding reagent is used at an anti-idiotype antibody concentration that is 10 ng/mL to 100 µg/mL, 100 ng/mL to 1.0 µg/mL, 250 ng/mL to 10 mcg/mL, 250 ng/mL to 1 mcg/mL, 1 mcg/mL to 10 mcg/mL, 250 ng to 2.5 mcg/mL, or 1 mcg/mL to 10 mcg/mL. In some embodiments, the solid surface or support is incubated with an anti-idiotypic antibody at a concentration of 250 ng/mL to 10 μg/mL. In some embodiments, the solid surface or substrate is incubated at or about 0.25 µg/mL, 0.5 µg/mL, 1.0 µg/mL, 1.25 µg/mL, 2 µg/mL, 2 .5 µg/ml, 5 µg/ml or 10 µg/ml.

In some embodiments, a sample from a patient who has received a cell therapy comprising cells engineered with a chimeric antigen receptor is or contains any sample of the patient's bodily fluid. In some aspects, the sample is or contains whole blood, serum, or plasma. In some embodiments, the sample is obtained from the patient within or about 1 hour to 1 year after the start of the cell therapy or cell dose, e.g., within or about 6 hours, 12 hours, 24 hours, one week, two weeks , three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or twelve months. In some aspects, the sample is obtained from the patient at or about 1 month to 6 months from the start of cell therapy administration, e.g., 2 months to 6 months, or 2 months to 4 months, e.g., about or about 2 months, 3 months , 4 months, 5 months or 6 months after the start of cell therapy.

VI. Products

Articles or kits containing the proposed anti-idiotypic antibodies and/or compositions are also provided. In some embodiments, an article of manufacture is provided that contains an anti-idiotypic antibody, or an antigen-binding fragment thereof. In some instances, the anti-idiotypic antibody binds to an anti-CD19 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor containing an anti-CD19 antibody or antigen-binding fragment thereof. In some examples, the anti-CD19 antibody is SJ25C1 or FMC63. In some aspects, the articles or kits provide a conjugate containing the anti-idiotypic antibodies described herein.

In some embodiments, the kit or article of manufacture comprises an anti-idiotypic antibody or an antigen-binding fragment thereof and a binding reagent containing the extracellular domain or part of the extracellular domain of a chimeric antigen receptor (CAR) to which the anti-idiotypic antibody binds, e.g., specifically binds. In some embodiments, the CAR extracellular domain is or includes an anti-CD19 antibody (eg, FMC63 or SJ25C1) or an antigen-binding fragment thereof.

In some embodiments, the implementation of the binding agent is the first binding agent, and the kit or article of manufacture further includes a second binding agent. In such examples, the second coupling agent is an agent that is capable of binding to the same or similar molecule as the first coupling agent. In some embodiments, the implementation of the second binding reagent contains the extracellular domain of CAR or part thereof. In some aspects, the extracellular domain of the CAR, or a portion thereof, of the first binding agent and the second binding agent are the same or substantially the same.

In some embodiments, a binding reagent, or at least one of the first and second binding reagents, is attached to a label (eg, a detectable label), such as the label described herein. In some embodiments, at least one of the first and second coupling reagents is attached to a solid support or is capable of being attached to a solid support, such as described herein. In some aspects, one of the first and second binding reagents is detectably labeled or capable of generating a detectable signal, and the other of the first and second binding reagents is attached or immobilized on a solid support. In some embodiments, binding reagents are provided as a kit or as part of a system, as described elsewhere herein, for use in connection with an immunoassay (eg, sandwich or bridge assay). In some embodiments, the implementation of the first binding reagent is associated with a solid support, optionally with a solid support coated with streptavidin. In some embodiments, the second soluble protein is directly or indirectly linked to a detectable label, such as a SULFO label.

In some embodiments, the kit further comprises an anti-idiotype antibody or antigen-binding fragment. In some aspects, the anti-idiotypic antibody binds to an anti-CD19 antibody, or antigen-binding fragment thereof, or a chimeric antigen receptor comprising the anti-CD19 antibody, or antigen-binding fragment thereof. In some examples, the anti-CD19 antibody is SJ25C1 or FMC63. In some embodiments, an anti-idiotypic antibody or antigen-binding fragment thereof is provided as a positive control sample. In some examples, a positive control sample is complexed with first and second soluble proteins or reagents that contain regions of the extracellular domain of a chimeric antigen receptor (CAR) containing an anti-CD19 antibody or antigen-binding fragment thereof.

In some embodiments, the kit or article contains reagents or components for carrying out any of the proposed methods. In some embodiments, the product or kit contains one or more commercially, therapeutically, and user-friendly reagents or other materials, including secondary antibodies, affinity labels, capture reagents, buffers, diluents, signal detection agents, filters, needles, syringes. , capillary tubes and package inserts with instructions for use.

In some embodiments, kits may be provided as articles that include packaging materials for packaging antibodies or compositions thereof, or one or more additional reagents, such as binding reagents or components. For example, kits may contain containers, bottles, test tubes, vials, and any packaging material suitable for separating or organizing kit components.

In some embodiments, the kit includes one or more containers. Suitable containers include, for example, bottles, vials (eg dual chamber vials), syringes (such as single chamber or dual chamber syringes) and test tubes. One or more containers may be made from various materials such as glass or plastic. One or more containers contain a composition containing the antibody or other reagents, such as binding reagents, for use in the methods. The product or kit may contain antibodies or reagents in separate containers or in the same container. In some embodiments, the one or more containers containing the composition may be a disposable vial or a refillable vial, which in some cases may allow reuse of the reconstituted composition.

In some embodiments, the product or kit may further comprise a second container containing a suitable diluent. The article or kit may additionally include other materials suitable from a commercial, therapeutic and user point of view, including other buffers, diluents, filters, needles, syringes, therapeutic agents and/or package inserts with instructions for use.

Articles may include a container and a label or insert within or associated with the package. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. The containers can be made from various materials such as glass or plastic. The container, in some embodiments, contains a composition comprising an anti-idiotypic antibody provided herein, which alone or in combination with another composition is effective in treating, preventing, and/or diagnosing a disease or condition. In some embodiments, the container has a sterile access port. Typical containers include intravenous solutions, vials, including those with stoppers pierced by an injection needle. The article of manufacture may include a first container containing a composition, wherein the composition comprises an anti-idiotypic antibody. Alternatively or additionally, the product may further include another or the same container containing an acceptable buffer. In addition, it may include other materials such as other buffers, diluents, filters, needles and/or syringes.

In some embodiments, the product or kit comprises a solid support, including a solid support formed from glass (e.g., porous glass with controlled pore size), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, nitrocellulose, cellulose, nylon, silicone and other materials well known in the art that are used in a solid support for direct or indirect attachment of a binding agent as described herein. The solid supports included in the articles or kits provided herein include, but are not limited to, a microsphere, a column (e.g., a chromatographic column, etc.), a chip (e.g., a microchip, a nanochip, etc.), assay plate, cartridge, stick, filter, strip, or any other solid support described herein.

In some embodiments, the product or kit may further comprise a second container containing a suitable diluent. The article or kit may additionally include other materials suitable from a commercial, therapeutic and user point of view, including other buffers, diluents, filters, needles, syringes, therapeutic agents and/or package inserts with instructions for use.

In some embodiments, the kit may optionally include instructions. The instructions typically include a literal term describing the antibodies and optionally other components included in the kit, such as a binding reagent, and methods for using the antibodies and/or other components in or in combination with any of the uses or methods as described. in this document. In some embodiments, the instructions are provided in the form of a label or package insert that is on or associated with the container. In some embodiments, the instructions may indicate directions for reconstitution and/or use of the composition.

In some embodiments, instructions are provided for using an anti-idiotypic antibody to detect an SJ25C1 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising an SJ25C1 antibody or antigen-binding fragment thereof, for example, according to or in combination with any of the methods or assays as described in this document. In some examples, instructions are given for using an anti-idiotypic antibody to select or enrich engineered cells from a cell population that express a chimeric antigen receptor (CAR) containing an SJ25C1 antibody or antigen-binding fragment thereof. In some examples, instructions are given for using an anti-idiotypic antibody to stimulate an initial composition containing cells expressing a chimeric antigen receptor containing an SJ25C1 antibody or antigen-binding fragment thereof.

In some embodiments, instructions are provided for using an anti-idiotype antibody to detect an FMC63 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising an FMC63 antibody or antigen-binding fragment thereof, for example, according to or in combination with any of the methods or assays as described in this document. In some examples, instructions are given for using an anti-idiotypic antibody to select or enrich engineered cells from a cell population that express a chimeric antigen receptor (CAR) containing the FMC63 antibody or antigen-binding fragment thereof. In some examples, instructions are given for using an anti-idiotypic antibody to stimulate an initial composition containing cells expressing a chimeric antigen receptor containing the FMC63 antibody or antigen-binding fragment thereof.

In some embodiments, instructions are provided for using a kit provided for detecting a molecule that binds to a cell therapy chimeric antigen receptor receptor, such as an antibody, such as an antibody produced in a humoral immune response to a chimeric antigen receptor (CAR). In some embodiments, instructions are provided for contacting a binding reagent with a sample of a patient who has been administered a cell therapy comprising cells constructed using a CAR containing a target antibody that is an anti-CD19 antibody (e.g., FMC63 or SJ25C1), or an antigen binding thereof. fragment, where the binding reagent contains the extracellular domain of CAR or part of the extracellular domain containing the target antibody, or its antigen-binding fragment. In some aspects, the instructions also define the detection of the presence or absence of a complex comprising binding reagents and a molecule from the sample that binds to both the first and second binding reagent, where the molecule optionally is or contains an antibody. In some aspects, the anti-CD19 antibody is SJ25C1 or FMC63. In some additional embodiments, instructions are provided for the use of binding reagents and a positive control sample.

VII. Definitions

Unless otherwise indicated, all terms of the technical field, remarks and other technical and scientific terms used in the present description have the same meaning as is usually understood by a person skilled in the art to which the present invention pertains. In some instances, terms with conventional meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a significant difference from what is commonly understood in the art.

As used herein, the term "corresponding form" of an antibody means that when a property or activity of two antibodies is compared, that property is compared using the same form of the antibody. For example, if an antibody is said to be more active than the corresponding form of the first antibody, this means that the particular form, such as the scFv of that antibody, is more active than the scFv form of the first antibody.

As used herein, the expression that nucleotide or amino acid positions "correspond" to nucleotide or amino acid positions in a disclosed sequence, such as that presented in a sequence listing, refers to the nucleotide or amino acid positions identified when aligned with the disclosed sequence to maximize identity using a standard algorithm. alignment such as the GAP algorithm. By aligning sequences, one of skill in the art can identify appropriate residues, for example, using conserved and identical amino acid residues as guides. In general, to identify relevant positions, amino acid sequences are aligned to obtain the highest order match (see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects , Smith, D.W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New.Jersey, 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073).

"Effector functions" refers to those biological activities that are related to the Fc region of the antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg B-cell receptor); and B cell activation.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes a native sequence Fc region and variants of Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The terms "full antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody, or having heavy chains that contain an Fc region as defined herein. document.

An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange HPLC or reverse phase HPLC). ) methods. For a review of methods for assessing the purity of antibodies, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

"Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal arrangement that differs from its natural chromosomal arrangement.

"An isolated nucleic acid encoding an anti-idiotypic antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and wherein such a molecule( s) nucleic acids are present at one or more locations in the host cell.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its derived progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected originally transformed cells are included in this description.

As used herein, the terms "percent (%) amino acid sequence identity" and "percent identity" when used in relation to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., antibody or fragment thereof) that are identical amino acid residues in the sequence of the reference polypeptide after aligning sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Suitable parameters for sequence alignment can be determined by those skilled in the art, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences.

An amino acid substitution may include the substitution of one amino acid in a polypeptide for another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions can be introduced into the binding molecule, eg, the antibody of interest, and into products screened for activity of interest, eg, preserved/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can generally be grouped according to the following general side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) main: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

In some embodiments, conservative substitutions may include replacing a member of one of these classes with another member of the same class. In some embodiments, non-conservative amino acid substitutions may include replacing a member of one of these classes with another class.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector inserted into the genome of the host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "package insert" is used to refer to instructions commonly included in commercial packaging of therapeutic products that contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

As used herein, the singular forms include references to the plural unless the context clearly dictates otherwise. For example, the singular forms mean "at least one" or "one or more". It is understood that the aspects and variants described herein include "consisting of" and/or "consisting essentially of" the aspects and variants.

Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, when a range of values is proposed, it is understood that every intermediate value between the upper and lower limit of that range and any other declared or intermediate value in the specified range is included in the claimed object. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the claimed subject matter, subject to any specifically excluded limit in the specified range. If the claimed range includes one or both of these limits, ranges excluding one or both of these included limits are also included in the claimed subject matter. This applies regardless of the latitude of the range.

Used in the present description, the term "about" refers to the usual range of errors for the corresponding value, well known to a person skilled in the art. Reference to "about" in relation to a value or parameter herein includes (and describes) embodiments that refer to that value or parameter as such. For example, a description referring to "about X" includes a description of "X".

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the proposed antibodies and antibody chains and other peptides, such as linkers, may include amino acid residues, including natural and/or non-natural amino acid residues. The terms also include modifications to the polypeptide after expression, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications in relation to the native or natural sequence, provided that the protein maintains the target activity. These modifications may be deliberate, such as introduced by site-directed mutagenesis, or may be accidental, such as mutations in hosts that produce proteins, or errors due to PCR amplification.

Used in the present description, the term "composition" refers to any mixture of two or more products, substances or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, the statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or within a cell of a particular marker, usually a surface marker. When referring to a surface marker, the term refers to the presence of surface expression detectable by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting said antibody, where the staining is detected by flow cytometry at a level substantially greater than the staining detected. in the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially similar to that of a cell known to be positive for the marker and/or at a level substantially higher than that for cells known to be negative for the marker.

As used herein, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of a detectable significant presence on or within the cell of a particular marker, usually a surface marker. When referring to a surface marker, the term refers to the absence of surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and by detecting said antibody, where the staining is not detected by flow cytometry at a level substantially higher than detected. staining by performing the same procedure with an isotype matched control under otherwise identical conditions, and/or at a level substantially lower than that of a cell known to be positive for the marker and/or at a level substantially similar to level for a cell known to be negative for the marker.

VIII. Exemplary Embodiments

The embodiments proposed in this document are:

1. An anti-idiotypic antibody, or antigen-binding fragment thereof, that specifically binds to a target antibody, which is an SJ25C1 antibody, or antigen-binding fragment thereof.

2. Anti-idiotypic antibody or antigen-binding fragment according to embodiment 1, characterized in that the antibody or antigen-binding fragment contains:

a light chain variable region (VL) that is at least 90% identical to the VL region with the amino acid sequence of SEQ ID NO: 5; and/or

a heavy chain variable region (VH) that is at least 90% identical to the VH region of the amino acid sequence of SEQ ID NO: 1.

3. The antibody or antigen-binding fragment, where the antibody or antigen-binding fragment contains:

a VL region that is at least 90% identical to the VL region with the amino acid sequence of SEQ ID NO: 5; and/or

a VH region that is at least 90% identical to the VH region with the amino acid sequence of SEQ ID NO: 1.

4. Anti-idiotypic antibody or antigen-binding fragment according to embodiments 2 or 3, characterized in that

the VH region contains a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence of SEQ ID NO: 11 or 84, or containing a CDR-H3 contained in the VH sequence shown in SEQ ID NO: 1; and/or

the VL region contains a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 14 or 87, or containing a CDR-L3 contained in the VL sequence of SEQ ID NO: 5,

5. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 2-4, characterized in that:

the VH region contains CDR-H1 and CDR-H2, respectively, containing the amino acid sequences of the CDR-H1 and CDR-H2 sequences contained in the VH region with the amino acid sequence of SEQ ID NO: 1; and/or

the VL region contains CDR-L1 and CDR-L2, respectively, containing the amino acid sequences of the CDR-L1 and CDR-L2 sequences contained in the VL region with the amino acid sequence of SEQ ID NO: 5.

6. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 2-5, characterized in that:

the VH region contains a CDR-H1 listed in SEQ ID NO: 9, 78, 79, or 80, a CDR-H2 listed in SEQ ID NO: 10, 81, 82, or 83, and a set of CDR-H3 further in SEQ ID NO: 11 or 84; and/or

the VL region contains CDR-L1 with SEQ ID NO: 12 or 85, CDR-L2 with SEQ ID NO: 13 or 86, and CDR-L3 with SEQ ID NO: 14 or 87.

7. Anti-idiotypic antibody or its antigen-binding fragment, containing:

CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 1; and/or

CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 5.

8. Anti-idiotypic antibody or its antigen-binding fragment, containing:

CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, 78, 79 or 80, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, 81, 82 or 83, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 11 or 84; and/or

CDR-L1 containing the amino acid sequence of SEQ ID NO: 12 or 85, CDR-L2 containing the amino acid sequence of SEQ ID NO: 13 or 86, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 14 or 87.

9. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-8, characterized in that

the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 1; and/or

the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 5.

10. Anti-idiotypic antibody or antigen-binding fragment thereof according to embodiment 9, characterized in that the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 1, and the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 5.

11. An anti-idiotypic antibody or antigen binding fragment according to any one of embodiments 1-10, wherein the target antibody or antigen binding fragment comprises the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24.

12. An anti-idiotypic antibody, or antigen-binding fragment thereof, that specifically binds to the target antibody, which is the FMC63 antibody, or antigen-binding fragment thereof.

13. An anti-idiotypic antibody or antigen-binding fragment thereof according to embodiment 12, characterized in that the antibody or antigen-binding fragment contains:

a light chain variable region (VL) that is at least 90% identical to the VL region with the amino acid sequence of SEQ ID NO: 40 or 62; and/or

a heavy chain variable region (VH) that is at least 90% identical to the VH region with the amino acid sequence of SEQ ID NO: 36 or 58.

14. An antibody or its antigen-binding fragment, characterized in that the antibody or its antigen-binding fragment contains:

a VL region that is at least 90% identical to the VL region with the amino acid sequence of SEQ ID NO: 40 or 62; and/or

a (VH) region that is at least 90% identical to the VH region with the amino acid sequence of SEQ ID NO: 36 or 58.

15. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-14, characterized in that

the VH area contains:

heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence GYX ₃ FX ₅ X ₆ YX ₈ MX ₁₀ (SEQ ID NO: 108), where 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;

heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence WIGX ₄ IX ₆ PX ₈ X ₉ X ₁₀ X ₁₁ TX ₁₃ X ₁₄ NQX ₁₇ FKX ₂₀ (SEQ ID NO: 109), where X ₄ is D or M, X ₆ is N or H, X ₈ is N or S, X ₉ is N or D, X ₉ is N or DX 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;

heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence 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 ), where 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 none , X ₈ is G or absent, X ₉ is S or absent, X ₁₀ is R or absent, X ₁₁ is D or absent, X ₁₂ is A or absent, X ₁₃ is M or absent, X ₁₄ is D or E and X ₁₅ is Y or A; and/or

the VL area contains:

light chain complementarity determining region 3 (CDR-L1) comprising the amino acid sequence X ₁ AX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ YX ₁₀ X ₁₁ WY (SEQ ID NO: 111), where 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 absent, X ₁₀ is M or L and X ₁₁ is Y or A;

light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence X ₁ X ₂ X ₃ YX ₅ X ₆ X ₇ X ₈ LAX ₁₁ (SEQ ID NO: 112), where 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;

light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence QX ₂ X ₃ X ₄ X ₅ X ₆ PX ₈ T (SEQ ID NO: 113), where 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.

16. Anti-idiotypic antibody or antigen-binding fragment according to embodiment 15, characterized in that

complementarity determining region 3 (CDR-H3) contains the amino acid sequence of SEQ ID NO: 94 or 104, or contains a CDR-H3 contained in the VH sequence of SEQ ID NO: 36 or 58; and/or

light chain complementarity determining region 3 (CDR-L3) contains the amino acid sequence of SEQ ID NO: 97 or 107, or contains a CDR-L3 contained in the VL sequence of SEQ ID NO: 40 or 62.

17. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-16, characterized in that

the VH region contains CDR-H1 and CDR-H2, respectively, containing the amino acid sequences of CDR-H1 and CDR-H2 contained in the VH region with the amino acid sequence of SEQ ID NO: 36 or 58; and/or

the VL region contains CDR-L1 and CDR-L2, respectively, containing the amino acid sequences of CDR-L1 and CDR-L2 contained in the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

18. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-17, characterized in that

the VH region contains CDR-H1 with SEQ ID NO: 88, 89, 90, 98, 99, or 100, CDR-H2 with SEQ ID NO: 91, 92, 93, 101, 102, or 103, and CDR-H3 with SEQ ID NO: 94 or 104; and/or

the VL region contains CDR-L1 with SEQ ID NO: 95 or 105, CDR-L2 with SEQ ID NO: 96 or 106, and CDR-L3 with SEQ ID NO: 97 or 107.

19. Anti-idiotypic antibody or its antigen-binding fragment, containing:

CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 36 or 58; and/or

CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 40 or 62.

20. Anti-idiotypic antibody or its antigen-binding fragment, containing:

CDR-H1 containing the amino acid sequence of SEQ ID NO: 88, 89, 90, 98, 99 or 100, CDR-H2 containing the amino acid sequence of SEQ ID NO: 91, 92, 93, 101, 102 or 103, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 94 or 104; and/or

CDR-L1 containing the amino acid sequence of SEQ ID NO: 95 or 105, CDR-L2 containing the amino acid sequence of SEQ ID NO: 96 or 106, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 97 or 107.

21. Anti-idiotypic antibody or antigen-binding fragment according to any of embodiments 13-21, characterized in that

the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 36 or 58; and/or

the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 40 or 62.

22. Anti-idiotypic antibody or antigen-binding fragment thereof according to embodiment 21, characterized in that the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 36 or 58, and the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 40 or 62.

23. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-22, characterized in that

the VH region contains CDR-H1 with SEQ ID NO: 44, 88, 89 or 90, CDR-H2 with SEQ ID NO: 45, 91, 92 or 93, and CDR-H3 with SEQ ID NO: 46 or 94; and/or

the VL region contains CDR-L1 with SEQ ID NO: 47 or 95, CDR-L2 with SEQ ID NO: 48 or 96, and CDR-L3 with SEQ ID NO: 49 or 97.

24. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-23, characterized in that

the VH region contains CDR-H1 with SEQ ID NO: 65, 98, 99 or 100, CDR-H2 with SEQ ID NO: 66, 101, 102 or 103, and CDR-H3 with SEQ ID NO: 67 or 104; and/or

the VL region contains CDR-L1 with SEQ ID NO: 68 or 105, CDR-L2 with SEQ ID NO: 69 or 106, and CDR-L3 with SEQ ID NO: 100 or 107.

25. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-24, characterized in that

the VH region contains CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 36; and/or

the VL region contains CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 40.

26. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-24, characterized in that

the VH region contains CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained in the VH region with the amino acid sequence of SEQ ID NO: 58; and/or

the VL region contains CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the VL region with the amino acid sequence of SEQ ID NO: 62.

27. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-25, characterized in that

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 contains the amino acid sequence of SEQ ID NO: 40.

28. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 13-24 and 26, characterized in that

the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 58; and/or

the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID NO: 62.

29. An anti-idiotypic antibody or antigen binding fragment according to any one of embodiments 1-28, wherein the target antibody or antigen binding fragment is a single chain fragment.

30. An anti-idiotypic antibody or antigen-binding fragment according to embodiment 29, characterized in that the fragment contains antibody variable regions connected by a flexible linker.

31. Anti-idiotypic antibody or antigen-binding fragment according to embodiment 29 or 30, characterized in that the fragment contains scFv.

32. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-11 and 29-31, wherein the target antibody or antigen-binding fragment:

contains the variable region of the heavy chain of SEQ ID NO: 23, and/or the variable region of the light chain of SEQ ID NO: 24; and/or

is an scFv containing the amino acid sequence of SEQ ID NO: 28.

33. Anti-idiotypic antibody or antigen-binding fragment according to claims 12-31, characterized in that the target antibody or antigen-binding fragment:

contains the variable region of the heavy chain of SEQ ID NO: 30, and/or the variable region of the light chain of SEQ ID NO: 31; and/or

is an scFv containing the amino acid sequence of SEQ ID NO: 34.

34. An anti-idiotypic antibody or antigen-binding fragment thereof, wherein the anti-idiotypic antibody or antigen-binding fragment specifically binds to the same or overlapping epitope of the target antibody or antigen-binding fragment thereof as the epitope specifically bound by the anti-idiotypic antibody or antigen-binding fragment. a binding fragment according to any one of embodiments 1-33.

35. Anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-34, characterized in that

the target antibody or antigen-binding fragment is within or included in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR); and/or

the anti-idiotypic antibody or antigen-binding fragment specifically binds to a target antibody or antigen-binding fragment contained in or included in the antigen-binding domain of the extracellular portion of the CAR.

36. An anti-idiotypic antibody or antigen-binding fragment according to embodiment 35, wherein the target antibody or antigen-binding fragment is an scFv and the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope in the scFv CAR.

37. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, 29-32 and 35, characterized in that the antibody or fragment specifically binds to a single-chain variable fragment (scFv) derived from the SJ25C1 antibody contained in the extracellular parts of a chimeric antigen receptor, where optionally the scFv derived from the SJ25C1 antibody contains the heavy chain variable region of SEQ ID NO: 23 and/or the light chain variable region of SEQ ID NO: 24; and/or contains the amino acid sequence of SEQ ID NO: 28.

38. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 12-31 and 33-35, characterized in that the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from the FMC63 antibody or containing variable regions, derived from FMC63 contained in the extracellular portion of the chimeric antigen receptor, where optionally the scFv derived from the FMC63 antibody comprises the heavy chain variable region of SEQ ID NO: 30 and/or the light chain variable region of SEQ ID NO: 31; and/or contains the amino acid sequence of SEQ ID NO: 34.

39. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-38, wherein the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope within or within all or part of the complementarity determining region (CDR) of the antibody- target or antigen-binding fragment.

40. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 35-39, wherein the CAR further comprises a transmembrane domain coupled to the antigen-binding domain via a spacer.

41. An anti-idiotypic antibody according to embodiment 40, wherein the spacer contains an extracellular portion from CD28, which is optionally human CD28.

42. An anti-idiotypic antibody or antigen-binding fragment according to embodiment 41, characterized in that the extracellular portion of CD28 contains the amino acid sequence of SEQ ID NO: 27.

43. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 40-42, wherein the transmembrane domain contains the transmembrane portion of CD28, which is optionally human CD28.

44. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 40-43, wherein the antibody or fragment does not bind to an epitope in a CAR spacer domain.

45. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-44, wherein the antibody or fragment does not bind or bind specifically to CD28 or a portion thereof, which is optionally human CD28, which optionally contains an extracellular portion of CD28, which optionally contains the amino acid sequence of SEQ ID NO: 27.

46. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-45, wherein the antibody or fragment does not bind to an epitope in an Fc domain, which is optionally a human IgG1 Fc domain.

47. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-46, wherein the target antibody or antigen-binding fragment specifically binds to human CD19.

48. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-47, wherein the anti-idiotypic antibody or fragment does not cross-react with another anti-CD19 antibody that is optionally contained in the extracellular antigen-binding domain of another CAR.

49. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-48, wherein the anti-idiotypic antibody or fragment does not cross-react with another CAR.

50. The anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-49, wherein the anti-idiotypic antibody or fragment is an agonist antibody for a CAR containing a target antibody or antigen-binding fragment.

51. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-49, wherein the antibody or fragment is an antagonist for a CAR containing a target antibody or antigen-binding fragment.

52. An anti-idiotypic antibody, or antigen-binding fragment thereof, according to any one of Embodiments 1-51, which are humanized.

53. An anti-idiotypic antibody or antigen-binding fragment thereof, according to any one of embodiments 1-52, which are recombinant.

54. An anti-idiotypic antibody, or antigen-binding fragment thereof, according to any one of embodiments 1-53, which is monoclonal.

55. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-54, which is an antigen-binding fragment.

56. Anti-idiotypic antibody or antigen-binding fragment according to embodiment 55, characterized in that the antigen-binding fragment is selected from antigen-binding fragments (Fab), F(ab') ₂ , Fab', Fv, single chain variable fragment (scFv) or a single domain antibody.

57. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 1-54, comprising at least a portion of an immunoglobulin constant region.

58. An anti-idiotypic antibody or antigen-binding fragment according to embodiment 57, characterized in that at least a portion of the immunoglobulin constant region contains an Fc region or an Fc portion containing CH2 and CH3 domains.

59. Anti-idiotypic antibody or antigen-binding fragment according to embodiment 57 or 58, characterized in that the constant region is derived from human IgG.

60. An anti-idiotypic antibody or antigen-binding fragment according to any one of embodiments 57-59, which is an intact antibody or a full-length antibody.

61. A conjugate comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-60 and a heterologous molecule or fragment.

62. A conjugate according to embodiment 61, wherein the heterologous molecule or fragment is a label.

63. The conjugate of embodiment 62, 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, streptavidin, biotin, a luminescent compound, or an oligonucleotide.

64. A conjugate according to embodiment 62, wherein the heterologous molecule or fragment is a protein, peptide, nucleic acid, or small molecule, which optionally is or contains a toxin or Strep tag.

65. Nucleic acid molecule(s) encoding the heavy chain and/or light chain of an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of embodiments 1-60.

66. A nucleic acid molecule according to embodiment 65, comprising

a nucleotide sequence encoding (i) a heavy chain variable region of SEQ ID NO: 15, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 15; or (iii) a degenerate sequence of (i) or (ii); and/or

a nucleotide sequence encoding (iv) a light chain variable region of SEQ ID NO: 19, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 19; or (vi) a degenerate sequence (iv) or (v).

67. A nucleic acid molecule according to embodiment 65 or 66, comprising

a nucleotide sequence encoding (i) a heavy chain of SEQ ID NO: 17, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 17; or (iii) a degenerate sequence of (i) or (ii); and/or

a nucleotide sequence encoding (iv) a light chain of SEQ ID NO: 21, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 21; or (vi) a degenerate sequence (iv) or (v).

68. A nucleic acid molecule according to embodiment 65, comprising

a nucleotide sequence encoding (i) a heavy chain variable region of SEQ ID NO: 50 or 71, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 50 or 71; or (iii) a degenerate sequence of (i) or (ii); and/or

a nucleotide sequence encoding (iv) a light chain variable region of SEQ ID NO: 54 or 75, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 54 or 75; or (vi) a degenerate sequence (iv) or (v).

69. A nucleic acid molecule according to embodiment 65 or 68, comprising

a nucleotide sequence encoding (i) a heavy chain of SEQ ID NO: 52 or 73, (ii) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 52 or 73; or (iii) a degenerate sequence of (i) or (ii); and/or

a nucleotide sequence encoding (iv) a light chain of SEQ ID NO: 56 or 76, (v) a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 56 or 76; or (vi) a degenerate sequence (iv) or (v).

70. A nucleic acid molecule according to any one of embodiments 65-69, characterized in that the nucleotide sequence encoding the heavy chain and/or light chain contains a signal sequence.

71. A vector containing a nucleic acid molecule according to any one of embodiments 65-70.

72. A cell comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 1-41 or a nucleic acid molecule according to any one of embodiments 65-70.

73. A method for producing an anti-idiotypic antibody or antigen-binding fragment thereof, comprising expressing the heavy and/or light chain encoded by the nucleic acid molecule according to any of embodiments 65-70 or the vector according to embodiment 71 in a suitable host cell and recovery or isolation antibodies.

74. A method for producing an anti-idiotypic antibody or an antigen-binding fragment thereof, comprising culturing a cell according to Embodiment 72 under conditions in which a heavy chain and/or a light chain is expressed, and recovering or isolating the antibody.

75. An anti-idiotypic antibody, or antigen-binding fragment thereof, obtained by the method of embodiment 73 or 74.

76. A composition comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 1-60, a conjugate according to any of embodiments 61-64, or a cell according to embodiment 72.

77. Composition according to embodiment 76 further comprising a pharmaceutically acceptable excipient.

78. A kit containing one or more anti-idiotypic antibodies or antigen-binding fragments thereof according to any of embodiments 1-60, a conjugate according to any of embodiments 161-64, a nucleic acid according to any of embodiments 65-70, and optionally instructions by application.

79. A kit according to embodiment 78 further comprising a reagent or support for immobilizing an anti-idiotypic antibody or an antigen-binding fragment or conjugate thereof, wherein said reagent or support is a microsphere, column, microwell, rod, filter, strip, or soluble streptavidin mutein oligomeric reagent.

80. A method for detecting a target antibody or its antigen-binding fragment, comprising:

(a) contacting a composition comprising a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment, with an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 1-11 and 29-32, 34-37 and 39- 60 or a conjugate according to any one of embodiments 61-64 that specifically binds to a target antibody, which is an SJ25C1 antibody or antigen-binding fragment thereof; and

(b) detecting an anti-idiotypic antibody bound to the target antibody or antigen-binding fragment.

81. A method for detecting a target antibody or its antigen-binding fragment, comprising:

(a) contacting a composition comprising a target antibody, which is an FMC63 antibody, or an antigen-binding fragment, with an anti-idiotypic antibody or an antigen-binding fragment thereof according to any of embodiments 12-31, 33-36 and 38-60 or a conjugate according to any of embodiments 61-64 that specifically bind to a target antibody, which is an FMC63 antibody or antigen-binding fragment thereof; and

(b) detecting an anti-idiotypic antibody bound to the target antibody or antigen-binding fragment.

82. The method according to embodiment 81, wherein the target antibody or antigen-binding fragment is bound to or expressed on the cell surface, and the detection in (b) includes detection of cells bound to the anti-idiotypic antibody.

83. The method according to embodiment 82, wherein the cell expresses on its surface a CAR containing a target antibody or antigen binding fragment.

84. A method for detecting a CAR containing a target antibody or its antigen-binding fragment, comprising:

(a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, according to any of embodiments 1-11 and 29-32, 34-37 and 39- 60, or a conjugate according to any one of embodiments 61-64, which specifically binds to a target antibody, which is an SJ25C1 antibody or antigen-binding fragment thereof; and

(b) detection of cells associated with the anti-idiotypic antibody.

85. A method for detecting a CAR containing a target antibody or its antigen-binding fragment, comprising:

(a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an FMC63 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof, according to any of embodiments 12-31, 33- 36, 34-37, and 38-60, or with a conjugate according to any of embodiments 61-64, which specifically binds to a target antibody, which is an FMC63 antibody or an antigen-binding fragment thereof; and

(b) detection of cells associated with the anti-idiotypic antibody.

86. The method according to any one of embodiments 80-85, wherein the anti-idiotype antibody or antigen-binding fragment thereof is directly or indirectly labeled for detection.

87. A method for selecting cells from a cell population, including:

(a) contacting a population of cells expressing a chimeric antigen receptor (CAR) containing the target antibody, or cells associated with the target antibody, with an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 1-11 and 29-32 , 34-37 and 39-60 or with a conjugate according to any of embodiments 61-64 that specifically binds to a target antibody that is an SJ25C1 antibody or an antigen-binding fragment thereof, wherein the target antibody is an SJ25C1 antibody or antigen thereof -connecting fragment; and

(b) selecting cells associated with the anti-idiotypic antibody.

88. A method for selecting cells from a cell population, including:

(a) contacting a population of cells expressing a chimeric antigen receptor (CAR) containing the target antibody, or cells associated with the target antibody, with an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 12-31, 33-36 and 38-60 or with a conjugate according to any one of embodiments 61-64 that specifically binds to a target antibody that is an FMC63 antibody or antigen-binding fragment thereof, wherein the target antibody is an FMC63 antibody or antigen-binding fragment thereof; and

(b) selecting cells associated with the anti-idiotypic antibody.

89. The method according to embodiment 87 or 88, wherein cells bound to the anti-idiotypic antibody are selected by affinity separation.

90. The method according to embodiment 89, wherein the affinity separation is an immunoaffinity separation.

91. The method according to embodiment 89 or 90, wherein the affinity separation is performed by flow cytometry.

92. The method according to embodiment 89 or 90, wherein the affinity separation is carried out using magnetically activated cell sorting.

93. The method according to embodiment 89 or 90, wherein the affinity separation comprises affinity chromatography.

94. The method according to embodiment 92 or 93, wherein the anti-idiotypic antibody reversibly binds or immobilizes on a support or in stationary phase.

95. A method for stimulating cells, comprising incubating an initial composition containing cells expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an SJ25C1 antibody or an antigen-binding fragment, with an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 1-11 and 29-32, 34-37 and 39-60, or with a conjugate according to any of embodiments 61-64, which specifically binds to a target antibody, which is an SJ25C1 antibody or antigen-binding fragment thereof, to obtain thereby the final composition containing the stimulated cells.

96. A method for stimulating cells, comprising incubation of an initial composition containing cells expressing a chimeric antigen receptor (CAR) containing a target antibody, which is an FMC63 antibody or an antigen-binding fragment, with an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 12-31, 33-36, and 38-60, or with a conjugate according to any of embodiments 61-64, which specifically binds to the target antibody, which is the FMC63 antibody or antigen-binding fragment thereof, thereby obtaining the final composition containing stimulated cells.

97. A method for obtaining a cell composition, including:

(a) introducing into the cells a nucleic acid molecule encoding a chimeric antigen receptor (CAR), whereby the initial composition is generated; and

(b) incubating the parent composition with an anti-idiotypic antibody or antigen-binding fragment thereof specific for the CAR antigen receptor, thereby obtaining a cell composition.

98. The method according to embodiment 97, wherein the CAR comprises a target antibody that specifically binds to CD19.

99. The method according to embodiment 98, wherein the target antibody is an SJ25C1 antibody or an antigen-binding fragment thereof.

100. The method according to any of embodiments 97-99, wherein the anti-idiotypic antibody or antigen-binding fragment thereof is an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 1-11 and 29-32, 34-37 and 39-60, which specifically bind to the target antibody, which is the SJ25C1 antibody or antigen-binding fragment thereof.

101. The method according to embodiment 98, wherein the target antibody is an FMC63 antibody or an antigen-binding fragment thereof.

102. The method according to any one of embodiments 97-98 and 101, wherein the anti-idiotypic antibody, or antigen-binding fragment thereof, specifically binds to a target antibody, which is an FMC63 antibody according to any one of embodiments 12-31, 33-36, and 38-60 that specifically binds to the target antibody, which is the FMC63 antibody or antigen-binding fragment thereof.

103. The method according to any one of embodiments 97-102, wherein the introduction in (a) comprises introducing the nucleic acid molecule into the cells by viral transduction, transposition, electroporation, or chemical transfection.

104. The method according to any one of embodiments 97-103, wherein the introduction in (a) comprises introducing the nucleic acid molecule into the cells by transduction with a viral vector containing the nucleic acid molecule, where the viral vector is optionally a retroviral vector or a lentiviral vector.

105. The method according to any one of embodiments 97-103, wherein the administration in (a) comprises introducing the nucleic acid molecule into the cells by transposition with a transposon containing the nucleic acid molecule.

106. The method according to any one of embodiments 97-103, wherein the administration in (a) comprises introducing the nucleic acid molecule into cells by electroporation or transfection of a vector containing the nucleic acid molecule.

107. The method according to any one of embodiments 97-106, further comprising the step of activating the cells prior to step (a).

108. The method of embodiment 107 wherein the step of activating the cells comprises contacting the cells with a CD3 agonist and optionally a CD28 agonist.

109. The method of embodiment 108, wherein the step of activating the cells comprises contacting the cells with a reagent containing anti-CD3 and anti-CD28 agonist antibodies.

110. The method according to any one of embodiments 95-109, wherein the incubation is carried out under conditions in which the anti-idiotypic antibody, or antigen-binding fragment thereof, binds to CAR, thereby inducing or modulating a signal in one or more cells in the original composition.

111. The method according to any one of embodiments 95-110, wherein the cells contain T cells.

112. The method according to embodiment 111, wherein the T cells contain CD4+ and/or CD8+ T cells.

113. The method according to any one of embodiments 95-112, wherein the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on a solid support, which optionally contains or is conjugated with a reagent containing a plurality of binding sites capable of reversibly binding to the anti-idiotypic antibody or its antigen-binding fragment.

114. The method according to any one of embodiments 95-112, wherein the anti-idiotypic antibody or antigen-binding fragment thereof is immobilized on a soluble reagent, which optionally is or contains a plurality of binding sites capable of reversibly binding to the anti-idiotypic antibody or antigen-binding fragment thereof. fragment.

115. The method of embodiment 113 or 114 wherein the reagent contains a streptavidin mutein.

116. The method according to any one of embodiments 95-115, wherein the incubation is at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or 96 hours.

117. The method according to any one of embodiments 95-116, wherein the starting 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 number of cells in the composition.

118. The method according to any one of embodiments 95-117, characterized in that

the number of CAR-expressing cells in the final composition is increased by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more compared to the amount of CAR- expressing cells in the original composition; and/or

the percentage of CAR-expressing cells in the final composition compared to the total number of cells in the composition is increased by more than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more.

119. The method according to any one of embodiments 95-118, wherein cells are not selected or enriched for CAR-expressing cells prior to administration and/or incubation.

120. The method according to any one of embodiments 80, 82-84, 86, 87, 89-95, 97-100, 103-119, wherein the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region of SEQ ID NO: 23, and/or light chain variable region of SEQ ID NO: 24.

121. The method according to any one of embodiments 81, 82, 83, 85, 86, 88-94, 96-99, 101, and 102-119, wherein the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region SEQ. ID NO: 30, and/or light chain variable region of SEQ ID NO: 31.

122. A method for purifying an antibody or its antigen-binding fragment, including:

(a) contacting a composition comprising a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof, according to any of embodiments 1-11 and 29-32, 34-37 and 39 -60 or a conjugate according to any one of embodiments 61-64 that specifically binds to the target antibody, which is the SJ25C1 antibody or antigen-binding fragment thereof; and

(b) isolating complexes containing the anti-idiotypic antibody.

123. A method for purifying an antibody or its antigen-binding fragment, including:

(a) contacting a composition comprising a target antibody, which is an FMC63 antibody, or an antigen-binding fragment thereof, with an anti-idiotypic antibody, or an antigen-binding fragment thereof according to any of embodiments 12-31, 33-36 and 38-60, or a conjugate according to any of embodiments 61-64 that specifically bind to a target antibody, which is an FMC63 antibody or antigen-binding fragment thereof; and

(b) isolating complexes containing the anti-idiotypic antibody.

124. The method of embodiment 122 or 123, wherein complexes containing the anti-idiotypic antibody are isolated by affinity resolution.

125. The method according to embodiment 124, wherein the affinity separation is an immunoaffinity separation.

126. The method of embodiment 124, wherein the affine separation is magnetic separation.

127. The method of embodiment 124, wherein the affinity separation comprises affinity chromatography.

128. A method for identifying an anti-idiotypic antibody or its antigen-binding fragment, including:

(a) administering to the patient a soluble immunizing reagent comprising an antigen-binding fragment of the target antibody fused to a solubilizing fragment; and

(b) identifying an antibody in the patient that specifically binds to the target antibody or antigen-binding fragment thereof.

129. The method of embodiment 128, wherein the antigen binding fragment comprises the heavy chain variable region and/or the light chain variable region of the target antibody.

130. The method according to embodiment 128 or 129, wherein the antigen binding fragment is a single chain fragment.

131. The method of embodiment 130 wherein the antigen binding fragment is scFv.

132. The method according to any one of embodiments 128-131, wherein the antigen-binding moiety is within or included in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR).

133. The method according to any one of embodiments 128-132, wherein the solubilizing fragment is an Fc domain or fragment thereof, which is optionally a human IgG1 Fc.

134. The method of Embodiment 133 wherein the solubilizing fragment is an Fc domain lacking a hinge region.

135. The method according to embodiment 134, wherein the solubilizing fragment contains the amino acid sequence of SEQ ID NO: 32.

136. The method according to any one of embodiments 128-135, wherein identifying the antibody comprises:

(i) isolating B cells from the patient's spleen and fusing them with immortalized B cells to create hybridomas;

(ii) screening hybridomas for the production of antibodies that specifically bind to a target antibody or antigen-binding fragment thereof, or to a chimeric antigen receptor containing an antigen-binding fragment; and

(iii) sequencing an antibody from the hybridoma producing an antibody that specifically binds, thereby identifying an anti-idiotypic antibody.

137. The method of any one of embodiments 128-136, wherein the target antibody binds to CD19.

138. The method according to any one of embodiments 128-137, characterized in that the antigen-binding fragment of the target antibody is derived from the antibody SJ25C1, where the antigen-binding fragment of the target antibody optionally contains the heavy chain variable region of SEQ ID NO: 23, and/ or a light chain variable region of SEQ ID NO: 24.

139. The method according to any one of embodiments 128-138, wherein the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from the SJ25C1 antibody, wherein the scFv optionally contains the amino acid sequence of SEQ ID NO: 28.

140. The method according to any one of embodiments 128-137, wherein the antigen-binding fragment of the target antibody is derived from the FMC63 antibody, wherein the antigen-binding fragment of the target antibody optionally comprises the heavy chain variable region of SEQ ID NO: 30 and/or light chain variable region of SEQ ID NO: 31.

141. The method according to any one of embodiments 128-137 and 140, wherein the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from the FMC63 antibody, wherein the scFv optionally contains the amino acid sequence of SEQ ID NO: 34 .

142. A cell depletion method comprising administering to a patient a composition comprising an anti-idiotype antibody or an antigen-binding fragment thereof according to any one of embodiments 1-11 and 29-32, 34-37 and 39-60 or a conjugate according to any one of embodiments 61-64 , which specifically binds to the target antibody, which is the SJ25C1 antibody, or to its antigen-binding fragment, where the patient was injected with a cell expressing a chimeric antigen receptor (CAR) containing the target antibody, which is the SJ25C1 antibody, or its antigen-binding fragment.

143. A cell depletion method comprising administering to a patient a composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 12-31, 33-36 and 38-60 or a conjugate according to any one of embodiments 61-64 that specifically binds with the target antibody, which is the FMC63 antibody, or with its antigen-binding fragment, where the patient was injected with a cell expressing a chimeric antigen receptor (CAR) containing the target antibody, which is the FMC63 antibody, or its antigen-binding fragment.

144. The method according to embodiment 137 or 138, wherein the depletion occurs via antibody-dependent cell-mediated cytotoxicity (ADCC).

145. A method for determining the presence or absence of a molecule that binds to a chimeric antigen receptor (CAR), including:

(a) contacting a binding reagent with a sample of a patient who has been administered a cell therapy comprising cells constructed with a CAR containing a target antibody, which is the SJ25C1 antibody, or an antigen-binding fragment thereof, under conditions of formation of a complex containing a binding reagent and a molecule from a sample that binds to a binding reagent, wherein the binding reagent contains an extracellular domain of CAR or a portion thereof containing the target antibody or antigen-binding fragment thereof; and

(b) detecting the presence or absence of the complex, thereby determining the presence or absence of a molecule that binds to the CAR.

146. The method of Embodiment 145, further comprising performing steps (a) and (b) with a positive control sample and optionally determining the presence or absence of a molecule by comparison with a positive control, wherein the positive control sample contains an anti-idiotypic antibody or antigen-binding antibody thereof. a fragment according to any of embodiments 1-11 and 29-32, 34-37 and 39-60 or a conjugate according to any of embodiments 61-64 that specifically binds to the target antibody or antigen-binding fragment thereof.

147. A method for determining the presence or absence of a molecule that binds to a chimeric antigen receptor (CAR), including:

(a) contacting a binding reagent with a sample of a patient who has been administered cell therapy, including cells constructed with a CAR containing a target antibody, which is the FMC63 antibody, or an antigen-binding fragment thereof, under conditions of formation of a complex containing a binding reagent and a molecule from a sample that binds to a binding reagent, wherein the binding reagent contains an extracellular CAR domain or a portion thereof containing the target antibody or antigen-binding fragment thereof;

(b) detecting the presence or absence of the complex.

148. The method of embodiment 147 further comprising performing steps (a) and (b) with a positive control sample and optionally determining the presence or absence of a molecule by comparison with a positive control, wherein the positive control sample contains an anti-idiotypic antibody or antigen-binding antibody thereof. a fragment according to any of embodiments 12-31, 33-36 and 38-60 or a conjugate according to any of embodiments 61-64 that specifically binds to the target antibody or antigen-binding fragment thereof.

149. The method according to any one of embodiments 145-148, wherein the molecule that binds to the binding reagent is or contains an antibody.

150. The method of any one of embodiments 145-149, wherein the binding reagent is detectably labeled or capable of generating a detectable signal.

151. The method according to any one of embodiments 145-150, wherein the binding agent is bound to a solid support or is soluble.

152. The method according to any one of embodiments 145-153, wherein the complex is detected by immunoassay.

153. The method of embodiment 152, wherein the immunoassay is an enzyme immunoassay (ELISA), chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR) biosensor (eg, BIAcore), flow cytometry, or Western blotting.

154. The method according to embodiment 152 or 153, wherein the immunoassay comprises detection of mesoscales.

155. The method according to any one of embodiments 152-154, wherein the immunoassay is a sandwich or bridge assay.

156. The method according to any one of embodiments 145-155, wherein the binding reagent is the first binding reagent, and detection of the presence or absence of the complex comprises:

(i) contacting the complex formed in step (a) with a second binding reagent, wherein the second binding reagent (1) contains the CAR extracellular domain or a portion thereof containing the target antibody or antigen-binding fragment thereof, and (2) is detectably labeled or capable of generating a detectable signal; and

(ii) assessing the presence or absence of the detected signal.

157. The method according to embodiment 156, wherein

the first binding reagent is associated with a solid support, where the first binding reagent is optionally associated, directly or indirectly, with biotin and/or associated with the solid support through streptavidin; and/or

the second binding agent is soluble.

158. The method according to embodiment 156 or 157, wherein the CAR extracellular domain, or portion thereof, of the first and second binding reagents are the same.

159. The method according to any one of embodiments 150-158, characterized in that

the detectable label is or contains a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label; and/or

the detected signal is or contains a fluorescent signal, a chemiluminescent signal, an electroluminescent signal, a colorimetric signal, a bioluminescent signal, or a radioactive signal.

160. The method according to any one of embodiments 150-159, wherein the detectable label is or contains a SULFO label.

161. The method of any one of embodiments 145-160, wherein the antigen-binding fragment of the target antibody comprises a heavy chain variable region and/or a light chain variable region of the target antibody.

162. The method of claims 145-161, wherein the antigen-binding fragment of the target antibody is a single chain fragment.

163. The method according to any one of embodiments 145-162, wherein the antigen-binding fragment of the target antibody is scFv.

164. The method according to any one of embodiments 145-163, wherein the sample contains whole blood, serum, or plasma.

165. An article of manufacture comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of embodiments 1-11 and 29-32, 34-37 and 39-60 or a conjugate according to any one of embodiments 61-64 and instructions for use of an anti-idiotypic antibody for detecting an SJ25C1 antibody or an antigen-binding fragment thereof or a chimeric antigen receptor comprising an SJ25C1 antibody or an antigen-binding fragment thereof; for selection or enrichment from a cell population of engineered cells expressing a chimeric antigen receptor (CAR) containing the SJ25C1 antibody or antigen-binding fragment; to stimulate the original composition containing cells expressing a chimeric antigen receptor containing the SJ25C1 antibody or its antigen-binding fragment.

166. An article of manufacture containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any of embodiments 12-31, 33-36 and 38-60 or a conjugate according to any of embodiments 61-64 and instructions for using the anti-idiotypic antibody to detect an FMC63 antibody or its antigen-binding fragment or a chimeric antigen receptor containing the FMC63 antibody or its antigen-binding fragment; for selection or enrichment from a cell population of engineered cells expressing a chimeric antigen receptor (CAR) containing the FMC63 antibody or an antigen-binding fragment thereof; to stimulate the original composition containing cells expressing a chimeric antigen receptor containing the FMC63 antibody or its antigen-binding fragment.

167. A product containing:

a binding reagent comprising an extracellular domain of a chimeric antigen receptor (CAR) containing a target antibody, which is the FMC63 antibody, or an antigen-binding fragment thereof, said extracellular domain or portion thereof comprising the target antibody or antigen-binding fragment thereof; and

an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 12-, 33-36 and 38-60 or a conjugate according to any of embodiments 61-64.

168. The article of manufacture according to embodiment 167, wherein the binding reagent is a first binding reagent, and the article further comprises a second binding reagent containing a CAR extracellular domain or a portion thereof.

169. The article of manufacture according to embodiment 167 or 168, wherein the extracellular domain of CAR or a portion thereof of the first and second binding reagents are the same.

170. An article of manufacture according to any one of embodiments 167-169, further comprising instructions for using a binding reagent, optionally a first and second binding reagent, to analyze a sample for the presence or absence of a molecule that binds to the binding reagent, using an immunoassay, where the immunoassay optionally represents is a bridging or sandwich immunoassay, where the sample is optionally taken from a patient who has been administered a cell therapy containing cells engineered with a CAR containing a target antibody that is an FMC63 antibody, or an antigen-binding fragment thereof.

171. A product containing:

a binding reagent comprising an extracellular domain of a chimeric antigen receptor (CAR) containing a target antibody, which is an SJ25C1 antibody, or an antigen-binding fragment thereof, said extracellular domain or portion thereof comprising the target antibody or antigen-binding fragment thereof; and

an anti-idiotypic antibody or antigen-binding fragment thereof according to any of embodiments 1-11 and 29-32, 34-37 and 39-60 or a conjugate according to any of embodiments 61-64.

172. The article of manufacture according to embodiment 171, wherein the binding reagent is a first binding reagent, and the article further comprises a second binding reagent containing a CAR extracellular domain or a portion thereof.

173. The article of manufacture according to embodiment 171 or 172, wherein the CAR extracellular domain or part thereof of the first and second binding reagents are the same.

174. The article of manufacture according to any one of embodiments 171-173, further comprising instructions for using a binding reagent, optionally a first and second binding reagent, to analyze a sample for the presence or absence of a molecule that binds to the binding reagent, using an immunoassay, where the immunoassay optionally represents is a bridge or sandwich immunoassay, where the sample is optionally taken from a patient who has been administered a cell therapy containing cells engineered with a CAR containing a target antibody, which is the SJ25C1 antibody, or an antigen-binding fragment thereof.

175. An article of manufacture according to any one of embodiments 167-174, wherein the binding reagent, optionally the first and/or second binding reagent, is detectably labeled or capable of generating a detectable signal.

176. An article of manufacture according to any one of embodiments 168-170 and 172-175, characterized in that one of the first and second binding reagents is attached to a solid support or is capable of attaching to a solid support, and the other of the first and second binding reagents is a detectable label or capable of generating a detectable signal.

177. The method of embodiment 176, wherein the article further comprises a solid support wherein one of the first and second binding reagents is optionally directly or indirectly bound to biotin, and the solid support comprises a streptavidin-coated surface.

IX. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Preparation of Anti-Idiotypic Antibodies Against Antibodies Derived from SJ25C1 Variable Region

This example describes the generation of anti-idiotypic antibodies (anti-IDs) recognizing an exemplary anti-CD19 chimeric antigen receptor (CAR) scFv portion containing an anti-CD19 scFv with heavy and light chain variable regions derived from SJ25C1 (antibody (in this case scFv) having the variable region sequences of SEQ ID NO: 23 and 24 separated by a linker shown in SEQ ID NO: 25), an extracellular portion derived from human CD28, a transmembrane domain derived from human CD28, an intracellular signaling domain derived from human CD28, and a signal domain derived from human CD3 zeta.

A. Hybridoma Generation and Antibody Screening

Mice were immunized with the extracellular domain (ECD) portion of CAR (containing anti-CD19 scFv with variable regions derived from SJ25C1). Part of the ECD contained the sequence

EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR (SEQ ID NO: 28) и внеклеточную часть из CD28 (SEQ ID NO: 27).

Serum isolated from immunized mice was tested by ELISA for its ability to bind to the recombinant soluble portion of the ECD by detection with a secondary antibody. Hybridoma fusion clones were generated and further characterized by ELISA for ECD binding, and five (5) positive clones were selected. Each selected hybridoma clone was grown and the antibodies were purified. To enable detection of the antibody in subsequent assays, each antibody was conjugated to Alexa647.

Antibodies were evaluated for their ability to specifically bind to T cells generated by the anti-CD19 CAR (derived from SJ25C1) (assessed by comparison with binding to mock-transduced control T cells). T cells were isolated by human immunoaffinity enrichment and the cells were activated and transduced with a viral vector encoding an anti-CD19 CAR (derived from SJ25C1). Two-fold serial dilution sequences (starting at 20 μg/ml and diluted to 0.0195 μg/ml) of each of the anti-idiotypic antibodies were individually used to detect surface CAR expression by flow cytometry. As a positive control, cells were also evaluated for surface expression of CAR using similar concentrations of goat anti-mouse antibody ("GAM"), which was able to detect the murine portion of the variable region of the ECD portion of CAR. Control antibody was not tested. The dose-response curve for the percentage of cells positive for the Alexa647 signal in CAR-transduced T cells was similar for each of the anti-idiotypic antibodies tested and comparable to that for the GAM positive control antibody, none of which recognized decoy-transduced T cells. empty vector. The staining index, which is the difference between the positive and background (false) peak values and the background peak spread of anti-idiotypic antibodies and GAM antibodies, was also determined and found to be comparable between anti-idiotypic antibodies and a GAM positive control antibody. Clone A-1 anti-idiotypic antibody (anti-ID A-1) was selected for further characterization.

B. Functional activity

Phosphorylation of Erk1/2 in Jurkat cells engineered to express the CAR described above was assessed by flow cytometry after stimulation with anti-ID A-1 antibody or anti-CD3 antibody, in each case in the presence or absence of cross-linking antibody. After incubation, the cells were fixed with formaldehyde, permeabilized, incubated with an antibody specific for phosphorylated Erk1/2, and analyzed by flow cytometry. As shown in FIG. 1, after cell incubation in the presence of anti-ID A-1 antibody, an increase in Erk1/2 phosphorylation was observed at a level similar to that observed for stimulation with anti-CD3 antibody in the presence of cross-linking antibody. Both antibodies were found to induce the same degree of Erk1/2 phosphorylation even in the absence of crosslinking.

Western blot analysis was further used to compare Erk phosphorylation in CAR-expressing Jurkat cells or parental Jurkat cells not expressing CAR after stimulation with anti-ID A-1, isotype control, or anti-CD3, or in the absence of stimulus, in the presence or in no cross-linking antibody. The results revealed that stimulation with anti-idiotypic antibody specifically increased Erk1/2 phosphorylation in Jurkat cells transduced with anti-CD19 CAR (derived from SJ25C1), but not in parental Jurkat cells (in which only background signal was observed, similar to unstimulated cells and cells stimulated with isotypic control), to a degree similar to the degree of phosphorylation induced by the anti-CD3 antibody. In contrast, the CD3 antibody induced phosphorylation in a non-specific manner, ie in both the CAR-expressing and parental Jurkat cells.

C. Sequence Identification

Anti-ID A-1 antibody sequences were determined. Total RNA was extracted from hybridoma cells containing a hybridoma clone expressing anti-ID A-1 and cDNA was prepared using isotype-specific antisense primers or universal primers using the PrimeScript™ 1st strand cDNA synthesis kit (Takara, Cat. No. .6110A). RACE-PCR was performed to amplify the variable (heavy and light chains) and constant regions of the antibody, which were then cloned separately into a cloning vector and sequenced. Table 2 lists the corresponding SEQ ID NOs of the nucleotide or amino acid sequence of the antibody.

Table 2. Sequence of anti-ID A-1

light chain heavy chain Complete variable constant Complete variable constant Nucleotide SEQ ID NO: 21 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 17 SEQ ID NO: 15 SEQ ID NO: 16 Amino acid SEQ ID NO: 7 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 3 SEQ ID NO: 1 SEQ ID NO: 2

Example 2 Preparation of anti-idiotypic antibodies against antibodies derived from FMC63

This example describes the generation of anti-idiotypic antibodies recognizing the binding domain portion (scFv) of an exemplary anti-CD19 chimeric antigen receptor (CAR) containing an anti-CD19 scFv with VH or VL domains derived from FMC63 (an antibody containing a variable domain), variable sequences heavy chain (VH) and light chain (VL) shown in SEQ ID NOs: 30 and 31, respectively). The ScFv is shown in SEQ ID NO: 34 and contains VH or VL regions separated by a linker shown in SEQ ID NO: 33.

A. Hybridoma Generation and Antibody Screening

Mice were immunized with a soluble protein containing the scFv portion of this CAR (SEQ ID NO: 34) fused to the Fc domain of human IgG1 (SEQ ID NO: 32; the protein lacked a hinge region). The soluble protein reagent used for immunization is shown in SEQ ID NO: 35.

Serum isolated from immunized mice was tested by ELISA for the ability to bind to part of the scFv-Fc by detection with a secondary antibody. The clones were screened against an Fc-only peptide (SEQ ID NO: 32) to select for anti-idiotypic antibodies that did not cross-react with the Fc portion of the scFv-Fc used to immunize mice. Clones were also screened against a construct containing an scFv derived from another anti-CD19 antibody, SJ25C1 (variable region sequences of SEQ ID NO: 23 and 24 separated by a linker shown in SEQ ID NO: 25) fused to a hingeless Fc domain ( SEQ ID NO: 32) for further selection of anti-idiotypic antibodies that lack cross-reactivity with another anti-CD19 antibody.

Hybrid hybridoma clones were generated and 12 candidate clones were further characterized by flow cytometry. Peripheral blood mononuclear cells were isolated from humans and the cells were activated and transduced with a viral vector encoding the anti-CD19 CAR. For flow cytometry, approximately 1×10 ⁶ cells per 100 μl were incubated with 10 μl of biotin-conjugated anti-idiotypic antibody, followed by staining with PE-conjugated streptavidin. As a positive control for surface expression of CAR, cells were stained with an anti-EGFR antibody to test for the expression of the transduction marker EGFRt, which was a surrogate for CAR expression. As a false control, PBMCs were transduced with an empty vector that did not express CAR. Cells were selected for PBMC CD3+CD4+/CD8+ and the fluorescent signal was determined. The results indicated that candidate anti-idiotypic antibodies exhibited specific binding to CAR on the surface of PBMCs. To confirm that antibodies are specific for scFv derived from FMC63 derived anti-CD19 antibody, similar experiments were performed on cells transduced with anti-CD19 antibody derived from SJ25C1 as described in Example 1. None of the potential anti-idiotypic antibodies against the antibody derived from FMC63 showed no specific binding to cells expressing another anti-CD19 CAR containing scFv derived from SJ25C1. Anti-idiotypic antibody clones, B-1 (anti-ID B-1) and B-2 (anti-ID B-2) were selected for further characterization.

B. Sequence identification

Anti-ID B-1 and B-2 antibody sequences were determined. Total RNA was extracted from hybridoma cells containing hybridoma clones expressing anti-ID B-1 or anti-ID B-2 and cDNA was generated using isotype-specific antisense primers or universal primers using the PrimeScript 1st Strand cDNA Synthesis Kit ™ (Takara, Cat. No. 6110A). RACE-PCR was performed to amplify the variable (heavy and light chains) and constant regions of the antibodies, which were then cloned separately into a cloning vector and sequenced. Table 3 shows the corresponding SEQ ID NOs of the nucleotide or amino acid sequences of the antibodies.

Table 3 Anti-ID B-1 and B-2 sequences

light chain heavy chain Complete (SEQ ID NO) variable (SEQ ID NO) constant (SEQ ID NO) Complete (SEQ ID NO) variable (SEQ ID NO) constant (SEQ ID NO) Anti-ID B-1 Nucleotide 56 54 55 52 fifty 51 Anti-ID B-1 Amino Acid 42 40 41 38 36 37 Anti-ID B-2 Nucleotide 76 75 55 73 71 72 Anti-ID B-2 Amino Acid 63 62 41 60 58 59

Example 3 Effect of plate-bound anti-idiotypic antibody on T-cell stimulation

This example describes the results after incubation of CAR T cells in the presence of the SJ25C1-specific anti-idiotypic antibody (anti-ID A-1) described in Example 1 or in the presence of the scFv-specific anti-idiotypic antibody derived from the FMC63 antibody (anti-ID B-1) described in Example 2.

Peripheral blood mononuclear cells were isolated from human blood and the cells were activated and transduced with a viral vector encoding an anti-CD19 CAR having a binding domain including scFv with VH or VL domains derived from FMC63 or from SJ25C1. Such vectors were introduced into cells to generate T cells engineered to express scFv-containing CARs derived from FMC63 and T cells engineered to express scFv-containing CARs derived from SJ25C1, respectively. In the case of a CAR derived from FMC63, the CAR encoding construct further included a sequence that encoded a truncated EGFR (EGFRt) that served as a surrogate marker for transduction and for CAR expression; the EGFRt coding region was separated from the CAR coding sequence by a T2A skip sequence. The engineered cells were evaluated in various assays.

For proliferation studies, engineered T cells were labeled with 50 nM carboxyfluorescein succinimidyl ether (CFSE) or CELL TRACE VIOLET (CTV) dye. Where appropriate, EGFRt surrogate marker expression was determined by staining with an anti-EGFR antibody. Cells were plated at a fixed number of cells per well in wells precoated with 10, 5, 2.5 or 1.25 µg/ml OKT3 (anti-CD3 antibody) or anti-idiotypic antibodies recognizing SJ25C1 and FMC63 (anti-ID A-1 and anti -ID B-1, respectively) in carbonate-bicarbonate buffer solution (pH 9) overnight. Cells seeded in wells without antibody coating were included as negative controls. Cells were cultured for 4 days and evaluated for viability, proliferation and expression of CD69 and CD25.

The proliferation of anti-CD19 (SJ25C1 or FMC63) CAR-expressing T cells was assessed by dye dilution using flow cytometry. The percentage of CD3+/CFSElow cells observed after stimulation of anti-CD19 (SJ25C1) CAR-expressing T cells with anti-ID A-1 is shown in FIG. 2A. The percentage of CD3+/EGFR+/CFSElow cells observed after stimulation of anti-CD19 (FMC63) CAR-expressing T cells with anti-ID B-1 is shown in FIG. 2b. Incubation in the presence of scFv-specific anti-ID A-1 antibody derived from SJ25C1 was observed to result in proliferation of anti-CD19 (SJ25C1) CAR-expressing T cells at a level comparable to stimulation with anti-CD3 antibody (FIG. 2A) . In contrast, the proliferation seen in anti-CD19 (SJ25C1) CAR-expressing T cells incubated in the presence of another anti-ID specific for another anti-CD19-CAR binding domain, scFv-specific FMC63-derived anti-ID B- 1 was similar to that observed in cells in the absence of any stimulating agent. Similarly, it was observed that incubation in the presence of scFv-specific anti-idiotype antibody anti-ID B-1 derived from FMC63 resulted in the proliferation of anti-CD19 CAR-expressing T cells (binding domain derived from FMC63) at a level comparable to level when stimulated with an anti-CD3 antibody. In contrast, cells incubated in the presence of another anti-ID recognizing another anti-CD19 CAR (scFv-specific anti-ID A-1 antibody derived from SJ25C1) were similar to cells incubated in the absence of stimulatory agents. (Fig. 2B). These results demonstrated that anti-ID A-1 and anti-ID B-1 were each able to stimulate and induce proliferation of T cells expressing a CAR containing the respective scFv for which the antibody was specific and did not induce T cell proliferation. expressing different CARs that target the same antigen but do not contain the specific binding domain for which the antibody was specific. The results are consistent with the ability of anti-ID A-1 and anti-ID B-1 to specifically recognize their respective targets, and not CARs containing different binding domains against the same antigen.

The results of another assay confirmed the ability of the typical anti-IDs evaluated in this assay to provide the CAR-spec signal for T cells in a concentration dependent anti-ID manner, consistent with the usefulness of anti-IDs in tuning the amount of signal received via T cells. expressing CD19-specific CAR. In this assay, sham-transduced and CAR-transduced T cells were labeled with CELL TRACE VIOLET (CTV). Before seeding, the wells were coated with 0.25, 0.5, or 1 μg/ml of a typical anti-idiotypic antibody specific for the anti-CD19 CAR binding domain. Cells were cultured for 4 days and proliferation was assessed by evaluating the degree of dye dilution using flow cytometry. FIG. 2C shows the induction of CD8+ CAR-T cell proliferation as a function of anti-idiotypic antibody concentration. These results demonstrate the ability to tune the amount of typical plate-bound anti-idiotypic antibodies provided herein, for example, to adjust the amount of signal and/or provide a controlled level of stimulation and/or optimize the degree of stimulation of CAR-expressing T cells in different contexts.

To further investigate the stimulatory capacity of anti-ID A-1 and anti-ID B-1, T cells transduced with the targeted anti-CD19 CAR (derived from SJ25C1 or FMC63, respectively) were evaluated using flow cytometry for the expression of two T activation markers. β-cells, CD69 and CD25, after stimulation with plate-bound antibodies at concentrations including 1.25, 2.5, 5 and 10 μg/ml. Transduced T cells were selected for EGFR as a surrogate for CAR expression, and CD4+ or CD8+ EGFR+ cells were evaluated for CD69 or CD25 expression.

As shown in FIG. 3 for T cells transduced with an anti-CD19 CAR derived from SJ25C1, anti-ID A-1 induced more CD25 expression in both CD4+ and CD8+ T cells than in cells stimulated with anti-CD3 antibody. Anti-ID A-1 also induced CD69 expression in CD4+ and CD8+ T cells at levels that were similar to or slightly less than when cells were stimulated with anti-CD3 antibody. Expression of CD69 and CD25 was not observed in unstimulated cells or cells treated with scFv-specific anti-ID B-1 derived from FMC63. As shown in FIG. 4 for T cells transduced with anti-CD19 CAR derived from FMC63, anti-ID B-1 induced greater expression of CD25 in both CD4+ and CD8+ T cells than in cells stimulated with anti-CD3 antibody. Compared to stimulation with anti-CD3 antibody, anti-ID B-1 antibody induced CD69 expression at a higher level in CD4+/EGFR+ T cells and at a level that was similar or slightly higher in CD8+/EGFRT+ T cells. CD69 expression was similar or slightly higher at higher antibody concentrations in CD8+/EGFR+ T cells. Expression of CD69 and CD25 was not observed in unstimulated cells or in cells treated with SJ25C1-specific anti-ID A-1 antibody. These results indicate that both anti-ID A-1 and anti-ID B-1 were able to specifically stimulate T cells expressing a CAR containing their scFv target.

Example 4 Analysis of Host Immune Responses Specific to the Transgenic Product

An Anti-Therapeutic Antibody (ATA) bridging assay was developed to detect the presence or absence of antibodies in the sera of treated patients recognizing the administered anti-CD19 CARs. Certain anti-ID antibodies described in Examples 1-3 were used as positive controls and to confirm the assay's ability to detect the presence of such antibodies.

A biotinylated human Fc fusion protein (biotinylated ECD fusion protein) containing the scFv portion of CAR with variable regions derived from FMC63 (shown in SEQ ID NO: 34) was used. The biotinylated ECD fusion protein was added to streptavidin coated wells; plates were incubated under conditions allowing fusion protein binding and washed. Various concentrations of anti-ID B-1 or anti-ID B-2 were added to wells coated with the biotinylated fusion protein and left to incubate under conditions allowing specific binding of the antibody. After washing, a sulfo-labeled variant of the ECD fusion protein (sulfo-ECD fusion protein) containing Fc-scFv derived from FMC63 was obtained. The wells were washed and the electrochemiluminescence (ECL) signal was read on a Meso Scale Discovery (MSD) sector imaging apparatus.

As shown in FIG. 5, the ECL signal increased with increasing concentrations of anti-ID B-1 and anti-ID B-2, indicating that the assay can be used to assess the presence or absence and level of antibodies in serum samples with any of the exemplary anti-ID antibodies. used as a positive control.

In some embodiments, an ATA assay using anti-ID B-1 and/or anti-ID B-2 antibodies as a positive control is used to evaluate the presence or absence of anti-CAR ATA antibodies in samples from patients receiving infusion doses of cell therapy containing anti-CD19 (FMC63) CAR-expressing T cells. Such ATAs can, in some cases, potentially indicate a host humoral immune response against the introduced CAR. In a typical assay, plasma samples are obtained from patients at various time points, such as prior to infusion and/or days 14 and 28, and in some cases 3, 6 and/or 12 months after the start of cell therapy administration. Samples obtained from such plasma samples are used along with control samples containing anti-ID antibodies in the assay as described above.

A similar bridging assay was also performed to evaluate patient samples receiving infusion doses of cell therapy containing anti-CD19 (SJ25C1) CAR-expressing T cells. In this assay, an anti-ID A-1 antibody was used as a positive control. The assay was observed to have a sensitivity of less than 100 ng/mL as determined by a positive anti-ID antibody control and a >4-5 fold signal in the plasma background.

Example 5 Conjugation of Anti-Idiotype Antibody to Microspheres

Either anti-ID B-1 or anti-ID-B1, as described in Example 2, was covalently attached to the surface of commercially available tosyl-activated magnetic microspheres (ThermoFisher, Waltham MA), which are superparamagnetic, non-porous, monodisperse, tosyl-activated microspheres. The microspheres covalently bind primary amino and sulfhydryl groups. Conjugation was performed using microspheres having a diameter of approximately 2.8 µm (denoted M-280) or 4.5 µm (denoted M-450).

200 μg of anti-ID antibody was added to approximately 1 ml of tosyl-activated microspheres (e.g., approximately 4×10 ⁹ tosyl-activated microspheres having a diameter of 2.8 μm or approximately 4×10 ⁸ tosyl-activated microspheres having a diameter of 4.5 μm) and covalent bonding was performed by overnight incubation at 37° C. in phosphate buffered saline (PBS) containing 0.1% human serum albumin (HSA). The microspheres were washed and resuspended in 1 ml PBS with 0.1% HSA. After conjugation, the concentration of the microspheres was determined using a Cellometer.

To evaluate the stability of anti-ID conjugated microspheres, the microspheres were pelleted, the supernatant removed and applied to a 4-12% Bis-Tris SDS-PAGE gel and the gel stained with Coomassie blue. As a control for total protein conjugated to the microspheres, the precipitated microspheres were boiled in LDS 4X (lithium dodecyl sulfate) sample buffer at about 70°C for 20 minutes, and about 12.5 µl or 25 µl of the boiled supernatant was also run on the SDS gel. -PAGE and assessed with Kumasi blue. Approximately 2.5 µg or 5 µg of anti-ID antibody that was not conjugated to myrospheres (positive control) or 5 µl of 0.1% HSA (negative control) was also evaluated by SDS-PAGE and Coomassie staining. No anti-ID antibodies were detected in the supernatant from the conjugated samples that were not boiled, indicating that the conjugation was stable, while anti-ID antibody was detected in the supernatant from the conjugated samples that were boiled.

Example 6 Evaluation of stimulation of T cells cultured with microspheres conjugated with anti-idiotypic antibodies

FMC63-derived scFv-specific anti-ID B-1 conjugated microspheres were incubated with T cells. CD3-purified T cells were isolated by immunoaffinity enrichment from leukopheresis samples from healthy donors. The isolated cells were transduced with a viral vector encoding an anti-CD19 CAR having a scFv derived from FMC63. The viral vector construct additionally encoded a truncated EGFR (EGFRt) that served as a surrogate marker for CAR expression; The EGFRt coding region was separated from the CAR sequence by a T2A skipping sequence. After transduction, cells were expanded in culture and frozen by cryopreservation.

For T cell stimulation studies, thawed CD4+ or CD8+ CAR-expressing cells were plated separately at approximately 50,000 total cells per well. In some cases, additional cytokines were added to the culture medium as follows: for CD4+ cells, approximately 1200 IU/ml recombinant IL-7, 20 IU/ml recombinant IL-15, and 100 IU/ml recombinant IL-2; for CD8+ cells, approximately 200 IU/ml recombinant IL-2 and 20 IU/ml recombinant IL-15. Anti-ID B-1 conjugated microspheres were added to cells at a ratio of 1:1 or 1:5 cells:microspheres and incubated for up to 14 days with 50% media change every 2-3 days. As a positive control, cells were cultured with anti-CD3/anti-CD28 magnetic beads at a cell:bead ratio of 3:1 in the presence or absence of the indicated cytokines.

At various culture times, CD4+ or CD8+ transduced cells (as detected by anti-EGFR for surrogate marker expression) were assessed for expansion, PD-1 expression, and viability.

As shown in FIGS. 6 and 7, expansion of CD4+ and CD8+ T cells, respectively, was observed when cells were cultured with anti-ID conjugated microspheres at a ratio of 1:1 or 1:5 cell:microspheres, especially in the presence of cytokines. The degree of expansion was higher than when cells were cultured with control anti-CD3/anti-CD28 magnetic microspheres.

Surface expression of PD-1 in a subset of CD4+ cells was assessed by flow cytometry on days 3, 7, 10 and 14 of culture. As shown in FIG. 8, PD-1 expression was high on cells cultured with anti-CD3/anti-CD28 magnetic microspheres, however PD-1 levels were significantly lower or not detected in cells cultured with anti-ID conjugated microspheres.

As shown in FIG. 9, the percent viability of CD4+ and CD8+ T cells cultured with 1:1 or 1:5 ratio of anti-ID conjugated microspheres or control anti-CD3/anti-CD28 conjugated microspheres remained consistently high during the culture period. when the cells were additionally cultured in the presence of cytokines. However, under all conditions, cell viability decreased in the absence of added cytokines, with the greatest loss of cell viability occurring on later days of cell culture.

Example 7 Evaluation of Cytokine Production by T Cells Cultured with Anti-Idiotype Antibody Conjugated Microspheres

Anti-CD19 CAR engineered T cells having an FMC63-derived scFv were generated essentially as described in Example 6. Thawed CD8+ T cells were cultured with anti-ID B-1 conjugated microspheres for 4 hours in the presence of a Golgi inhibitor. Intracellular levels of the cytokines TNFα, IFNγ, and IL-2 were determined by flow cytometry in the CAR ⁺ T cell subset (as defined by positive surface staining for anti-EGFR surrogate transduction marker EGFRt) or in the CAR ^- T cell subset (as defined by negative surface staining for EGFRt with anti-EGFR). For comparison, CAR ⁺ T cells (EGFR+) were also cultured with CD19 expressing target cells (K562 cells transduced to express CD19, K562-CD19) at an effector:T cell ratio of 1:2.

As shown in FIG. 10A, intracellular levels of the cytokines TNFα, IFNγ and IL2 were induced in CAR ⁺ T cells (EGFRt+) but not in CAR ⁻ T cells (EGFR−) when the cells were cultured in the presence of anti-ID B-1 conjugated microspheres. In this study, the degree of stimulation observed in the presence of anti-ID-conjugated microspheres was similar to stimulation of CAR ⁺ T cells using antigen-expressing K562-CD19 cells, which is an alternative CAR-specific stimulatory reagent (FIG. 10B). These results demonstrate that microsphere-conjugated anti-IDs are agonistic and specifically stimulate T cells expressing a CAR having an antigen-binding domain recognized by the anti-ID antibody. In addition, the microsphere reagent provides a better CAR-specific stimulating reagent than cell lines that require culture and are subject to lot-to-lot variability.

Example 8 Evaluation of expansion after serial restimulation

The ability of cells to expand ex vivo after repeated stimulations may be a potential surrogate for the ability of CAR ⁺ T cells to persist (eg, after initial activation) and/or be an indicator of in vivo function (Zhao et al. (2015) Cancer Cell, 28: 415-28 ). CAR ⁺ T cells were generated as described above and thawed CD4+ or CD8+ CAR-expressing T cells were plated separately at 50,000 CAR ⁺ cells/well. Anti-ID B-1 conjugated microspheres were added to cells in a ratio of 1:1 or 1:5 cell:microspheres in the presence or absence of cytokines as described in Example 6. As a control, anti-CD3 magnetic microspheres were added to the cells. /anti-CD28 in a ratio of 3:1 cell:microspheres in the presence or absence of cytokines. Cells were harvested every 3-4 days and counted and restimulated with new target cells using the same culture conditions after resetting the cell number to the initial seeding density for each round. A total of 4 stimulation rounds were performed during the 14 day culture period. For each stimulation round, the number of doublings was determined.

As shown in FIG. 11, CAR-expressing (EGFR+) CD4+ cells continued to expand after restimulation with anti-ID conjugated microspheres, although the degree of expansion was higher when the cells were cultured in the presence of cytokines. In addition, the degree of expansion was higher than when cells were cultured with anti-CD3/anti-CD28 microspheres. For CD8+ T cells, similar expansion kinetics were observed when cells were cultured in the presence of either anti-ID-conjugated microspheres or anti-CD3/anti-CD28 microspheres, although expansion was somewhat greater when cells were cultured with anti-ID conjugated microspheres. ID, especially in the absence of added recombinant cytokines.

Example 9 Additional analysis of CAR-specific cell expansion using microspheres conjugated with anti-idiotypic antibody

Studies were conducted similar to those described in Examples 7 and 8, except that used CAR-expressing T cells obtained from two different donor patients. CD3-purified T cells were isolated from peripheral blood mononuclear cells (PBMCs) from two donor patients, transduced with a viral vector encoding an anti-CD19 CAR having an FMC63-derived scFv, expanded in culture, frozen, and thawed.

Thawed CD4+, CD8+, or CD4/CD8 co-cultures (1:1 ratio) were plated in wells of a 6-well plate at approximately 5×10 ⁶ cells/well in culture medium that had been further supplemented with cytokines as follows: for CD4+ cells or co-cultures of CD4+/CD8+ media were supplemented with approximately 1200 IU/ml recombinant IL-7, 20 IU/ml recombinant IL-15, and 100 IU/ml recombinant IL-2; for CD8+ cells, 200 IU/ml recombinant IL-2 and 20 IU/ml recombinant IL-15 were added to the medium. Anti-ID B-1 conjugated microspheres were added to cells at a 1:1 cell:microspheres ratio and incubated for up to 9 days with 50% media change every 2-3 days.

At different culture times, the number of CD4+ or CD8+ transduced cells (as detected by anti-EGFR for surrogate marker expression) present in culture for each condition was assessed and the expansion fold or frequency of CAR-expressing cells was determined as a percentage of total cells. PD-1 and CD25 expression and cell viability were also determined.

As shown in FIG. 12A, a greater than 60-fold increase in CAR-expressing (EGFRt+) CD4+ T cells was observed when CD4+ cells were cultured alone with anti-ID conjugated microspheres. With regard to CD8+ T cells, in the presence of anti-ID conjugated microspheres, there was a significantly higher expansion of CAR-expressing (EGFRt+) CD8+ T cells when CD8+ T cells were co-cultured with CD4+ cells. As shown in FIG. 12B, the frequency of CAR-expressing (EGFRt+) CD4+ or CD8+ cells increased during 9 days of culture in the presence of anti-ID conjugated microspheres with >90% transduced cells (EGFRt+) present in culture at day 9. The viability of CD4+ and CD8+ cells also remained close to 100% during culture, with somewhat greater viability of CD8+ T cells being observed when co-cultured with CD4+ T cells (FIG. 12C). Similar results were observed for both donors.

Surface expression of PD-1 and CD25 on transduced (EGFRt+) CD4+ or CD8+ cells was assessed by flow cytometry on days 5, 7 and 9 of culture with anti-ID conjugated microspheres. As shown in FIG. 13A, PD-1 expression on both CD4+ and CD8+ T cells significantly decreased over time when cultured with anti-ID conjugated microspheres. As shown in FIG. 13B, CD25 expression also decreased after 9 days of culture in the presence of anti-ID conjugated microspheres. Similar results were observed for both donors.

Example 10 Comparison of cytokine levels and phenotype after culture with anti-idiotypic antibody-conjugated microspheres

CD3-purified T cells were isolated from peripheral blood mononuclear cells (PBMCs) of two donor patients, transduced with a viral vector encoding an anti-CD19 CAR having a scFv derived from FMC63, and expanded in culture with microspheres coated with each of the anti-CD3 antibodies. and against CD28. After expansion, the expanded T cells were frozen by cryopreservation. For studies, frozen T cells were thawed and CD4+ and CD8+ T cells were assessed for intracellular cytokine levels or surface phenotype (d=0), or thawed CD4+, CD8+ T cells, or co-cultured CD4+/CD8+ T cells were cultured for additional 9 days in the presence of anti-ID B-1-conjugated microspheres to evaluate intracellular cytokine levels and surface marker phenotype after stimulation with PMA/ionomycin or CD19-transduced K562 cells (d=9).

To assess the levels of intracellular cytokines, the Golgi inhibitor was added for 4 hours, and then assessed TNFα, IFNγ and IL-2 using flow cytometry. For all conditions, the degree of intracellular cytokine expression was significantly higher when cells were stimulated with PMA/ionomycin compared to CAR-specific stimulation with CD19-transduced K562 cells. As shown in FIG. 14A, the levels of TNFα and IL-2 cytokines were similar in CD4+ or CD8+ cells immediately after thawing compared to the corresponding level in CD4+ or CD8+ cells or in co-cultures of CD4/CD8 T cells additionally cultured in the presence of microspheres conjugated with anti- ID, within 9 days. Elevated IFNγ levels were observed in thawed CD4+, CD8+ T cells or co-cultured CD4/CD8 T cells that were additionally cultured in the presence of anti-ID conjugated microspheres for 9 days compared to IFNγ levels in CD4+ or CD8+ T cells. cells immediately after thawing. These results demonstrated that T cell function was maintained after 9 days of expansion in the presence of anti-ID conjugated microspheres. Similar results were obtained in cells from two donors.

Surface expression of the activation marker CD25, surface expression of inhibitory receptors PD-1 and LAG-3, and nuclear expression of the proliferation marker Ki-67 were also assessed in CD4+ or CD8+ cells immediately after thawing (d=0) or in CD4+ or CD8+ T cells that expanded alone or as a co-culture of CD4/CD8 T cells for an additional 9 days in the presence of anti-ID-conjugated microspheres (d=9). As shown in FIG. 14B, reduced expression of CD25, but not Ki-67, was observed in cells that were additionally cultured in the presence of anti-ID-conjugated microspheres for 9 days compared to T cells immediately after thawing. The reduced expression of CD25 was significantly higher in CD8+ cells than in CD4+ cells. In addition, reduced expression of PD-1 and LAG-3 was also observed in both CD4+ and CD8+ cells cultured alone or as CD4/CD8 co-culture after 9 days of incubation with anti-ID conjugated microspheres. compared to expression in cells immediately after thawing. This result demonstrated that after incubation with anti-ID conjugated microspheres, the previously frozen transduced cells retained functional capacity, as evidenced by the high percentage of cells that were positive for the Ki-67 marker, indicative of cell proliferation, but also showed another an activation state characterized by low surface expression of the activation marker CD25 and inhibitory receptor markers PD-1 and LAG-3.

Example 11 Detection of CAR Expressing Cells Using Anti-Idiotype Antibodies.

Compositions of T cells were obtained containing CAR-T cells expressing anti-CD19 CAR containing anti-CD19 scFv with variable regions derived from the FMC63 antibody, an immunoglobulin spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4 -1BB, and the CD3-zeta intracellular signaling domain. The viral construct encoding CAR additionally encoded an EGFRt surrogate marker that was separated from the CAR sequence by a T2A skip sequence.

Cells expressing anti-CD19 CAR were introduced into a sample of cells containing healthy human peripheral blood mononuclear cells (PBMCs). The resulting cell compositions were incubated with various concentrations of anti-CD19 FMC63 scFv-specific anti-ID B1 or anti-ID B2 antibodies. As a control, samples of each condition were also incubated with a concentration of an anti-EGFR antibody capable of detecting the EGFRt surrogate marker on the transduced cells. Control samples included samples containing only PBMC without the addition of T cells expressing CAR. Geometric mean fluorescence intensity (MFI) was quantified in positively labeled cells to assess antibody staining.

As shown in FIG. 15A, both anti-idiotypic antibodies detected anti-CD19 CAR T cells having FMC63-derived variable region scFvs in a concentration dependent manner. As shown in FIG. 15B, anti-ID B1 and anti-ID B2 antibodies detected positive cells in compositions that contained CAR-expressing cells, and not in compositions that contained only PBMC. Staining for EGFRt showed that the ratio of cells expressing the transgene and PBMC was constant under all conditions. The results confirmed the ability of anti-ID B1 and anti-ID B2 antibodies to specifically detect anti-CD19 FMC63 scFv CAR-expressing cells even among samples containing human PBMCs that did not express CAR. The results are consistent with the interpretation that anti-idiotypic antibodies can be used to detect CAR-expressing cells in samples from patients injected with CAR-expressing cells recognized by antibodies, e.g., in peripheral blood samples collected from humans, e.g. to measure expansion, directional migration, and /or the persistence of such cells over time in various tissues and/or fluids of the patient.

The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and teachings provided herein. Such variations may be made without departing from the true scope and spirit of the disclosure, and are intended to be embraced by the scope of the present disclosure.

SEQUENCES

# SUBSEQUENCE ANNOTATION one QVQLQQPGSELVRPGGSVKLSCKASDYTFTSYWMHWVRQRPGQGLEWIGNIYPGSGGTNYDEKFKRKATLTVDTSSSTAYMQLRSLTSEDSAVYYCTREVTTVAYYYSMDYWGQGTSVTVSS anti-ID VH 2 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK anti-ID CH 3 QVQLQQPGSELVRPGGSVKLSCKASDYTFTSYWMHWVRQRPGQGLEWIGNIYPGSGGTNYDEKFKRKATLTVDTSSSTAYMQLRSLTSEDSAVYYCTREVTTVAYYYSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK anti-ID heavy chain four MGWSSIILFLVATASGVHS anti-ID HC signal sequence 5 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTVPFTFGSGTKLEIK anti-ID VL 6 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNNYTCEATHKTSTSPIVKSFNRNEC anti-ID CL 7 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTVPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERRNHNNYTCSFNTSPI anti-ID light chain eight MMSSAQFLGLLLLCFQGTRC anti-ID LC signal sequence 9 SYWMH anti-ID HC-CDR1 ten NIYPGSGGTNYDEKFKR anti-ID HC-CDR2 eleven EVTTVAYYYSMDY anti-ID HC-CDR3 12 RASQDISNYLN anti-ID LC-CDR1 13 YTSRLHS anti-ID LC-CDR2 fourteen QQGKTVPFT anti-ID LC-CDR3 fifteen CAGGTCCAACTGCAACAACCTGGGTCTGAGCTGGTGAGGCCTGGAGGTTCAGTGAAGCTGTCCTGCAAGGCTTCTGACTACACTTTCACCAGCTACTGGATGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTGGTACTAACTACGATGAGAAGTTCAAGAGGAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGAGGTTACTACAGTAGCTTATTACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA anti-ID VH 16 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA anti-ID CH 17 CAGGTCCAACTGCAACAACCTGGGTCTGAGCTGGTGAGGCCTGGAGGTTCAGTGAAGCTGTCCTGCAAGGCTTCTGACTACACTTTCACCAGCTACTGGATGCACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTGGTACTAACTACGATGAGAAGTTCAAGAGGAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGAGGTTACTACAGTAGCTTATTACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAA CCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA anti-ID heavy chain eighteen ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCCTCAGGTGTCCACTCC anti-ID HC signal sequence 19 GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGTCAGCAGGGTAAAACGGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA anti-ID VL twenty CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAACTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG anti-ID CL 21 GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGTCAGCAGGGTAAAACGGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAACTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG anti-ID light chain 22 ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGT anti-ID LC signal sequence 23 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS SJ25C1 VH 24 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 VL 25 GGGGSGGGGSGGGGS Linker 26 GGGGS 4GS linker 27 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Extracellular portion of CD28 28 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 scFv 29 GGGS 3GS linker thirty EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 VH 31 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT FMC63 VL 32 PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQALNHVCSK IgG1 Fc without hinge 33 GSTSGSGKPGSGEGSTKG Linker 34 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 scFv 35 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FMC63 reagent 36 EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQCHGKSLEWIGDINPNNGGTDYNQNFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCAREGNNYGSRDAMDYWGQGTSVTVSS Anti-ID B-1 VH 37 AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Anti-ID B-1 CH 38 EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQCHGKSLEWIGDINPNNGGTDYNQNFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCAREGNNYGSRDAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK anti-ID B-1 heavy chain 39 MGWSWIFLFLLSGTAGVLS anti-ID B-1 HC signal sequence 40 QIVLTQSPALMSASPGEKVTMTCSASSGVIYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK Anti-ID B-1 VL 41 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Anti-ID B-1 CL 42 QIVLTQSPALMSASPGEKVTMTCSASSGVIYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCSFEATHKTSTSPIVKTSTS anti-ID B-1 light chain 43 MDFQVQIFSFLLMASVIMSRG anti-ID B-1 LC signal sequence 44 DYYMK anti-ID B-1 HC-CDR1 45 DINPNNGGTDYNQNFKG anti-ID B-1 HC-CDR2 46 EGNNYGSRDAMDY anti-ID B-1 HC-CDR3 47 SASSGVIYMY anti-ID B-1 LC-CDR1 48 LTSNLAS anti-ID B-1 LC-CDR2 49 QQWSSNPLT anti-ID B-1 LC-CDR3 fifty GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGAAGTGGGTGAAGCAGTGTCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTGACTACAACCAGAACTTTAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGGGGAATAACTACGGTAGTAGAGATGCTATGGACTACTGGGGTCAAGGAACGTCAGTCACCGTCTCCTCA Anti-ID B-1 VH 51 GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA anti-ID B-1 CH 52 GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTACATGAAGTGGGTGAAGCAGTGTCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTGACTACAACCAGAACTTTAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGGGGAATAACTACGGTAGTAGAGATGCTATGGACTACTGGGGTCAAGGAACGTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCC CAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA anti-ID B-1 heavy chain 53 ATGGGATGGAGCTGGATCTTTCTCTTCCTCTTGTCAGGAACTGCAGGTGTCCTCTCT anti-ID B-1 HC signal sequence 54 CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAGGTGTAATTTACATGTACTGGTACCAACAGAAGCCAAGATCCTCCCCCAAACCCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGCACCAAGCTGGAGCTGAAA anti-ID B-1 VL 55 CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT anti-ID B-1 CL 56 CAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAGGTGTAATTTACATGTACTGGTACCAACAGAAGCCAAGATCCTCCCCCAAACCCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGCACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT anti-ID B-1 light chain 57 ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATAATGTCCAGGGGA anti-ID B-1 LC signal sequence 58 QVQLQQPGAELVRPGASVKLSCKTSGYSFTRYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDNSSSTAYMQLSSPTSEDSAVYYCASIYYEEAWGQGTLVTVSA anti-ID B-2 VH 59 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK anti-ID B-2 CH 60 QVQLQQPGAELVRPGASVKLSCKTSGYSFTRYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDNSSSTAYMQLSSPTSEDSAVYYCASIYYEEAWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK anti-ID B-2 heavy chain 61 MGWSSIILFLVATATGVHS anti-ID B-2 HC signal sequence 62 DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADSVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPYTFGGGTKLEIK anti-ID B-2 VL 63 DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADSVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLRNPISFECEATHKEATHKEATHK anti-ID B-2 light chain 64 MSVLTQVLALLLLWLTGARC anti-ID B-2 LC signal sequence 65 RYWMN anti-ID B-2 HC-CDR1 66 MIHPSDSETRLNQKFKD anti-ID B-2 HC-CDR2 67 IYYEEA anti-ID B-2 HC-CDR3 68 RASGNIHNYLA anti-ID B-2 LC-CDR1 69 NAKTLAD anti-ID B-2 LC-CDR2 70 QHFWSTPYT anti-ID B-2 LC-CDR3 71 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACTCCTTCACCAGGTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGCATCTACTATGAAGAGGCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA anti-ID B-2 VH 72 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA anti-ID B-2 CH 73 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACTCCTTCACCAGGTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGCATCTACTATGAAGAGGCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCA GACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA anti-ID B-2 heavy chain 74 ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC anti-ID B-2 HC signal sequence 75 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATAGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA anti-ID B-2 VL 76 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATAGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT anti-ID B-2 light chain 77 ATGAGTGTGCTCACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGT anti-ID B-2 LC signal sequence 78 DYTFTSY anti-ID HC-CDR1 79 DYTFTSYWMH anti-ID HC-CDR1 80 TSYWMH anti-ID HC-CDR1 81 YPGSGG anti-ID HC-CDR2 82 NIYPGSGGTN anti-ID HC-CDR2 83 WIGNIYPGSGGTN anti-ID HC-CDR2 84 TREVTVAYYYSMD anti-ID HC-CDR3 85 SNYLNWY anti-ID LC-CDR1 86 LLIYYTSRLH anti-ID LC-CDR2 87 QQGKTVPF anti-ID LC-CDR3 88 GYTFTDY anti-ID B-1 HC-CDR1 89 GYTFTDYYMK anti-ID B-1 HC-CDR1 90 TDYYMK anti-ID B-1 HC-CDR1 91 NPNNGG anti-ID B-1 HC-CDR2 92 DINPNNGGTD anti-ID B-1 HC-CDR2 93 WIGDINPNNGGTD anti-ID B-1 HC-CDR2 94 AREGNNYGSRDAMD anti-ID B-1 HC-CDR3 95 IYMYWY anti-ID B-1 LC-CDR1 96 PWIYLTSNLA anti-ID B-1 LC-CDR2 97 QQWSSNPL anti-ID B-1 LC-CDR3 98 GYSFTRY anti-ID B-2 HC-CDR1 99 GYSFTRYWMN anti-ID B-2 HC-CDR1 100 TRYWMN anti-ID B-2 HC-CDR1 101 HPSDSE anti-ID B-2 HC-CDR2 102 MIHPSDSETR anti-ID B-2 HC-CDR2 103 WIGMIHPSDSETR anti-ID B-2 HC-CDR2 104 ASIYYEE anti-ID B-2 HC-CDR3 105 HNYLAWY anti-ID B-2 LC-CDR1 106 LLVYNAKTLA anti-ID B-2 LC-CDR2 107 QHFWSTPY anti-ID B-2 LC-CDR3 108 GYX ₃ FX ₅ X ₆ YX ₈ MX ₁₀ HC-CDR1 Consensus X ₃ =T or S; X ₅ =T or S; X ₆ =D or R; X ₈ =Y or W; X ₁₀ =K or N 109 WIGX ₄ IX ₆ PX ₈ X ₉ X ₁₀ X ₁₁ TX ₁₃ X ₁₄ NQX ₁₇ FKX ₂₀ HC-CDR2 Consensus X ₄ =D or M; X ₆ =N or H; X ₈ =N or S; X ₉ =N or D; X ₁₀ =G or S; X ₁₁ =G or E; X ₁₃ =D or R; X ₁₄ =Y or L; X ₁₇ =N or K; X ₂₀ =G or D 110 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 HC-CDR3 Consensus X ₂ =R or S; X ₃ =E or I; X ₄ =G or Y; X ₅ =N or Y; X ₆ =N or E; X ₇ =Y or missing; X ₈ =G or missing; X ₉ =S or missing; X ₁₀ =R or missing; X ₁₁ =D or missing; X ₁₂ =A or absent; X ₁₃ =M or missing; X ₁₄ =D or E; X ₁₅ =Y or A 111 X ₁ AX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ YX ₁₀ X ₁₁ WY LC-CDR1 Consensus X ₁ =S or R; X ₃ =S or R; X ₄ =S or G; X ₅ =G or N; X ₆ =V or I; X ₇ =I or H; X ₈ =N or missing; X ₁₀ =M or L; X ₁₁ =Y or A 112 X ₁ X ₂ X ₃ YX ₅ X ₆ X ₇ X ₈ LAX ₁₁ LC-CDR2 Consensus X ₁ =P or L; X ₂ =W or L; X ₃ =I or V; X ₅ =L or N; X ₆ =T or A; X ₇ =S or K; X ₈ =N or T; X ₁₁ =S or D 113 QX ₂ X ₃ X ₄ X ₅ X ₆ PX ₈ T LC-CDR3 Consensus X ₂ =Q or H; X ₃ =W or F; X ₄ =S or W; X ₅ =S or W; X ₆ =N or T; X ₈ =L or Y 114 SYWMN SJ25C1HC-CDR1 115 QIYPGDGDTNYNGKFKG SJ25C1HC-CDR2 116 KTISSVVDFYFDY SJ25C1HC-CDR3 117 KASQNVGTNVA SJ25C1 LC-CDR1 118 SATYRNS SJ25C1 LC-CDR2 119 QQYNRYPYT SJ25C1 LC-CDR3 120 DYGVS FMC63HC-CDR1 121 VIWGSETTYYNSALKS FMC63HC-CDR2 122 HYYYGGSYAMDY FMC63HC-CDR3 123 RASQDISKYLN FMC63 LC-CDR1 124 HTSRLHS FMC63 LC-CDR2 125 QQGNTLPYT FMC63 LC-CDR3 126 LEGGGEGRGSLTCGDVEENPGPR T2A 127 EGRGSLLTCGDVEENPGP T2A 128 GSGATNFSLLKQAGDVEENPGP P2A 129 ATNFSLLKQAGDVEENPGP P2A 130 QCTNYALLKLAGDVESNPGP E2A 131 VKQTLNFDLLKLAGDVESNPGP F2A 132 QVQLKQSGPGLVQPSQSLSITCTVS DYTFTSY GVHWVRQSPGKGLEWLGVI YPGSGG TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR EVTTVAYYYSMDY WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-SJ25C1 heavy chain 133 DILLTQSPVILSVSPGERVSFSC RASQDISNYLN WYQQRTNGSPRLLIK YTSRLHS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQGKTVPFT FGAGTKLELKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKPVNGSFTHQQ Illustrative humanized anti-SJ25C1 light chain 134 QVQLKQSGPGLVQPSQSLSITCTVSGFSL TSYWMH WVRQSPGKGLE WIGNIYPGSGGTN YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC TREVTTVAYYYSMD YWGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-SJ25C1 heavy chain 135 DILLTQSPVILSVSPGERVSFSCRASQSI SNYLNWY QQRTNGSPR LLIYYTSRLH SGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQGKTVPF TFGAGTKLELKRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSPVACE Illustrative humanized anti-SJ25C1 light chain 136 QVQLKQSGPGLVQPSQSLSITCTVSGFSLT SYWMH WVRQSPGKGLEWLG NIYPGSGGTNYDEKFKR RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR EVTTVAYYYSMDY WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-SJ25C1 heavy chain 137 DILLTQSPVILSVSPGERVSFSC RASQDISNYLN WYQQRTNGSPRLLIK YTSRLHS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQGKTVPFT FGAGTKLELKRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVYACEVACE Illustrative humanized anti-SJ25C1 light chain 138 QVQLKQSGPGLVQPSQSLSITCTVS GYTFTDY GVHWVRQSPGKGLEWLGVI NPNNGG TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR EGNNYGSRDAMDY WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 139 DILLTQSPVILSVSPGERVSFSC SASSGVIYMY WYQQRTNGSPRLLIK LTSNLAS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQWSSNPLT FGAGTKLELKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL Illustrative humanized anti-FMC63 light chain 140 QVQLKQSGPGLVQPSQSLSITCTVSGFSLT DYYMK WVRQSPGKGLEWLG DINPNNGGTDYNQNFKG RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR EGNNYGSRDAMDY WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 141 DILLTQSPVILSVSPGERVSFSC SASSGVIYMY WYQQRTNGSPRLLIK LTSNLAS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQWSSNPLT FGAGTKLELKRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVYACENSFTHQ Illustrative humanized anti-FMC63 light chain 142 QVQLKQSGPGLVQPSQSLSITCTVSGFSL TDYYMK WVRQSPGKGLE WIGDINPNNGGTD YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC AREGNNYGSRDAMD YWGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 143 DILLTQSPVILSVSPGERVSFSCRASQSI IYMYWY QQRTNGSPR PWIYLTSNLA SGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQWSSNPL TFGAGTKLELKRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVYACEVTHQ Illustrative humanized anti-FMC63 light chain 144 QVQLKQSGPGLVQPSQSLSITCTVS GYSFTRY GVHWVRQSPGKGLEWLGVI HPSDSE TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR IYYEEA WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 145 DILLTQSPVILSVSPGERVSFSC RASGNIHNYLA WYQQRTNGSPRLLIK NAKTLAD GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QHFWSTPYT FGAGTKLELKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL Illustrative humanized anti-FMC63 light chain 146 QVQLKQSGPGLVQPSQSLSITCTVSGFSLT RYWMN WVRQSPGKGLEWLG MIHPSDSETRLNQKFKD RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR IYYEEA WGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 147 DILLTQSPVILSVSPGERVSFSC RASGNIHNYLA WYQQRTNGSPRLLIK NAKTLAD GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QHFWSTPYT FGAGTKLELKRTV AAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Illustrative humanized anti-FMC63 light chain 148 QVQLKQSGPGLVQPSQSLSITCTVSGFSL TRYWMN WVRQSPGKGLE WIGMIHPSDSETR YNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ASIYYEE YWGQGTLVTVSA TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Illustrative humanized anti-FMC63 heavy chain 149 DILLTQSPVILSVSPGERVSFSCRASQSI HNYLAWY QQRTNGSPR LLVYNAKTLA SGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QHFWSTPY TFGAGTKLELKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVYACEVTHQ Illustrative humanized anti-FMC63 light chain 150 ESKYGPPCPPCP spacer (IgG4 hinge) (AK) Homo sapiens 151 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4 hinge) (nt) Homo sapiens 152 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH3 spacer Homo sapiens 153 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH2-CH3 spacer Homo sapiens 154 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-Hinge-Fc Homo sapiens 155 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR artificial 156 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR artificial 157 FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179 Accession No. P10747) Homo sapiens 158 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 Accession No. P10747) Homo sapiens 159 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220, P10747) Homo sapiens 160 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL - GG) Homo sapiens 161 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255, Q07011.1) Homo sapiens 162 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens 163 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens 164 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta Homo sapiens

--->

SEQUENCE LIST

<110> JUNO THERAPUTICS, INC.

HOSKINS, Colleen

Hapel, Mark, D.

SUTERLAND, Claire, L.

SATALIA, Taher

SMITH, Jeff

<120> ANTI-IDIOTYPE ANTIBODIES AND THEIR RELATED METHODS

<130> 735042006540

<140> not assigned

<141> -

<150> 62/369.008

<151> 2016-07-29

<160> 164

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID VH

<400> 1

Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe

50 55 60

Lys Arg Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp

100 105 110

Gly Glyn Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 2

<211> 324

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID CH

<400> 2

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 3

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID heavy chain

<400> 3

Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe

50 55 60

Lys Arg Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

210 215 220

Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

245 250 255

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

260 265 270

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

275 280 285

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

290 295 300

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

355 360 365

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

405 410 415

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

420 425 430

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210> 4

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC signal sequence

<400> 4

Met Gly Trp Ser Ser Ile Ile Leu Phe Leu Val Ala Thr Ala Ser Gly

1 5 10 15

Val His Ser

<210> 5

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID VL

<400> 5

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Val Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 6

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID CL

<400> 6

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Asn Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 7

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID light chain

<400> 7

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Val Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Asn Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 8

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC signal sequence

<400> 8

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys

twenty

<210> 9

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR1

<400> 9

Ser Tyr Trp Met His

fifteen

<210> 10

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR2

<400> 10

Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe Lys

1 5 10 15

Arg

<210> 11

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR3

<400> 11

Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr

1 5 10

<210> 12

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR1

<400> 12

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 13

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR2

<400> 13

Tyr Thr Ser Arg Leu His Ser

fifteen

<210> 14

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR3

<400> 14

Gln Gln Gly Lys Thr Val Pro Phe Thr

fifteen

<210> 15

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID VH

<400> 15

caggtccaac tgcaacaacc tgggtctgag ctggtgaggc ctggaggttc agtgaagctg 60

tcctgcaagg cttctgacta cactttcacc agctactgga tgcactggggt gaggcagagg 120

cctggacaag gccttgagtg gattggaaat atttatcctg gtagtggtgg tactaactac 180

gatgagaagt tcaagaggaa ggccacactg actgtagaca catcctccag cacagcctac 240

atgcagctcc gcagcctgac atctgaggac tctgcggtct attactgtac aagagaggtt 300

actacagtag cttattacta ttctatggac tactggggtc aaggaacctc agtcaccgtc 360

tcctca 366

<210> 16

<211> 975

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID CH

<400> 16

gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg ccctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc acccaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900

acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960

tctcctggta aatga 975

<210> 17

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID heavy chain

<400> 17

caggtccaac tgcaacaacc tgggtctgag ctggtgaggc ctggaggttc agtgaagctg 60

tcctgcaagg cttctgacta cactttcacc agctactgga tgcactggggt gaggcagagg 120

cctggacaag gccttgagtg gattggaaat atttatcctg gtagtggtgg tactaactac 180

gatgagaagt tcaagaggaa ggccacactg actgtagaca catcctccag cacagcctac 240

atgcagctcc gcagcctgac atctgaggac tctgcggtct attactgtac aagagaggtt 300

actacagtag cttattacta ttctatggac tactggggtc aaggaacctc agtcaccgtc 360

tcctcagcca aaacgacacc cccatctgtc tatccactgg cccctggatc tgctgcccaa 420

actaactcca tggtgaccct gggatgcctg gtcaagggct atttccctga gccagtgaca 480

gtgacctgga actctggatc cctgtccagc ggtgtgcaca ccttcccagc tgtcctgcag 540

tctgacctct acactctgag cagctcagtg actgtcccct ccagcacctg gcccagcgag 600

accgtcacct gcaacgttgc ccacccggcc agcagcacca aggtggacaa gaaaattgtg 660

cccagggatt gtggttgtaa gccttgcata tgtacagtcc cagaagtatc atctgtcttc 720

atcttccccc caaagcccaa ggatgtgctc accattactc tgactcctaa ggtcacgtgt 780

gttgtggtag acatcagcaa ggatgatccc gaggtccagt tcagctggtt tgtagatgat 840

gtggaggtgc acacagctca gacgcaaccc cgggaggagc agttcaacag cactttccgc 900

tcagtcagtg aacttcccat catgcaccag gactggctca atggcaagga gttcaaatgc 960

agggtcaaca gtgcagcttt ccctgccccc atcgagaaaa ccatctccaa aaccaaaggc 1020

agaccgaagg ctccacaggt gtacaccatt ccacctccca aggagcagat ggccaaggat 1080

aaagtcagtc tgacctgcat gataacagac ttcttccctg aagacattac tgtggagtgg 1140

cagtggaatg ggcagccagc ggagaactac aagaacactc agcccatcat ggacacagat 1200

ggctcttact tcgtctacag caagctcaat gtgcagaaga gcaactggga ggcaggaaat 1260

actttcacct gctctgtgtt acatgagggc ctgcacaacc accatactga gaagagcctc 1320

tcccactctc ctggtaaatg a 1341

<210> 18

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID HC signal sequence

<400> 18

atgggatgga gctctatcat cctcttcttg gtagcaacag cctcaggtgt ccactcc 57

<210> 19

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID VL

<400> 19

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgtcagcag ggtaaaacgg ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 20

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID CL

<400> 20

60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240

cgacataaca actatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg ttag 324

<210> 21

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID light chain

<400> 21

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgtcagcag ggtaaaacgg ttccattcac gttcggctcg 300

gggacaaagt tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540

ttgaccaagg acgagtatga acgacataac aactatacct gtgaggccac tcacaagaca 600

gttag 645

<210> 22

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID LC signal sequence

<400> 22

atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taccagatgt 60

<210> 23

<211> 122

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 VH

<400> 23

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 108

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 VL

<400> 24

Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr

85 90 95

Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 25

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 25

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 26

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> 4GS Linker

<400> 26

Gly Gly Gly Gly Ser

fifteen

<210> 27

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<223> Extracellular portion of CD28

<400> 27

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 28

<211> 245

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 scFv

<400> 28

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser

130 135 140

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe

210 215 220

Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 29

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> 3GS Linker

<400> 29

Gly Gly Gly Ser

one

<210> 30

<211> 120

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 VH

<400> 30

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 31

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 VL

<400> 31

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 32

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<223> IgG1 Fc without hinge

<400> 32

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

115 120 125

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

165 170 175

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

180 185 190

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210> 33

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 33

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 34

<211> 245

<212> Protein

<213> Artificial sequence

<220>

<223> FMC63 scFv

<400> 34

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 35

<211> 465

<212> Protein

<213> Artificial sequence

<220>

<223> FMC63 reagent

<400> 35

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

275 280 285

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

Lys

465

<210> 36

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 VH

<400> 36

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Lys Gln Cys His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp

100 105 110

Gly Glyn Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 37

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 CH

<400> 37

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

1 5 10 15

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 38

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 heavy chain

<400> 38

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Lys Gln Cys His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro

115 120 125

Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

180 185 190

Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro

210 215 220

Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu

245 250 255

Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

275 280 285

Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

290 295 300

Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

305 310 315 320

Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro

325 330 335

Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln

340 345 350

Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val

355 360 365

Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val

370 375 380

Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg

405 410 415

Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val

420 425 430

Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg

435 440 445

Thr Pro Gly Lys

450

<210> 39

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC signal sequence

<400> 39

Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

Val Leu Ser

<210> 40

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 VL

<400> 40

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Ile Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 41

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 CL

<400> 41

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 42

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 light chain

<400> 42

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Ile Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys

210

<210> 43

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC signal sequence

<400> 43

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Met Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly

twenty

<210> 44

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR1

<400> 44

Asp Tyr Tyr Met Lys

fifteen

<210> 45

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR2

<400> 45

Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe Lys

1 5 10 15

gly

<210> 46

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR3

<400> 46

Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr

1 5 10

<210> 47

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR1

<400> 47

Ser Ala Ser Ser Gly Val Ile Tyr Met Tyr

1 5 10

<210> 48

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR2

<400> 48

Leu Thr Ser Asn Leu Ala Ser

fifteen

<210> 49

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR3

<400> 49

Gln Gln Trp Ser Ser Asn Pro Leu Thr

fifteen

<210> 50

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 VH

<400> 50

gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgtaagg cttctggata cacattcact gactactaca tgaagtgggt gaagcagtgt 120

catggaaaga gccttgagtg gattggagat attaatccta acaatggtgg tactgactac 180

aaccagaact ttaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg 300

aataactacg gtagtagaga tgctatggac tactggggtc aaggaacgtc agtcaccgtc 360

tcctca 366

<210> 51

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 CH

<400> 51

gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 60

tcctcggtga ctctaggatg cctggtcaag ggttatttcc ctgagccagt gaccttgacc 120

tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacaccc tcagcagctc agtgactgta acctcgagca cctggcccag ccagtccatc 240

acctgcaatg tggcccaccc ggcaagcagc acccaaggtgg acaagaaaat tgagcccaga 300

gggcccacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga 360

ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc 420

atagtcacat gtgtggtggt ggatgtgagc gaggatgacc cagatgtcca gatcagctgg 480

tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 540

agtactctcc gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag 600

gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca 660

aaacccaaag ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag 720

atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt 780

tacgtggagt ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc 840

ctggactctg atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg 900

gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg 960

actaagagct tctcccggac tccgggtaaa 990

<210> 52

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 heavy chain

<400> 52

gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgtaagg cttctggata cacattcact gactactaca tgaagtgggt gaagcagtgt 120

catggaaaga gccttgagtg gattggagat attaatccta acaatggtgg tactgactac 180

aaccagaact ttaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg 300

aataactacg gtagtagaga tgctatggac tactggggtc aaggaacgtc agtcaccgtc 360

tcctcagcca aaacaacagc cccatcggtc tatccactgg cccctgtgtg tggagataca 420

actggctcct cggtgactct aggatgcctg gtcaagggtt atttccctga gccagtgacc 480

ttgacctgga actctggatc cctgtccagt ggtgtgcaca ccttcccagc tgtcctgcag 540

tctgacctct acaccctcag cagctcagtg actgtaacct cgagcacctg gcccagccag 600

tccatcacct gcaatgtggc ccacccggca agcagcacca aggtggacaa gaaaattgag 660

cccagagggc ccacaatcaa gccctgtcct ccatgcaaat gcccagcacc taacctcttg 720

ggtggaccat ccgtcttcat cttccctcca aagatcaagg atgtactcat gatctccctg 780

agccccatag tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc 840

agctggtttg tgaacaacgt ggaagtacac acagctcaga cacaaaccca tagagaggat 900

tacaacagta ctctccgggt ggtcagtgcc ctccccatcc agcaccagga ctggatgagt 960

ggcaaggagt tcaaatgcaa ggtcaacaac aaagacctcc cagcgcccat cgagagaacc 1020

1080

gaagagatga ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa 1140

gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 1200

ccagtcctgg actctgatgg ttcttacttc atgtacagca agctgagagt ggaaaagaag 1260

aactgggtgg aaagaaatag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 1320

cacacgacta agagcttctc ccggactccg ggtaaa 1356

<210> 53

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC signal sequence

<400> 53

atgggatgga gctggatctt tctcttcctc ttgtcaggaa ctgcaggtgt cctctct 57

<210> 54

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 VL

<400> 54

caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aggtgtaatt tacatgtact ggtaccaaca gaagccaaga 120

tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggc 300

accaagctgg agctgaaa 318

<210> 55

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 CL

<400> 55

60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240

cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg t 321

<210> 56

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 light chain

<400> 56

caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aggtgtaatt tacatgtact ggtaccaaca gaagccaaga 120

tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggc 300

accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360

agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420

aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480

agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 540

accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600

acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639

<210> 57

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC signal sequence

<400> 57

atggattttc aagtgcagat tttcagcttc ctgctaatga gtgcctcagt cataatgtcc 60

aggga 66

<210> 58

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 VH

<400> 58

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Arg Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 59

<211> 324

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 CH

<400> 59

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 60

<211> 439

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 heavy chain

<400> 60

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Arg Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Ala Lys Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro

115 120 125

Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val

130 135 140

Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser

145 150 155 160

Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu

165 170 175

Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser

180 185 190

Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val

195 200 205

Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys

210 215 220

Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val

245 250 255

Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp

260 265 270

Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe

275 280 285

Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys

325 330 335

Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu Gln Met Ala Lys

340 345 350

Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp

355 360 365

Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys

370 375 380

Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser

385 390 395 400

Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr

405 410 415

Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser

420 425 430

Leu Ser His Ser Pro Gly Lys

435

<210> 61

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC signal sequence

<400> 61

Met Gly Trp Ser Ser Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 62

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 VL

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Asp Ser Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 63

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 light chain

<400> 63

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Asp Ser Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 64

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC signal sequence

<400> 64

Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Gly Ala Arg Cys

twenty

<210> 65

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR1

<400> 65

Arg Tyr Trp Met Asn

fifteen

<210> 66

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR2

<400> 66

Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys

1 5 10 15

asp

<210> 67

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR3

<400> 67

Ile Tyr Tyr Glu Glu Ala

fifteen

<210> 68

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR1

<400> 68

Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala

1 5 10

<210> 69

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR2

<400> 69

Asn Ala Lys Thr Leu Ala Asp

fifteen

<210> 70

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR3

<400> 70

Gln His Phe Trp Ser Thr Pro Tyr Thr

fifteen

<210> 71

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 VH

<400> 71

caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60

tcctgcaaga cttctggcta ctccttcacc aggtactgga tgaactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180

aatcagaagt tcaaggacaa ggccacattg actgtagaca attcctccag cacagcctac 240

atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagcatctac 300

tatgaagagg cctggggcca agggactctg gtcactgtct ctgca 345

<210> 72

<211> 972

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 CH

<400> 72

gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg ccctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc acccaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900

acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960

tctcctggta aa 972

<210> 73

<211> 1317

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 heavy chain

<400> 73

caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60

tcctgcaaga cttctggcta ctccttcacc aggtactgga tgaactggggt gaagcagagg 120

cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180

aatcagaagt tcaaggacaa ggccacattg actgtagaca attcctccag cacagcctac 240

atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagcatctac 300

tatgaagagg cctggggcca agggactctg gtcactgtct ctgcagccaa aacgacaccc 360

ccatctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg 420

ggatgcctgg tcaagggcta ttttccctgag ccagtgacag tgacctggaa ctctggatcc 480

ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt ctgacctcta cactctgagc 540

agctcagtga ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc 600

cacccggcca gcagcaccaa ggtggacaag aaaattgtgc ccagggattg tggttgtaag 660

ccttgcatat gtacagtccc agaagtatca tctgtcttca tcttcccccc aaagcccaag 720

gatgtgctca ccattactct gactcctaag gtcacgtgtg ttgtggtaga catcagcaag 780

gatgatcccg aggtccagtt cagctggttt gtagatgatg tggaggtgca cacagctcag 840

acgcaaccc gggaggagca gttcaacagc actttccgct cagtcagtga acttcccatc 900

atgcaccagg actggctcaa tggcaaggag ttcaaatgca gggtcaacag tgcagctttc 960

cctgccccca tcgagaaaac catctccaaa accaaaggca gaccgaaggc tccacaggtg 1020

tacaccattc cacctcccaa ggagcagatg gccaaggata aagtcagtct gacctgcatg 1080

ataacagact tcttccctga agacattact gtggagtggc agtggaatgg gcagccagcg 1140

gagaactaca agaacactca gcccatcatg gacacagatg gctcttactt cgtctacagc 1200

aagctcaatg tgcagaagag caactgggag gcaggaaata ctttcacctg ctctgtgtta 1260

catgaggggcc tgcacaacca ccatactgag aagagcctct cccactctcc tggtaaa 1317

<210> 74

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC signal sequence

<400> 74

atgggatgga gctctatcat cctcttcttg gtagcaacag ctacaggtgt ccactcc 57

<210> 75

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 VL

<400> 75

gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtgg gaatattcac aattatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagatag tgtgccatca 180

aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240

gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 76

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 light chain

<400> 76

gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtgg gaatattcac aattatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagatag tgtgccatca 180

aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240

gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtacac gttcggaggg 300

gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360

tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420

cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480

aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540

ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600

tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642

<210> 77

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC signal sequence

<400> 77

atgagtgtgc tcactcaggt cctggcgttg ctgctgctgt ggcttacagg tgccagatgt 60

<210> 78

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR1

<400> 78

Asp Tyr Thr Phe Thr Ser Tyr

fifteen

<210> 79

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR1

<400> 79

Asp Tyr Thr Phe Thr Ser Tyr Trp Met His

1 5 10

<210> 80

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR1

<400> 80

Thr Ser Tyr Trp Met His

fifteen

<210> 81

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR2

<400> 81

Tyr Pro Gly Ser Gly Gly

fifteen

<210> 82

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR2

<400> 82

Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn

1 5 10

<210> 83

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR2

<400> 83

Trp Ile Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn

1 5 10

<210> 84

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID HC-CDR3

<400> 84

Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp

1 5 10

<210> 85

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR1

<400> 85

Ser Asn Tyr Leu Asn Trp Tyr

fifteen

<210> 86

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR2

<400> 86

Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His

1 5 10

<210> 87

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID LC-CDR3

<400> 87

Gln Gln Gly Lys Thr Val Pro Phe

fifteen

<210> 88

<211> 7

<212> Protein

<213> anti-ID B-1 HC-CDR1

<400> 88

Gly Tyr Thr Phe Thr Asp Tyr

fifteen

<210> 89

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR1

<400> 89

Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys

1 5 10

<210> 90

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR1

<400> 90

Thr Asp Tyr Tyr Met Lys

fifteen

<210> 91

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR2

<400> 91

Asn Pro Asn Asn Gly Gly

fifteen

<210> 92

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR2

<400> 92

Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp

1 5 10

<210> 93

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR2

<400> 93

Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp

1 5 10

<210> 94

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 HC-CDR3

<400> 94

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp

1 5 10

<210> 95

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR1

<400> 95

Ile Tyr Met Tyr Trp Tyr

fifteen

<210> 96

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR2

<400> 96

Pro Trp Ile Tyr Leu Thr Ser Asn Leu Ala

1 5 10

<210> 97

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-1 LC-CDR3

<400> 97

Gln Gln Trp Ser Ser Asn Pro Leu

fifteen

<210> 98

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR1

<400> 98

Gly Tyr Ser Phe Thr Arg Tyr

fifteen

<210> 99

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR1

<400> 99

Gly Tyr Ser Phe Thr Arg Tyr Trp Met Asn

1 5 10

<210> 100

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR1

<400> 100

Thr Arg Tyr Trp Met Asn

fifteen

<210> 101

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR2

<400> 101

His Pro Ser Asp Ser Glu

fifteen

<210> 102

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR2

<400> 102

Met Ile His Pro Ser Asp Ser Glu Thr Arg

1 5 10

<210> 103

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR2

<400> 103

Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg

1 5 10

<210> 104

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 HC-CDR3

<400> 104

Ala Ser Ile Tyr Tyr Glu Glu

fifteen

<210> 105

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR1

<400> 105

His Asn Tyr Leu Ala Trp Tyr

fifteen

<210> 106

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR2

<400> 106

Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala

1 5 10

<210> 107

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> anti-ID B-2 LC-CDR3

<400> 107

Gln His Phe Trp Ser Thr Pro Tyr

fifteen

<210> 108

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> HC-CDR1 Consensus

<220>

<221> OPTION

<222> 3

<223> Xaa = T or S

<220>

<221> OPTION

<222> 5

<223> Xaa = T or S

<220>

<221> OPTION

<222> 6

<223> Xaa = D or R

<220>

<221> OPTION

<222> 8

<223> Xaa = Y or W

<220>

<221> OPTION

<222> 10

<223> Xaa = K or N

<400> 108

Gly Tyr Xaa Phe Xaa Xaa Tyr Xaa Met Xaa

1 5 10

<210> 109

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> HC-CDR2 Consensus

<220>

<221> OPTION

<222> 4

<223> Xaa = D or M

<220>

<221> OPTION

<222> 6

<223> Xaa = N or H

<220>

<221> OPTION

<222> 8

<223> Xaa = N or S

<220>

<221> OPTION

<222> 9

<223> Xaa = N or D

<220>

<221> OPTION

<222> 10

<223> Xaa = G or S

<220>

<221> OPTION

<222> (11)...(11)

<223> Xaa = G or E

<220>

<221> OPTION

<222> (13)...(13)

<223> Xaa = D or R

<220>

<221> OPTION

<222> (14)...(14)

<223> Xaa = Y or L

<220>

<221> OPTION

<222> (17)...(17)

<223> Xaa = N or K

<220>

<221> OPTION

<222> (20)...(20)

<223> Xaa = G or D

<400> 109

Trp Ile Gly Xaa Ile Xaa Pro Xaa Xaa Xaa Xaa Thr Xaa Xaa Asn Gln

1 5 10 15

Xaa Phe Lys Xaa

twenty

<210> 110

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> HC-CDR3 Consensus

<220>

<221> OPTION

<222> 2

<223> Xaa = R or S

<220>

<221> OPTION

<222> 3

<223> Xaa = E or I

<220>

<221> OPTION

<222> 4

<223> Xaa = G or Y

<220>

<221> OPTION

<222> 5

<223> Xaa = N or Y

<220>

<221> OPTION

<222> 6

<223> Xaa = N or E

<220>

<221> OPTION

<222> (7)...(7)

<223> Xaa = Y or missing

<220>

<221> OPTION

<222> (8)...(8)

<223> Xaa = G or missing

<220>

<221> OPTION

<222> (9)...(9)

<223> Xaa = S or missing

<220>

<221> OPTION

<222> (10)...(10)

<223> Xaa = R or missing

<220>

<221> OPTION

<222> (11)...(11)

<223> Xaa = D or missing

<220>

<221> OPTION

<222> (12)...(12)

<223> Xaa = A or missing

<220>

<221> OPTION

<222> (13)...(13)

<223> X13 = M or missing

<220>

<221> OPTION

<222> (14)...(14)

<223> X14 = D or E

<220>

<221> OPTION

<222> (15)...(15)

<223> X15 = Y or A

<400> 110

Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

<210> 111

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> LC-CDR1 Consensus

<220>

<221> OPTION

<222> (1)...(1)

<223> Xaa = S or R

<220>

<221> OPTION

<222> (3)...(3)

<223> Xaa = S or R

<220>

<221> OPTION

<222> (4)...(4)

<223> Xaa = S or G

<220>

<221> OPTION

<222> (5)...(5)

<223> Xaa = G or N

<220>

<221> OPTION

<222> (6)...(6)

<223> Xaa = V or I

<220>

<221> OPTION

<222> (7)...(7)

<223> Xaa = I or H

<220>

<221> OPTION

<222> (8)...(8)

<223> Xaa = N or missing

<220>

<221> OPTION

<222> (10)...(10)

<223> Xaa = M or L

<220>

<221> OPTION

<222> (11)...(11)

<223> Xaa = Y or A

<400> 111

Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Trp Tyr

1 5 10

<210> 112

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> LC-CDR2 Consensus

<220>

<221> OPTION

<222> (1)...(1)

<223> Xaa = P or L

<220>

<221> OPTION

<222> (2)...(2)

<223> Xaa = W or L

<220>

<221> OPTION

<222> (3)...(3)

<223> Xaa = I or V

<220>

<221> OPTION

<222> (5)...(5)

<223> Xaa = L or N

<220>

<221> OPTION

<222> (6)...(6)

<223> Xaa = T or A

<220>

<221> OPTION

<222> (7)...(7)

<223> Xaa = S or K

<220>

<221> OPTION

<222> (8)...(8)

<223> Xaa = N or T

<220>

<221> OPTION

<222> (11)...(11)

<223> Xaa = S or D

<400> 112

Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Leu Ala Xaa

1 5 10

<210> 113

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> LC-CDR3 Consensus

<220>

<221> OPTION

<222> (2)...(2)

<223> Xaa = Q or H

<220>

<221> OPTION

<222> (3)...(3)

<223> Xaa=W or F

<220>

<221> OPTION

<222> (4)...(4)

<223> Xaa = S or W

<220>

<221> OPTION

<222> (5)...(5)

<223> Xaa = S or W

<220>

<221> OPTION

<222> (6)...(6)

<223> Xaa = N or T

<220>

<221> OPTION

<222> (8)...(8)

<223> Xaa = L or Y

<400> 113

Gln Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr

fifteen

<210> 114

<211> 5

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 HC-CDR1

<400> 114

Ser Tyr Trp Met Asn

fifteen

<210> 115

<211> 17

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 HC-CDR2

<400> 115

Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys

1 5 10 15

gly

<210> 116

<211> 13

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 HC-CDR3

<400> 116

Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr

1 5 10

<210> 117

<211> 11

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 LC-CDR1

<400> 117

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 118

<211> 7

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 LC-CDR2

<400> 118

Ser Ala Thr Tyr Arg Asn Ser

fifteen

<210> 119

<211> 9

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 LC-CDR3

<400> 119

Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr

fifteen

<210> 120

<211> 5

<212> Protein

<213> Mus musculus

<220>

<223> SJ25C1 LC-CDR3

<400> 120

Asp Tyr Gly Val Ser

fifteen

<210> 121

<211> 16

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 HC-CDR2

<400> 121

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 122

<211> 12

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 HC-CDR3

<400> 122

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 123

<211> 11

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 LC-CDR1

<400> 123

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 124

<211> 7

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 LC-CDR2

<400> 124

His Thr Ser Arg Leu His Ser

fifteen

<210> 125

<211> 9

<212> Protein

<213> Mus musculus

<220>

<223> FMC63 LC-CDR3

<400> 125

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

fifteen

<210> 126

<211> 24

<212> Protein

<213> Artificial sequence

<220>

<223> T2A

<400> 126

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

twenty

<210> 127

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> T2A

<400> 127

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

GlyPro

<210> 128

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> P2A

<400> 128

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

twenty

<210> 129

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> P2A

<400> 129

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 130

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223>E2A

<400> 130

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

twenty

<210> 131

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> F2A

<400> 131

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210> 132

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 heavy chain

<400> 132

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Asp Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Tyr Pro Gly Ser Gly Gly Thr Asp Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 133

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 light chain

<400> 133

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 134

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 heavy chain

<400> 134

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 135

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 light chain

<400> 135

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 136

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 heavy chain

<400> 136

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe

50 55 60

Lys Arg Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 137

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-SJ25C1 light chain

<400> 137

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 138

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 138

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 139

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 139

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Ser Gly Val Ile Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu

65 70 75 80

Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 140

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 140

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 141

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 141

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Ser Gly Val Ile Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu

65 70 75 80

Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 142

<211> 459

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 142

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser

115 120 125

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

130 135 140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

145 150 155 160

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

165 170 175

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

180 185 190

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

195 200 205

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

355 360 365

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 143

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 143

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Ile Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu

65 70 75 80

Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 144

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 144

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Tyr Ser Phe Thr Arg Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile His Pro Ser Asp Ser Glu Thr Asp Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 145

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 145

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Asn Ala Lys Thr Leu Ala Asp Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 146

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 146

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 147

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 147

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Asn Ala Lys Thr Leu Ala Asp Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 148

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 heavy chain

<400> 148

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Asn Thr Pro Phe

50 55 60

Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe

65 70 75 80

Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ser Ile Tyr Tyr Glu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 149

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Illustrative humanized anti-FMC63 light chain

<400> 149

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 150

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<223> spacer (IgG4 hinge) (AK)

<400> 150

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 151

<211> 36

<212> DNA

<213> Homo sapiens

<220>

<223> spacer (IgG4 hinge) (NT)

<400> 151

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 152

<211> 119

<212> Protein

<213> Homo sapiens

<220>

<223> Hinge-CH3 spacer

<400> 152

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 153

<211> 229

<212> Protein

<213> Homo sapiens

<220>

<223> Hinge-CH2-CH3

<400> 153

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 154

<211> 282

<212> Protein

<213> Homo sapiens

<220>

<223> IgD-hinge-Fc

<400> 154

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 155

<211> 335

<212> Protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 155

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 156

<211> 357

<212> Protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 156

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 157

<211> 27

<212> Protein

<213> Homo sapiens

<220>

<223> CD28 (amino acids 153-179, available number no. P10747)

<400> 157

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 158

<211> 66

<212> Protein

<213> Homo sapiens

<220>

<223> CD28 (amino acids 114-179, Accession No. P10747)

<400> 158

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 159

<211> 41

<212> Protein

<213> Homo sapiens

<220>

<223> CD28 (amino acids 180-220, P10747)

<400> 159

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 160

<211> 41

<212> Protein

<213> Homo sapiens

<220>

<223> CD28 (LL - GG)

<400> 160

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 161

<211> 42

<212> Protein

<213> Homo sapiens

<220>

<223> 4-1BB (amino acids 214-255, Q07011.1)

<400> 161

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 162

<211> 112

<212> Protein

<213> Homo sapiens

<220>

<223> CD3 zeta

<400> 162

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 163

<211> 112

<212> Protein

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 163

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 164

<211> 112

<212> Protein

<213> Homo sapiens

<220>

<223> CD3-zeta

<400> 164

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<---

Claims (115)

1. Anti-idiotypic antibody or its antigen-binding fragment, containing: a heavy chain variable region (VH) containing CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained within the VH region with the amino acid sequence of SEQ ID NO: 36; and a light chain variable region (VL) containing CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained within the VL with the amino acid sequence of SEQ ID NO:40 , where the anti-idiotypic antibody or antigen-binding fragment specifically binds to the target antibody or antigen-binding fragment, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 2. Anti-idiotypic antibody or its antigen-binding fragment according to claim 1, where: CDR-H1 contains the sequence of SEQ ID NO:44, 88, 89 or 90; CDR-H2 contains the sequence of SEQ ID NO:45, 91, 92 or 93; CDR-H3 contains the sequence of SEQ ID NO:46 or 94; CDR-L1 contains the sequence SEQ ID NO:47 or 95; CDR-L2 contains the sequence SEQ ID NO:48 or 96; and CDR-L3 contains the sequence SEQ ID NO:49 or 97. 3. The anti-idiotypic antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are determined according to the Kabat numbering system. 4. Anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1-3, where: VL contains an amino acid sequence that is at least 90% identical to the sequence of the VL region c SEQ ID NO:40; and The VH contains an amino acid sequence that is at least 90% identical to the sequence of the VH region of SEQ ID NO:36. 5. Anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1-4, where: VL contains the amino acid sequence of SEQ ID NO:40, and VH contains the amino acid sequence of SEQ ID NO:36. 6. Anti-idiotypic antibody or its antigen-binding fragment, containing: VH containing CDR-H1, CDR-H2 and CDR-H3, respectively, containing the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence of the VH region with SEQ ID NO:58; and VL containing CDR-L1, CDR-L2 and CDR-L3, respectively, containing the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 contained in the amino acid sequence of the VL region of SEQ ID NO:62, where the anti-idiotypic antibody or antigen-binding fragment specifically binds to the target antibody or antigen-binding fragment, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 7. Anti-idiotypic antibody or antigen-binding fragment according to claim 6, where: CDR-H1 contains the sequence of SEQ ID NO:65, 98, 99 or 100; CDR-H2 contains the sequence of SEQ ID NO:66, 101, 102 or 103; CDR-H3 contains the sequence of SEQ ID NO:67 or 104; CDR-L1 contains the sequence SEQ ID NO:68 or 105; CDR-L2 contains the sequence of SEQ ID NO:69 or 106; and CDR-L3 contains the sequence SEQ ID NO:70 or 107. 8. The anti-idiotypic antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are defined according to the Kabat numbering system. 9. Anti-idiotypic antibody or antigen-binding fragment according to any one of claims 6-8, where: VL contains an amino acid sequence that is at least 90% identical to the sequence of the VL region c SEQ ID NO:62; and The VH contains an amino acid sequence that is at least 90% identical to the sequence of the VH region of SEQ ID NO:58. 10. Anti-idiotypic antibody or antigen-binding fragment according to any one of claims 6-9, where: VH contains the amino acid sequence of SEQ ID NO:58, and VL contains the amino acid sequence of SEQ ID NO:62. 11. The anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the target antibody or antigen-binding fragment thereof comprises a single chain variable fragment (scFv) containing the amino acid sequence of SEQ ID NO:34. 12. Anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1-11, where: the target antibody or antigen-binding fragment thereof is within or included in the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR); and/or the anti-idiotypic antibody, or antigen-binding fragment thereof, specifically binds to a target antibody, or antigen-binding fragment thereof, located within or included in the antigen-binding domain of the extracellular portion of the CAR. 13. The anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the anti-idiotypic antibody or antigen-binding fragment thereof is: a CAR agonist antibody, wherein the CAR contains the target antibody or antigen-binding fragment thereof; or a CAR antagonist, wherein the CAR contains the target antibody or antigen-binding fragment thereof. 14. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which is humanized and/or recombinant. 15. An anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is monoclonal. 16. The anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the anti-idiotypic antibody or antigen-binding fragment thereof is an antigen-binding fragment. 17. The anti-idiotypic antibody or antigen-binding fragment thereof according to claim 16, wherein the antigen-binding fragment is selected from the group consisting of antigen-binding (Fab) fragments, F(ab') ₂ fragments, Fab' fragments, Fv fragments, single chain variable fragments (scFv), and single domain antibodies (sdAb). 18. The anti-idiotypic antibody or antigen-binding fragment thereof according to claim 16 or 17, wherein the antigen-binding fragment contains at least a portion of an immunoglobulin constant region. 19. The anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the anti-idiotypic antibody is an intact antibody or a full length antibody. 20. A conjugate for detecting the binding of an anti-idiotypic antibody or an antigen-binding fragment thereof and a target antibody or an antigen-binding fragment thereof, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a heterologous molecule or group selected from the group consisting of an imaging agent, detectable agent and radioactive atom, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 21. A conjugate for visualizing the binding of an anti-idiotypic antibody or an antigen-binding fragment thereof and a target antibody or an antigen-binding fragment thereof, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a heterologous molecule or group selected from the group consisting of an imaging agent, detectable agent and radioactive atom, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 22. A conjugate for diagnosing a disease or disorder in which CD19 is expressed, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a heterologous molecule or group selected from the group consisting of an imaging agent, a detectable agent and a radioactive atom. 23. A conjugate for the treatment of a disease or disorder in which CD19 is expressed, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a heterologous molecule or group selected from the group consisting of a cytotoxic agent, a drug, and a radioactive atom. 24. A nucleic acid molecule encoding the heavy chain of an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-19. 25. Nucleic acid molecule encoding the light chain of an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-19. 26. A nucleic acid molecule encoding the heavy chain and light chain of an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-5 and 11-19. 27. A nucleic acid molecule encoding the heavy chain and light chain of an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 6-19. 28. A vector for expressing nucleic acid molecules, comprising a nucleic acid molecule according to any one of claims 24-27. 29. A method for producing an anti-idiotypic antibody or antigen-binding fragment thereof, comprising expressing the heavy and light chains encoded by the nucleic acid molecule according to claim 26 or 27 in a suitable host cell and recovering or isolating the antibody. 30. A method for producing an anti-idiotypic antibody or an antigen-binding fragment thereof, comprising expressing the heavy chain encoded by the nucleic acid molecule of claim 24 and the light chain encoded by the nucleic acid molecule of claim 25 in a suitable host cell and recovering or isolating the antibody. 31. A cell for expressing an anti-idiotypic antibody or an antigen-binding fragment thereof, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19. 32. A composition for the treatment of a disease or disorder in which CD19 is expressed, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient. 33. A detection composition comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient. 34. An imaging composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19 and a pharmaceutically acceptable excipient. 35. A diagnostic composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient. 36. A composition for stimulating cells expressing a target antibody or an antigen-binding fragment thereof, containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 37. A cell selection composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient. 38. A kit for the treatment of a disease or disorder in which CD19 is expressed, comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use. 39. A detection kit containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use. 40. An imaging kit comprising an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use. 41. A diagnostic kit containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use. 42. A kit for stimulating cells expressing a target antibody or an antigen-binding fragment thereof, containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 43. A cell selection kit containing an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19 and instructions for use. 44. A method for detecting an anti-idiotypic antibody or an antigen-binding fragment thereof that binds to a target antibody or an antigen-binding fragment thereof, comprising: (a) contacting a composition comprising a target antibody or antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, which specifically binds to the target antibody or antigen-binding fragment thereof, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and (b) detecting an anti-idiotypic antibody or antigen-binding fragment thereof that has bound to the target antibody or antigen-binding fragment thereof. 45. A method for detecting cells expressing a CAR containing a target antibody or its antigen-binding fragment, comprising: (a) contacting a cell expressing a CAR containing the target antibody or antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1 to 19, which specifically binds to the target antibody or antigen-binding fragment thereof, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and (b) detecting cells associated with the anti-idiotypic antibody or antigen-binding fragment thereof. 46. A method for selecting cells expressing a CAR containing a target antibody or an antigen-binding fragment thereof from a population of cells, comprising: (a) contacting a cell population expressing a CAR containing the target antibody or antigen-binding fragment thereof, or a cell associated with the target antibody or antigen-binding fragment thereof, with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and (b) selecting cells associated with the anti-idiotypic antibody or antigen-binding fragment thereof. 47. A method for stimulating cells expressing a CAR containing a target antibody or its antigen-binding fragment, comprising incubation of the initial composition containing cells expressing a CAR containing a target antibody or antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, thereby obtaining a final composition containing stimulated cells, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 48. A method for obtaining a cell composition, including: (a) introducing into the cells a nucleic acid molecule encoding a CAR, thereby obtaining the original composition; and (b) incubating the original composition with an anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1 to 19, thereby obtaining a cell composition. 49. A method for purifying an antibody or its antigen-binding fragment, including: (a) contacting a composition comprising a target antibody or antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1-19, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and (b) isolating complexes containing the anti-idiotypic antibody or antigen-binding fragment. 50. A method for depleting cells expressing a CAR containing a target antibody or its antigen-binding fragment, comprising administering to an individual a composition comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1-19, where the individual is injected with a cell expressing a CAR containing a target antibody or antigen-binding fragment, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 51. A composition for depleting cells in an individual, comprising an anti-idiotypic antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the individual has been administered a cell expressing a CAR containing the target antibody or antigen-binding fragment thereof, where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31. 52. A kit for detecting a target antibody or its antigen-binding fragment, containing: (a) one or more containers containing the anti-idiotypic antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, and instructions for using the anti-idiotypic antibody to detect the target antibody or antigen-binding fragment or chimeric antigen receptor containing the target antibody or its antigen-binding fragment; where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and instructions for selecting or enriching a cell population, engineered cells expressing a CAR containing said target antibody or antigen-binding fragment thereof; or instructions for stimulating an administered composition comprising cells expressing a chimeric antigen receptor containing a target antibody or antigen-binding fragment thereof; or (b) one or more containers containing a binding reagent containing a CAR extracellular domain containing a target antibody or an antigen-binding fragment, said extracellular domain or a portion thereof contains a target antibody or an antigen-binding fragment thereof; where the target antibody or antigennegative fragment contains VH with the sequence of SEQ ID NO:30 and VL with the sequence of SEQ ID NO:31; and one or more containers containing an anti-idiotypic antibody or antigen-binding fragment according to any one of claims 1-19.

Images