Antigen-binding molecules and methods of use

Abstract

The present disclosure describes antigen-binding molecules, including antibodies that specifically bind to anti-CD20 scFv-14 or gibbon ape leukemia virus gp70 protein, as well as molecules comprising the described sequences and cells that present such molecules. The antigen-binding molecules can be used in research, diagnostic, clinical, and other applications.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 489,373, filed March 9, 2023, and U.S. Provisional Patent Application No. 63 / 620,111, filed January 11, 2024, each of which is incorporated herein in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on February 8, 2024 is named K-1137-WO-PCT_SL.xml and is 312,108 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure also relates to antigen-binding molecules, such as antibodies, that specifically bind to targets, comprising an anti-CD20 scFv-14 binding domain or the gibbon ape leukemia virus (GALV) protein gp70, as well as molecules comprising these sequences and cells that present such molecules, polynucleotides encoding such antigen-binding molecules, and humanized forms of and methods of using the antigen-binding molecules. [Background technology]

[0004] Antigen-binding molecules, including antibodies and fragments such as Fab, F(ab')2, scFvs, etc., are used in immunotherapy and solid-phase-based applications such as biosensors, affinity chromatography, and immunoassays. These antibodies and other antigen-binding molecules gain utility due to their ability to specifically bind to their targets.

[0005] Anti-idiotypic antibodies are a subset of antibodies that are produced in response to an immune antibody. These anti-idiotypic antibodies show specific binding to the idiotope (a unique antigenic determinant on the surface of the antibody) of the immune antibody. Anti-idiotypic antibodies can generally be classified into three different groups: (1) antibodies that recognize idiotopes that are different from the antigen-binding site (ABS) on the immune antibody; (2) antibodies that recognize epitopes within the ABS and mimic the structure, forming a so-called "internal image" of the nominal antigen; and (3) antibodies that recognize epitopes within the ABS without structural similarity to the nominal antigen (see, for example, Pan et al., (1995) FASEB J 9:43-49).

[0006] There is a further need for detection and quantification of viral particles. In particular, viral envelope proteins such as the gibbon ape leukemia virus (GALV) protein gp70 provide excellent targets for virus detection. Antigen-binding molecules specific for GALV gp70 would have many uses in assays, such as flow-based virus detection methods.

[0007] Disclosed herein are antigen-binding molecules comprising antibodies that specifically bind to anti-CD20 scFv-14 or GALV protein gp70, as well as molecules comprising these sequences and cells that present such molecules. Humanized forms of the disclosed antigen-binding molecules are also embodiments of the present disclosure. Applications and uses of these antigen-binding molecules are also disclosed. Summary of the Invention

[0008] In various aspects, an isolated antigen-binding molecule that binds to anti-CD20 is disclosed. In various other aspects, an isolated antigen-binding molecule that binds to Gibbon Ape Leukemia Virus (GALV) protein gp70 is disclosed. In various embodiments, the heavy chain variable (VH) sequence has at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-10. In various embodiments, the light chain variable (VL) sequence has at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 11-20. In various embodiments, a linker connects the VH to the VL.

[0009] In various aspects, an isolated antigen-binding molecule is disclosed, comprising a VH amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen-binding molecule described herein.

[0010] In various aspects, an isolated antigen-binding molecule is disclosed, comprising a VL amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VL of an antigen-binding molecule described herein.

[0011] In various embodiments, the isolated antigen-binding molecule comprises a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 21 to 41. In various embodiments, the isolated antigen-binding molecule comprises a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 42 to 65. In various embodiments, the isolated antigen-binding molecule comprises a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 66 to 85. In various embodiments, the isolated antigen-binding molecule comprises a light chain CDR1 selected from the group consisting of SEQ ID NOs: 86 to 99. In various embodiments, the isolated antigen-binding molecule comprises a light chain CDR2 selected from the group consisting of SEQ ID NOs: 100 to 111. In various embodiments, the antigen-binding molecule comprises a light chain CDR3 selected from the group consisting of SEQ ID NOs: 112 to 120.

[0012] In various embodiments, the linker comprises an amino acid sequence. In various embodiments, the amino acid sequence of the linker comprises SEQ ID NO: 121. In various embodiments, the amino acid sequence of the linker comprises SEQ ID NO: 126. In various embodiments, the isolated antigen-binding molecule comprises a linker amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VL of an antigen-binding molecule described herein.

[0013] In various embodiments, the isolated antigen-binding molecule comprises a detectable label selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

[0014] In various embodiments, the isolated antigen binding molecule comprises a heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 25, 32, and 39. In various embodiments, the isolated antigen binding molecule comprises a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 46, 54, and 62. In various embodiments, the isolated antigen binding molecule comprises a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 70 and 80. In various embodiments, the isolated antigen binding molecule comprises a light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 90 and 98. In various embodiments, the isolated antigen binding molecule comprises a light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 104 and 110. In various embodiments, the isolated antigen binding molecule comprises a light chain CDR3 sequence comprising SEQ ID NO: 116.

[0015] In a specific embodiment, the antigen binding system, antibody, or antigen-binding fragment thereof comprises a GALV gp70-binding motif, the GALV gp70-binding motif comprising the sequences of three heavy chain complementarity-determining regions (HCDRs) of any one of the heavy chain variable regions (HCVRs) selected from the group consisting of SEQ ID NOs: 303-314, and the sequences of three light chain CDRs (LCDRs) of the light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 315-324.

[0016] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises a first domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a second domain comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); (i) HCDR1 has a sequence according to any one of SEQ ID NOs: 127 to 138, 157 to 168, and 187 to 198; (ii) HCDR2 has a sequence according to any one of SEQ ID NOs: 139-150, 169-180, and 199-210; (iii) HCDR3 has a sequence according to any one of SEQ ID NOs: 151 to 156, 181 to 186, 211 to 222, and DYY; (iv) LCDR1 has a sequence according to any one of SEQ ID NOs: 223 to 232, 253 to 262, and 283 to 292; (v) LCDR2 has a sequence according to any one of SEQ ID NOs: 233-242, 263-272, SGS, GTN, RAS, DTS, and KVS; (vi) LCDR3 has a sequence according to any one of SEQ ID NOs: 243 to 252, 273 to 282, and 293 to 302.

[0017] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises an HCDR and an LCDR, wherein the HCDR is (i) an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, and an HCDR3 according to SEQ ID NO: 211 or DYY; (ii) an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, and an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; (iii) an HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189, an HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, and an HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; (iv) HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, and HCDR3 according to SEQ ID NO: 214 or DYY; (v) an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; (vi) an HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; and an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; (vii) an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; and an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; (viii) HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194, HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206, and HCDR3 according to SEQ ID NO: 218 or DYY; (ix) an HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; an HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; and an HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; (x) an HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196, an HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208, and an HCDR3 according to SEQ ID NO: 220 or DYY; (xi) an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, and an HCDR3 according to SEQ ID NO: 221 or DYY; or (xii) comprises an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, and an HCDR3 according to SEQ ID NO: 222 or DYY; and LCDR, (i) an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; (ii) an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; and an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; (iii) an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (iv) LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (v) an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; and an LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; (vi) an LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; an LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; (vii) LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; (viii) an LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; an LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299; (ix) an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291; an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301; or (x) comprises an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0018] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs); HCDR and LCDR are (i) an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292, an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN, an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302, (ii) an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; (iii) an HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; an HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; an HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; and an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (iv) an HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; an HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; an HCDR3 according to SEQ ID NO: 214 or DYY; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (v) an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (vi) an HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (vii) an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; an LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; (viii) an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194, an HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206, an HCDR3 according to SEQ ID NO: 218 or DYY, an LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, an LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, an LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; (ix) an HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; an HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; an HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; an LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; an LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; an LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; (x) an HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196, an HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208, an HCDR3 according to SEQ ID NO: 220 or DYY, an LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, an LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, an LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, (xi) an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NO: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or (xii) comprises an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NO: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, and 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0019] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs; (i) the heavy chain variable domain is at least 80% identical to one of SEQ ID NOs: 303-314; (ii) the light chain variable domain is at least 80% identical to one of SEQ ID NOs: 315-324;

[0020] In certain aspects, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs; (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 303 and the light chain variable domain is at least 80% identical to SEQ ID NO: 324; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 304 and the light chain variable domain is at least 80% identical to SEQ ID NO: 320; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 305 and the light chain variable domain is at least 80% identical to SEQ ID NO: 318; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 306 and the light chain variable domain is at least 80% identical to SEQ ID NO: 319; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 307 and the light chain variable domain is at least 80% identical to SEQ ID NO: 319; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 308 and the light chain variable domain is at least 80% identical to SEQ ID NO: 318; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 309 and the light chain variable domain is at least 80% identical to SEQ ID NO: 317; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 310 and the light chain variable domain is at least 80% identical to SEQ ID NO: 316; (ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 311 and the light chain variable domain is at least 80% identical to SEQ ID NO: 315; (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 312 and the light chain variable domain is at least 80% identical to SEQ ID NO: 321; (xi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 313 and the light chain variable domain is at least 80% identical to SEQ ID NO: 323; or (xii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 314 and the light chain variable domain is at least 80% identical to SEQ ID NO: 322.

[0021] In a particular embodiment, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is characterized in that the three HCDRs and three LCDRs are contained in a single polypeptide.

[0022] In a particular embodiment, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is characterized in that three HCDRs are comprised in a first polypeptide and three LCDRs are comprised in a second polypeptide.

[0023] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is characterized in that the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

[0024] In a particular embodiment, nucleic acids encoding at least one GALV gp70 binding polypeptide as described above are disclosed.

[0025] In one aspect, a vector containing such a nucleic acid is disclosed.

[0026] In certain aspects, methods of producing engineered cells are disclosed, the methods comprising transfecting or transducing cells with the nucleic acids or vectors described immediately above.

[0027] In particular aspects, cells encoding or expressing the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof are disclosed, optionally, the cells are immune cells.

[0028] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof further comprises a detectable label.

[0029] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises a detectable label selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

[0030] In certain embodiments, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is conjugated to a dye selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarin, aminocouramin, methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, and Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTruquoise 2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange Orange), mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, peridinin chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

[0031] In a particular aspect, a method for determining the number of viral particles expressing a Gibbon Ape Leukemia Virus (GALV) gp70 protein having the amino acid sequence of SEQ ID NO: 325 is disclosed, the method comprising: (a) providing a sample known or suspected to contain viral particles expressing GALV gp70 protein; (b) contacting the sample with an antigen-binding system, antibody, or antigen-binding fragment thereof under conditions that allow the formation of one or more binding complexes comprising the virus particles and the antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 21 to 29, wherein the antigen-binding system, antibody, or antigen-binding fragment thereof further comprises a detectable label; (c) detecting one or more binding complexes by detecting the detectable label; (d) determining the number of virus particles present in the sample based on the detecting of step (c).

[0032] In a particular aspect, a method for determining the presence or absence of a viral particle expressing a Gibbon Ape Leukemia Virus (GALV) gp70 protein having the amino acid sequence of SEQ ID NO: 325 is disclosed, the method comprising: (a) providing a sample known or suspected to contain viral particles expressing GALV gp70 protein; (b) providing an antigen-binding molecule that specifically binds to GALV gp70 protein, wherein the antigen-binding molecule further comprises a detectable label; (c) contacting the sample with the antigen-binding molecule under conditions that allow the formation of a binding complex between GALV gp70 and the antigen-binding protein; (d) separating any molecules from the binding complex that are not part of the binding complex; and (e) detecting the presence or absence of the binding complex.

[0033] In a particular aspect, the method for determining the number of viral particles or the method for determining the presence or absence of viral particles is characterized in that the antigen-binding molecule is disposed on a surface selected from the group consisting of agarose beads, magnetic beads, plastic well plates, glass well plates, ceramic well plates, and cell culture bags.

[0034] In a particular aspect, the method for determining the number of viral particles or the method for determining the presence or absence of viral particles is characterized in that the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

[0035] In certain embodiments, the methods for determining the number of viral particles or the methods for determining the presence or absence of viral particles include methods in which the fluorescent label is an Atto dye, an Alexafluor dye, a quantum dot, a hydroxycoumarin, an aminocouramin, a methoxycourmarin, a Cascade Blue, a Pacific Blue, a Pacific Orange, a Lucifer Yellow, NBD, R-Phycoerythrin (PE), a PE-Cy5 conjugate, a PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, or Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTruquoise 2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange Orange), mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, peridinin chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

[0036] In a particular aspect, the method for determining the number of viral particles or the method for determining the presence or absence of viral particles is characterized in that the detecting is carried out using a flow-based detection method.

[0037] In a particular embodiment, the method for determining the number of viral particles or the presence or absence of viral particles is characterized in that the flow-based detection method is flow virometry.

[0038] In a particular aspect, the method for determining the number of viral particles or the method for determining the presence or absence of viral particles is characterized in that the detecting is performed by ELISA, biolayer interferometry (BLI), Western blot, or any combination thereof. [Brief explanation of the drawings]

[0039] [Figure 1-1] Panels A-E are control experimental data for expression data from transduced T cells. [Figure 1-2] Panels A-E are control experimental data for expression data from transduced T cells.

[0040] [Figure 2] 1 shows experimental data showing CAR binding with scFv14 compared to a control.

[0041] [Figure 3A] Panel A shows experimental data for binding of supernatants to scFv14 for potential hybridoma candidates.

[0042] [Figure 3B] Panel B shows experimental data for binding of supernatants to scFv14 for potential hybridoma candidates.

[0043] [Figure 4]1 shows experimental data assessing specificity for scFv14. Supernatants were screened for binding to T cells expressing CAR with scFv14, CAR with irrelevant anti-CD20 scFv2, or CAR with Leu 16, or NTD T cell control.

[0044] [Figure 5] 1 shows experimental data evaluating scFv14 specificity.

[0045] [Figure 6A] Panel A shows experimental data for PE-conjugated clone 24C12 screened against healthy donor T cells transduced to express CAR either as NTD or with either scFv2, scFv14, or FMC63.

[0046] [Figure 6B] Panel B shows experimental data for FITC-conjugated clone 24C12 screened against healthy donor T cells transduced to express CAR either as NTD or with either scFv2, scFv14, or FMC63.

[0047] [Figure 7A] Panel A shows experimental data for PE-conjugated clone 24C12 screened against healthy donor T cells transduced to express CAR either as NTD or with either scFv2, scFv14, or FMC63.

[0048] [Figure 7B] Panel B shows experimental data for FITC-conjugated clone 24C12 screened against healthy donor T cells transduced to express CAR either as NTD or with either scFv2, scFv14, or FMC63.

[0049] [Figure 8] A schematic diagram of the recombinant GALV gp70 viral envelope protein design is shown. A secretion signal (SS) was included in all constructs. Various affinity tags (monoFc huIgG1, muIgG2a Fc, or 6x His (SEQ ID NO: 348)) were included for purification. The entire predicted viral surface-exposed portion of GALV gp70 was generated, including residues 42-616 (numbering according to Uniprot P21415) with an N-terminal affinity tag. Truncations including the coiled-coil region (residues 505-616) or only the receptor-binding domain (residues 42-474) were made using a C-terminal affinity tag.

[0050] [Figure 9A] K562 or CHO cells stained with anti-SLC20A1 antibody (Proteintech Cat. No. 12423-1-AP) and PE-conjugated secondary antibody are shown. Cells were stained with secondary antibody alone as a control. The percentage of cells positive for SLC20A1 surface expression is shown (94.7% for K562, 0% for CHO). [Figure 9B] Dilution series of recombinant GALV gp70 protein is shown, followed by staining of K562 or CHO cells with PE-conjugated secondary antibody. PE mean fluorescence intensity (MFI) increased in a dose-dependent manner when K562 cells were stained with gp70 (42-474) or (42-616), but not with (505-616). No staining was observed for CHO cells.

[0051] [Figure 10A] Indirect ELISA titers using immobilized GALV gp70(42-616) protein are shown, measured against immunized mouse sera or normal mouse sera as a negative control. Two mice (Ms6609 and Ms6423) with the highest OD450 readings were selected for hybridoma fusion. [Figure 10B]PG13 cells stably producing viral particles containing the GALV gp70 envelope protein were stained with a dilution series of serum from immunized mice selected for hybridoma fusion and a PE-conjugated secondary antibody (left and center). Negative controls including no staining (NS), normal mouse serum (non-immunized mice: NMS1 and NMS2), or isotype control antibody staining of PG13 cells are shown (right).

[0052] [Figure 11] Relative comparison of indirect ELISA titers using immobilized GALV gp70 proteins (42-616), (505-616), (42-616) or affinity-tagged negative control protein measured after incubation with hybridoma supernatants. 17 positive hybridoma clones are shown.

[0053] [Figure 12] PG13 cells stably producing viral particles containing GALV gp70 envelope protein are shown, incubated with a dilution series of each hybridoma supernatant and stained with a PE-conjugated secondary antibody. The relative PE signal for each hybridoma is shown.

[0054] [Figure 13] PG13 cells (top), which stably produce viral particles containing the GALV gp70 envelope protein, were incubated with a single dilution (1 / 10) of each antibody clone successfully purified on a small scale. NIH-3T3 cells (bottom) were stained with the same antibody clones as a negative control.

[0055] [Figure 14A]PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14B] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14C] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14D] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14E]PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14F] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14G] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14H] PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL. [Figure 14I]PG13 cells (left), which stably produce viral particles containing GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a top concentration of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same antibody clone dilution series as a negative control. Isotype controls were also included for both cell line stainings at a top concentration of 10 μg / mL.

[0056] [Figure 15A] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15B] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15C] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15D] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15E] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15F]Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15G] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474). [Figure 15H] Purified antibody clones loaded onto an AMC biosensor (2 μg / mL) are shown. Octet sensorgrams (association and dissociation) are shown for all clones showing binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474).

[0057] [Figure 16] Hybridoma supernatants that do not bind to FMC63-28z at the highest concentration tested are shown. Sample identities are indicated on the left side of each panel. Each figure shows a histogram of fluorescence intensity values ​​(x-axis) plotted against frequency (y-axis) normalized for the respective mode. DETAILED DESCRIPTION OF THE INVENTION

[0058] In a particular aspect, the present embodiment relates to an anti-idiotypic antigen-binding molecule comprising an antibody that specifically binds to an antigen-binding molecule that specifically binds to anti-CD20 scFv14 (see Kanyarat Thueng-in, Jeeraphong Thanongsaksrikul, Surasak Jittavisutthikul, Watee Seesuay, Monrat Chulanetra, Yuwaporn Sakolvaree, Potjanee Srimanote & Wanpen Chaicumpa (2014) Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase, (mAbs, 6:5, 1327-1339, DOI:10.4161 / mAbs.29978).

[0059] Anti-CD20 scFv-14 has the following amino acid sequence:

[0060] VH DNA-CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAAAGAATATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAGTGGTCACACATACTATGCACA GAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTGCGCCAGAGGGCCTCACTACGACGACTGGAGCGGATTTATCATATGGTTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (Sequence number 122)

[0061] VL DNA-GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAG TTTGCAAAGTGGGGTCCCTTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGGTTTCCTCCTACCTTTGGCCAAGGGACCAAGGTTGAGATCAAA (Sequence number 123)

[0062] VH protein - QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS (SEQ ID NO: 124)

[0063] VL protein -DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK (SEQ ID NO: 125)

[0064] Also provided are humanized forms of the antigen-binding molecule, molecules comprising anti-CD20 scFv 14, and cells that display molecules comprising anti-CD20 scFv 14. Also disclosed are polynucleotides encoding the antigen-binding molecule, vectors comprising the polynucleotides, and in vitro cells comprising the polynucleotides and vectors.

[0065] Methods using the disclosed antigen-binding molecules are provided. The antigen-binding molecules, polynucleotides, vectors, in vitro cells, and methods described herein can be used in a variety of applications, for example, as reagents for detecting the presence of anti-CD20 scFv14-containing moieties, molecules comprising this sequence, and cells presenting such molecules, for quantifying the amount of anti-CD20 scFv14-containing moieties, molecules comprising this sequence, and cells presenting such molecules, for screening for anti-CD20 scFv14-containing moieties, molecules comprising this sequence, and cells presenting such molecules, for purifying anti-CD20 scFv14-containing moieties, molecules comprising this sequence, and cells presenting such molecules, and for biomarker research focusing on anti-CD20 scFv-containing moieties, molecules comprising this sequence, and cells presenting such molecules. Therapeutic uses, e.g., applications in which the biological activity of moieties comprising anti-CD20 scFv14 and cells presenting such molecules are modulated (enhanced or inhibited), as well as therapeutic agents comprising anti-CD20 scFv14, and molecules comprising this sequence and cells presenting such molecules, and dose ranging studies relating to cells presenting such molecules are also provided.

[0066] The antigen-binding molecules (e.g., scFvs, antibodies, etc.) disclosed herein were produced from hybridomas generated using B cells of murine origin, but can be readily humanized using standard methods known to those skilled in the art, as well as the methods described herein. Representative humanized forms of the disclosed antigen-binding molecules can be produced as described herein.

[0067] In a further specific aspect, the present embodiment relates to an antigen-binding molecule, including an antibody, that specifically binds to a viral coat protein. In a specific aspect, the antigen-binding molecule binds to the gibbon ape leukemia virus gp70 envelope protein (GALV gp70). GALV gp70 is a surface protein that attaches the virus to host cells by binding to its receptor.

[0068] GALV gp70 has the amino acid sequence (SEQ ID NO: 325).

[0069] Also disclosed are polynucleotides encoding the GALV gp70 binding molecules, as well as vectors containing the polynucleotides, and in vitro cells containing the polynucleotides and vectors.

[0070] Methods using the disclosed antigen-binding molecules are provided. The antigen-binding molecules, polynucleotides, vectors, in vitro cells, and methods described herein can be used in a variety of applications, such as as reagents for detecting the presence of GALV gp70-containing moieties, molecules containing this sequence, and viral particles and cells displaying such molecules, to quantify the amount of GALV gp70-containing moieties, molecules containing this sequence, and cells displaying such molecules, to screen for GALV gp70-containing moieties, molecules containing this sequence, and cells displaying such molecules, to purify GALV gp70-containing moieties, molecules containing this sequence, and cells displaying such molecules, and for biomarker tests focusing on GALV gp70-containing moieties, molecules containing this sequence, and cells displaying such molecules. Therapeutic uses, such as applications in which the biological activity of GALV gp70-containing moieties and cells displaying such molecules are modulated (enhanced or suppressed), and dose-ranging studies related to therapeutic agents containing GALV gp70, molecules containing this sequence, and cells displaying such molecules, and cells displaying such molecules, are also provided. I. Definition

[0071] In order that this disclosure may be more readily understood, certain terms are first defined below. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. The headings provided herein are not limitations of the various aspects of the disclosure, but should be understood by reference to the specification as a whole.

[0072] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also presented.

[0073] Units, prefixes and symbols used herein are presented using the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Juo, The Concise Dictionary of Biomedicine and Molecular Biology, 2nd Edition, (2001), CRC Press; The Dictionary of Cell & Molecular Biology, 5th Edition, (2013), Academic Press, and The Oxford Dictionary of Biochemistry And Molecular Biology, edited by Cammack et al., 2nd Edition (2006), Oxford University Press provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0075] As used herein, the 20 conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunolo-yA Synthesis (2nd ed.), Golub and Green, eds., Sinauer Assoc., Sunderland, Mass. (1991), incorporated herein by reference for any purpose. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids, such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids, may also be suitable components for the polypeptides of the present invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0076] As used herein, the terms "a" and "an" are used according to standard convention to mean one or more, unless the context dictates otherwise.

[0077] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within one or more standard deviations as practiced in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 10% (i.e., ±10%). For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean a value that is up to an order of magnitude or up to five times greater. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range for that particular value or composition.

[0078] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, as appropriate (such as integer tenths and hundredths), unless otherwise specified.

[0079] As used herein, the term "and / or" is to be understood as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, for example, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0080] As used herein, the use of the term alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0081] As used herein, the term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

[0082] The term "antibody" includes both naturally occurring and non-naturally occurring antibodies, human and non-human antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies (e.g., Stocks, (2004) Drug Discovery Today 9(22):960-66), antibody fusions (which term encompasses antibody-drug conjugates, and may be referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments thereof. In certain embodiments, antibodies as described herein refer to polyclonal antibody populations.

[0083] The term "antibody," unless otherwise specified, also includes an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments containing the complementarity-determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen-binding properties on the polypeptide.

[0084] Non-human antibodies can be humanized using recombinant methods to reduce their immunogenicity in humans, as disclosed herein for antibodies that specifically bind to anti-CD20 scFv14, as well as molecules comprising this sequence and cells that present such molecules. Unless explicitly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments of any of the aforementioned immunoglobulin antigen-binding molecules, including monovalent and bivalent fragments or portions, as well as single-chain antibodies (i.e., scFvs).

[0085] The term "antigen" refers to any molecule that can elicit an immune response or be bound by an antibody or other antigen-binding molecule. The immune response can include either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily understand that any macromolecule, including virtually any protein or peptide (including anti-CD20 scFv14), as well as molecules containing the same sequence and cells presenting such molecules, can serve as an antigen. Generally, antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed, or chemically synthesized. In a specific embodiment, antigens include all or a portion of anti-CD20 scFv14 and molecules containing the same sequence, optionally conjugated to an adjuvant such as keyhole limpet hemocyanin (KLH) or to Fc for ease of screening.

[0086] As used herein, the term "antigen-binding molecule" refers to a protein comprising a portion that binds to an antigen or target protein, and, optionally, a scaffold or framework portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding molecule to the antigen. Representative types of antigen-binding molecules include scFv, human, mouse, or rabbit antibodies; humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; diabodies; triabodies; tetrabodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies, and fragments thereof.

[0087] Antigen-binding molecules can include alternative protein scaffolds or artificial scaffolds, for example, with grafted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding molecule, as well as fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Furthermore, peptide antibody mimetics ("PAMs") and scaffolds based on antibody mimetics that utilize various components (e.g., fibronectin) as scaffolds can be used. Antigen-binding molecules can have, for example, the structure of naturally occurring immunoglobulins.

[0088] An antigen-binding molecule may have one or more binding sites. When two or more binding sites are present, the binding sites may be identical to one another or may be different. For example, naturally occurring human immunoglobulins typically have two identical binding sites, whereas "bispecific" or "bifunctional" antibodies have two different binding sites and can specifically bind to two different antigens (e.g., anti-CD20 scFv14 and a cell surface activator molecule).

[0089] In various embodiments, the antigen-binding molecule is an antibody or fragment thereof comprising one or more of the complementarity-determining regions (CDRs) disclosed herein that specifically binds to anti-CD20 scFv14, a molecule comprising anti-CD20 scFv14, and a cell that presents such a molecule. In further embodiments, the antigen-binding molecule binds to a CAR comprising anti-CD20 scFv14 and a molecule comprising anti-CD20 scFv14, and can be expressed on immune cells such as T cells.

[0090] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves the collection of lymphocytes from a patient, which are then engineered to express a construct, such as a CAR construct, and then administered to the same patient.

[0091] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen, such as an antigen-binding molecule). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A K D is k off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art such as BIAcore® or KinExA or surface plasmon resonance.

[0092] As used herein, the term "complementarity determining region" or "CDR" refers to an amino acid sequence that contributes to antigen-binding specificity and affinity. Framework regions can help maintain proper identification of CDRs to promote binding between an antigen-binding molecule and an antigen. Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, IMGT numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the Kabat and Chothia systems and is used by Oxford Molec'lar's AbM antibody modeling software. Table 1 defines CDRs using each numbering system. The contact definition is based on an analysis of available complex crystal structures. TIFF2026507879000002.tif66136

[0093] Terms such as "Kabat numbering" are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding molecule thereof. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD 1991). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (optionally including one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). In some embodiments, the CDRs of the antibodies described herein can be described according to the Kabat numbering convention (although they can be readily interpreted in other numbering systems using Table 1 above). In some embodiments, the CDRs of the antibodies described herein can be described according to the Clothia numbering convention. In some embodiments, the CDRs of the antibodies described herein can be described according to the IGMT numbering convention.

[0094] In certain embodiments, the CDRs of an antibody can be determined according to the Chothia numbering convention, which refers to the position of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, using the Kabat numbering convention, the Chothia CDR-H1 loop is located at amino acids 26-32, 33, or 34 in the heavy chain, the Chothia CDR-H2 loop is located at amino acids 52-56 in the heavy chain, and the Chothia CDR-H3 loop is located at amino acids 95-102 in the heavy chain, while the Chothia CDR-L1 loop is located at amino acids 24-34 in the light chain, the Chothia CDR-L2 loop is located at amino acids 50-56 in the light chain, and the Chothia CDR-L3 loop is located at amino acids 89-97 in the light chain. The end of the Chothia CDR-HI loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). See Table 1. In some embodiments, the CDRs of the antibodies described herein have been determined according to the Chothia numbering convention.

[0095] As used herein, a "conservative amino acid substitution" refers to one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within the CDR(s) or framework region(s) of an antibody or antigen-binding molecule (or fragment thereof) provided herein can be replaced with an amino acid residue having a similar side chain.

[0096] Conservative amino acid substitutions encompassed by the present disclosure can include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other inverted or reversed forms of amino acid moieties. Naturally occurring residues can be grouped into classes based on common side chain properties. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, Acidification: Asp, Glu, Bases: His, Lys, Arg, Residues that influence chain orientation: Gly, Pro, Aromatic: Trp, Tyr, Phe.

[0097] Non-conservative substitutions can involve exchanging a member of one of these classes for a member of another class. Such substituted residues can be introduced, for example, into regions of the human antibody that are homologous with the non-human antibody, or into the non-homologous regions of the molecule. Exemplary conservative amino acid substitutions are shown in Table 2 below. TIFF2026507879000003.tif154162

[0098] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0099] As used herein, the term "cross-compete" refers to a situation in which the interaction between an antigen and a first antigen-binding molecule or its binding fragment blocks, limits, inhibits, or otherwise reduces the ability of a reference antigen-binding molecule or its binding fragment to interact with the antigen. Cross-competition can be complete, e.g., binding of a binding molecule to an antigen completely blocks the ability of the reference antigen-binding molecule to bind to the antigen, or it can be partial, e.g., binding of a binding molecule to an antigen reduces the ability of the reference antigen-binding molecule to bind to the antigen. In certain embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. Many types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another antigen-binding molecule: for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., (1983) Method Enzymol 9:242-53), solid-phase direct biotin-avidin EIA (Kirkland et al., (1986) J Immunol 137:3614-19), solid-phase direct label assay, solid-phase direct label sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), I 125 Label-based solid-phase direct labeling RIA (Morel et al., (1988) Molec Immunol 25:7-15), solid-phase direct biotin-avidin EIA (Cheung et al., (1990) Virology 176:546-52), and direct labeling RIA (Moldenhauer et al., (1990) Scand J Immunol 32:77-82).

[0100] The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative contains a covalent modification, including, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen-binding molecule (derivative) can have a longer circulating half-life than an antigen-binding molecule that is not chemically modified. In some embodiments, a derivative antigen-binding molecule is covalently modified to contain one or more water-soluble polymer linkages, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0101] As used herein, the term "diabody" or dAB refers to a bivalent antibody comprising two polypeptide chains, each of which comprises a VH and VL domain connected by a linker that is too short to allow pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., (1993) Proc Natl Acad Sci USA 90:6444-48, Poljak et al. (1994) Structure 2:1121-23, and Perisic et al., (1994) Structure 2(12):1217-26). When the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to generate diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0102] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, a polypeptide or two or more non-contiguous regions of a polypeptide (a conformational, non-linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (see, e.g., Giege et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson, (1990) Eur J Biochem 189:1-23; Chayen, (1997) Structure 5:1269-1274; McPherson (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations Inc., e.g., Meth Enzymol (1985) Vols 114 & 115, eds. Wyckoff et al.), and BUSTER (see Bricogne, (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne, (1997) Meth Enzymol 276A:361-423, ed. Carter; Roversi et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323).Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe et al., (1995) J Biol Chem 270:1388-94, and Cunningham & Wells, (1989) Science 244:1081-85.

[0103] As used herein, the term "Fab fragment" refers to a monovalent fragment containing the VL, VH, CL, and CH domains. An "F(ab')2 fragment" is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, an "Fv fragment" contains the VH and VL domains of a single antibody arm, and a "dAb fragment" contains the VH domain, the VL domain, or an antigen-binding fragment of the VH or VL domain.

[0104] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are used interchangeably in the context of antigen-binding molecules and mean that a given molecule preferentially binds to an antigen (e.g., an epitope or immune complex), as such binding is understood by those of skill in the art. For example, an antigen-binding molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, a molecule that specifically binds to an antigen has a K A At least 2 log, 2.5 log, 3 log, 4 log, or more than K A and binds to the antigen.

[0105] In another embodiment, the number of molecules that specifically bind to the antigen (e.g., anti-CD20 scFv14, and molecules that specifically bind to molecules containing the same sequence and cells presenting such molecules) is about 1 x 10 -7 Dissociation constant of M (K d In some embodiments, the antigen-binding molecule binds to an antigen (e.g., anti-CD20 scFv14, as well as molecules containing the same sequence and cells presenting such molecules) at a K d is about 1×10 -9 M ~ approx. 5×10 -9 In some embodiments, an antigen-binding molecule specifically binds with an antigen (e.g., anti-CD20 scFv14, as well as molecules containing the same sequence and cells presenting such molecules) with a K d is 1×10 -10 M ~ approx. 5×10 -10 When M, it binds specifically with "ultra-high affinity."

[0106] In yet another embodiment, molecules that specifically bind to an antigen (e.g., anti-CD20 scFv14, molecules comprising this sequence, and cells presenting such molecules) do not cross-react with other proteins under similar binding conditions. In some embodiments, molecules that specifically bind to an antigen (e.g., anti-CD20 scFv14, molecules comprising the same sequence, and cells presenting such molecules) do not cross-react with other proteins that do not include anti-CD20 scFv14, molecules comprising this sequence, and cells presenting such molecules. In some embodiments, provided herein are antibodies or fragments thereof that bind to anti-CD20 scFv14, molecules comprising the same sequence, and cells presenting such molecules with higher affinity than to another unrelated antigen. In certain embodiments, provided herein are antigen-binding molecules (e.g., antibodies) or fragments thereof that bind to anti-CD20 scFv14, as well as molecules comprising this sequence and cells presenting such molecules, with an affinity that is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher than the affinity for another unrelated antigen as measured by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In some embodiments, the degree of binding of an antigen-binding molecule, antibody, or antigen-binding fragment thereof that specifically binds to anti-CD20 scFv14 described herein, molecules comprising this sequence, and cells presenting such molecules, compared to an unrelated protein that does not contain anti-CD20 scFv14, molecules comprising this sequence, and cells presenting such molecules, is less than 10%, 15%, or 20% of the binding of the antibody to the linker fragment protein, as measured, for example, by radioimmunoassay.

[0107] As used herein, the term "heavy chain," when used in reference to an antibody, may refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which types give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0108] As used herein, the term "immunoglobulin" refers to an immune molecule derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. Many of the molecules described herein are immunoglobulins. As used herein, "isotype" refers to the antibody (e.g., IgM or IgG1) class or subclass encoded by heavy chain constant region genes.

[0109] Immunoglobulins are tetrameric molecules comprising two identical pairs of polypeptide chains, each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-130 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids. See generally, Berzofsky & Berkower, Ch. 7 in Fundamental Immunology (Paul, W., ed., Lippincott Williams & Wilkins (2012)), which chapter and volume are incorporated herein by reference in their entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact immunoglobulin has two primary binding sites.

[0110] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity-determining regions or "CDRs." From the N-terminus to the C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain can be made according to the Kabat definition (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991)) or the Chothia definition (Chothia as used herein) (see, e.g., Chothia & Lesk (1987), J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger & Pluckthun (2001), J. Mol. Biol. 309:657-670). The Kabat, Chothia, IGMT, and Abm (Oxford Molecular) numbering systems are described more fully herein.

[0111] As used herein, the term "in vitro cells" refers to any cells cultured ex vivo. In vitro cells can include human cells such as T cells or dendritic cells, or can include CHO, sP2 / 0, rabbit, and other non-human cells.

[0112] As used herein, the term "light chain," when used in reference to an antibody, may refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0113] The term "neutralizing" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand (e.g., a moiety comprising anti-CD20 scFv14, as well as molecules comprising this sequence and cells presenting such molecules) and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or otherwise alters the binding ability of the ligand by indirect means (such as a structural or energetic change within the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0114] As used herein, the term "patient" means any human being treated for an abnormal physiological condition such as cancer or formally diagnosed with a disorder, a human being without a formally recognized disorder, a human being receiving medical attention, a human being at risk for developing a disorder, etc. The terms "subject" and "patient" are used interchangeably herein and include both human and non-human animal subjects.

[0115] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. The term polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. The term "polypeptide" includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0116] In some embodiments, the polypeptides and / or proteins have deletions, additions, and / or substitutions of one or more amino acids of the antigen-binding molecule. Useful polypeptide fragments may include immunologically functional fragments of antigen-binding molecules, including, but not limited to, one or more CDR regions, heavy and / or light chain variable domains, portions of other portions of antibody chains, etc. Moieties that can be substituted for one or more amino acids of the antigen-binding molecule include, for example, D- or L-forms of amino acids, amino acids different from those normally found at the same position in antigen-binding molecules, deletions, non-naturally occurring amino acids, and chemical analogs of amino acids.

[0117] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide, and form an aspect of the present disclosure. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." See, e.g., Fauchere, (1986) Adv. Drug Res. (Testa, ed.) 15:29-69; Veber & Freidinger, (1985) TINS, p. 392; and Evans et al., (1987) J. Med. Chem, 30:1229-39, which are incorporated herein by reference for any purpose.

[0118] Polypeptides, peptides, proteins and similar molecules comprising anti-CD20 scFv14, as well as molecules comprising this sequence and cells presenting such molecules, are specifically encompassed by this term.

[0119] As used herein, the term "percent identity" means the percent of identical residues between amino acids or nucleotides in the compared molecules. For these calculations, gaps in the alignment, if any, must be addressed by a particular mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, ed.), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds.), 1994, New Jersey: Humana Press; von Heinje, (1987) Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov and Devereux, eds.), 1991, New York: M. Stockton Press; and Carillo et al., (1988) J. Applied Math. 48:1073.

[0120] When calculating percent identity, the sequences being compared are aligned in a way that maximizes the match between the sequences. Computer programs used to determine percent identity can be, for example, MOE (Chemical Computing Group) or DNASTAR (University of Wisconsin, Madison). The computer algorithm GAP can be used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. Sequences are aligned for optimal matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). A gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix being used, and "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (usually 1 / 10 of the gap opening penalty), as well as a comparison matrix such as PAM250 or BLOSUM62, are used with the algorithm. In certain embodiments, standard comparison matrices (e.g., for the PAM250 comparison matrix, see Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0121] Certain alignment schemes for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (e.g., the GAP program) can be adjusted, if desired, to result in an alignment spanning at least 50 consecutive amino acids of the target polypeptide.

[0122] As used herein, the terms "single-chain antibody" and "single-chain variable fragment (scFv)" are used interchangeably and refer to a V L and V H The domains are joined via a linker to form a continuous protein chain, where the linker is long enough to allow the protein chain to fold back on itself to form a monovalent antigen-binding site (see, e.g., Zuhaida Asra Ahmad, Swee Keong Yeap, Abdul Manaf Ali, Wan Yong Ho, Noorjahan Banu Mohamed Alitheen, Muhajir Hamid, "scFv Antibody: Principles and Clinical Application", Journal of Immunology Research, vol. 2012, article ID 980250, page 15, 2012, which is incorporated herein by reference in its entirety for all purposes). This refers to an antigen-binding molecule, and scFv14 is a specific example of an scFv.

[0123] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective administration amount" of a therapeutic agent (e.g., a moiety comprising anti-CD20 scFv14, as well as molecules comprising this sequence and cells presenting such molecules) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing the disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability resulting from disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0124] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Hartl and Jones (1997) "Genetics: Principles and Analysis," 4th ed., Jones & Bartlett). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0125] As used herein, the terms "variable region" and "variable domain" are used interchangeably and refer to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 100-130 amino acids of the mature heavy chain and approximately 90-115 amino acids of the mature light chain, which vary significantly in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its particular antigen. The sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen.

[0126] In certain embodiments, the variable region of the antigen-binding molecule is a human variable region. In further embodiments, the variable region comprises rodent, human, or mouse CDRs and human framework regions (FRs). In further embodiments, the variable region is a primate (e.g., non-human primate) variable region. In still further embodiments, the variable region is a rabbit variable region. In other embodiments, the variable region comprises human CDRs and non-human (e.g., rabbit, mouse, rat, or non-human primate) framework regions (FRs). In other embodiments, the variable region comprises non-human (e.g., rabbit, mouse, rat, or non-human primate) CDRs and human framework regions (FRs).

[0127] The terms "VH" and "VH domain," and "VH chain" are used interchangeably and refer to the heavy chain variable region of an antigen-binding molecule, antibody, or antigen-binding fragment thereof.

[0128] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antigen-binding molecule, antibody, or antigen-binding fragment thereof.

[0129] As used herein, the term "virus particle" refers to one or more complete virions, as well as any portion of one or more virions.

[0130] Various aspects of the invention are described in further detail in the following subsections. II. Overview

[0131] Immunotherapy using T cells engineered to express chimeric antigen receptors (CARs) has the potential to cure relapsed B-cell malignancies and has shown remarkable promise in the clinic. However, data from multiple clinical trials have identified a significant vulnerability of treatment with anti-CD19 CAR T cells: tumor cell susceptibility to antigen escape (i.e., downregulation or loss of detectable antigens on tumor cells), leading to tumor recurrence after treatment. For example, in a phase 1 / 2 clinical trial (ZUMA-1) of the autologous anti-CD19 CAR T cell product, axicabtagene ciloleucel, 39 of 88 responders (44%) subsequently relapsed after treatment. Of all patients with available post-relapse samples, 4 of 16 patients (25%) showed CD19-positive disease at diagnosis and CD19-negative disease after treatment. See, e.g., Locke et al., (2019) Lancet Oncol. 2019(1):31-42, and Neelapu et al., (2017) The New England journal of medicine 2017;377(26):2531-44, which are incorporated by reference in their entireties for any purpose. Additionally, patients receiving tisagenlecleucel, another anti-CD19 CAR T cell therapy, have suffered disease relapse caused by antigen loss. See, e.g., Maude et al., (2018) The New England Journal of Medicine 2018;378(5):439-48, and Maude et al., (2016) Journal of Clinical Oncology 2016;34(15_suppl):3011, which are incorporated by reference in their entireties for any purpose.

[0132] Non-clinical data demonstrate that a dual-targeting approach using CARs directed against two independent target cell surface antigens is more effective in vitro and in vivo compared to monovalent CAR T cells. See, e.g., Hegde et al., (2013) Mol. Ther. 2013;21(11):2087-101, Hegde et al., (2016) J. Clin. Invest 2016;126(8):3036-52, Ruella et al., (2018) Mol. Ther Oncolytics. 2018;11:127-37, and Zah et al., (2016) J. Clin. Invest Cancer Immunol. Res. 2016;4(6):498-508, each of which is incorporated by reference in its entirety for any purpose. Like CD19, CD20 is a cell surface antigen expressed on most healthy B cells, from pre-B cells to memory B cells, as well as on leukemia and lymphoma cells. Proof-of-concept targeting CD20 has been demonstrated in clinical settings in both monoclonal antibody therapy and CAR T cell therapy, and when combined with CD19 targeting, it may represent an effective strategy for reducing the likelihood of antigen escape. See, for example, Boye et al., (2003) Annals of Oncology 2003;14(4):520-35, Brudno et al., (2018) Nat. Rev. Clin. Oncol. 2018;15(1):31-46, and Zah et al., (2016) J. Clin. Invest Cancer Immunol. Res. 2016;4(6):498-508, the entire contents of which are incorporated herein by reference for any purpose.

[0133] Both single and dual antigen targeting anti-CD19 / CD20 CAR T cell therapies for the treatment of patients with relapsed or refractory B cell malignancies need to be well understood and characterized during the development and manufacturing process. More specifically, embodiments herein describe antibodies specific for scFv14 to characterize the specific protein expression of anti-CD20 CARs.

[0134] There is a further need for the detection and quantification of viral particles. In certain embodiments, detection of viral particles can be achieved by detection of viral envelope proteins, such as the gibbon ape leukemia virus (GALV) protein gp70. As disclosed herein, antigen binding molecules specific for GALV gp70 have many uses in assays, such as flow-based virus detection methods. III. Antigen-binding molecule: specific for anti-CD20 scFv14

[0135] The present disclosure relates to antigen-binding molecules, including antibodies that specifically bind to anti-CD20 scFv14, as well as molecules and cells that present such molecules and / or that cross-compete with one or more of the antigen-binding molecules described herein. A heavy chain antigen-binding molecule may comprise a unique set of CDR sequences as defined in Tables 3A, 3B, and 3C, and is exemplified by the provided light chain CDR1, CDR2, and CDR3 sequences listed in Tables 4A, 4B, and 4C. Related clones can be found in Tables 5 and 6. In various embodiments, an scFv form of an antigen-binding molecule may comprise a heavy chain binding molecule linked to a light chain binding molecule by a linker amino acid sequence (e.g., a "Whitlow" linker). Exemplary linker sequences are described herein.

[0136] In various embodiments, the antigen binding molecules described herein may be used in one or more methods (eg, methods described herein and methods in the art).

[0137] In various embodiments, an antigen-binding molecule may comprise one or more CDRs. In various embodiments, an antigen-binding molecule may comprise one or more framework regions. In various embodiments, an antigen-binding molecule may comprise three CDRs spaced apart between four framework regions.

[0138] In various embodiments, an antigen-binding molecule may comprise one or more CDRs incorporated into a variable heavy chain. In various embodiments, an antigen-binding molecule may comprise one or more CDRs incorporated into a variable light chain. In various embodiments, an antigen-binding molecule may comprise a variable heavy chain connected to a variable light chain by a linker.

[0139] In various embodiments, an antigen-binding molecule may comprise a light chain. In various embodiments, an antigen-binding molecule may comprise a heavy chain. In various embodiments, an antigen-binding molecule may comprise a light chain and a heavy chain connected by a disulfide bond. In various embodiments, an antigen-binding molecule may comprise a first heavy chain connected to a second heavy chain by a disulfide bond. In various embodiments, an antigen-binding molecule may comprise two light chains and two heavy chains.

[0140] The encoded antibody or antigen-binding molecule of the present disclosure can be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule may be single-chain. In certain embodiments, the antigen-binding molecule may be selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof. In one particular embodiment, the antibody or antigen-binding molecule may comprise an scFv.

[0141] In certain embodiments, an antigen-binding molecule such as an antibody may comprise a single chain, and the heavy chain variable region and the light chain variable region may be connected by a linker. H may be located at the N-terminus of the linker, and V L may be located at the C-terminus of the linker. L may be located at the N-terminus of the linker, and V H may be located at the C-terminus of the linker.

[0142] TIFF2026507879000004.tif82170

[0143] TIFF2026507879000005.tif118170

[0144] TIFF2026507879000006.tif114170

[0145] TIFF2026507879000007.tif72170

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[0148] TIFF2026507879000010.tif156170

[0149] TIFF2026507879000011.tif115170

[0150] In one embodiment, the antigen-binding molecules of the present disclosure are antibodies and antigen-binding fragments thereof. In one embodiment, antibodies specific for anti-CD20 scFvs of the present disclosure comprise at least one CDR set forth in Tables 3A-3C and 4A-4C. In another aspect, the present disclosure provides hybridomas capable of producing the antibodies disclosed herein, and methods of producing antibodies from hybridomas, as described herein and known in the art.

[0151] Humanized antibodies are described herein and can be prepared by known techniques. In one embodiment, a humanized monoclonal antibody comprises a variable domain (or all or part of its antigen-binding site) of a mouse or rabbit antibody and a constant domain derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen-binding site of a mouse or rabbit monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for producing engineered monoclonal antibodies include those described in Riechmann et al. (1988) Nature 332:323, Liu et al. (1987) Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al. (1989) Bio / Technology 7:934, and Winter et al. (1993) TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are described, for example, in U.S. Patent Nos. 5,869,619; 5,225,539, 5,821,337, 5,859,205, 6,881,557, Padlan et al., (1995) FASEB J. 9:133-39; Tamura et al., (2000) J. Immunol. 164:1432-41; Zhang et al., (2005) Mol. Immunol. 42(12):1445-1451; Hwang et al., Methods. (2005) 36(1):35-42; Dall'Acqua et al., (2005) Methods 36(1):43-60; and Clark, (2000) Immunology Today. 21(8):397-402.

[0152] The antigen-binding molecules of the present invention may also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any number of techniques familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (including, for example, B lymphocytes), in vitro immunization of human B cells, fusion of splenocytes from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other procedures known in the art and based on the disclosure herein.

[0153] Procedures for producing human monoclonal antibodies in non-human animals have been developed. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into mice to replace the inactivated mouse genes. In this technique, elements of human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci (see also Bruggemann et al., 1997) Curr. Opin. Biotechnol. 8:455-58).

[0154] Examples of techniques for the production and use of transgenic animals for the production of human or partially human antibodies are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al., Antibody Engineering: Methods and Protocols, (Lo, ed.) Humana Press, NJ, 191-200 (2003); Kellermann et al., (2002) Curr Opin Biotechnol. 13:593-97; Russell et al., (2000) Infect Immun. 68:1820-26; Gallo et al., (2000) Eur J. Immun. 30:534-40; Davis et al., (1999) Cancer Metastasis Rev. 18:421-25; Green, (1999) J Immunol Methods 231:11-23;Jakobovits,(1998)Advanced Drug Delivery Reviews 31:33-42;Green et al.,(1998)J Exp Med.188:483-95;Jakobovits,(1998)Exp.Opin.Invest.Drugs.7:607-14;Tsuda et al. al.,(1997)Genomics,42:413-21;Mendez et al.,(1997)Nat.Genet.15:146-56;Jakobovits,(1994)Curr Biol.4:761-63;Arbones et al.,(1994)Immunity 1:247-60;Green et al. al.,(1994)Nat.Genet.7:13-21;Jakobovits et al.,(1993)Nature 362:255-58;Jakobovits et al.,(1993)Proc Natl Acad Sci USA 90:2551-55;Chen et al.,(1993)Intl Immunol 5:647-656;Choi et al.,(1993)Nature Genetics 4:117-23;Fishwild et al.,(1996)Nature Biotechnology 14:845-51;Lonberg et al.,(1994)Nature 368:856-59;Lonberg,(1994)Handbook of Experimental Pharmacology 113:49-101;Neuberger,(1996)Nature Biotech 14:826;Taylor et al.,(1992)Nucleic Acids Research 20:6287-95;Taylor et al Proc Nat Acad Sci USA 97:722-27;Tomizuka et al.,(1997)Nature Genetics 16:133-43; 90:3720-24;Tuaillon et al.,(1994)J Immunol 152:2912-20;Lonberg et al.,(1994)Nature 368:856;Taylor et al.,(1994)Intl Immunol 6:579;Environment 5,877,397;Bruggemann et al.,(1997)Curr.Opin.Biotechnol.8:455-58;Jakobovits et al al.,(1995)Ann.NYacad.Sci.764:525-35.

[0155] Another method for obtaining the antigen-binding molecules of the present invention is by using phage display, which has been well established for this purpose. See, for example, Winter et al., (1994) Ann. Rev. Immunol. 12:433-55; Burton et al., (1994) Adv. Immunol 57:191-280. A combinatorial library of human or mouse immunoglobulin variable region genes can be generated in a phage vector that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind to scFv-14, as well as molecules containing this sequence and cells that display such molecules. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., (1989) Science 246:1275-81; Sastry et al., (1989) Proc. Natl. Acad. Sci. USA 86:5728-32; Alting-Mees et al., (1990) Strategies in Molecular Biology 3:1-9; Kang et al., (1991) Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., (1992) J. Mol. Biol. 227:381-388; Schlebusch et al., (1997) Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments can be inserted in frame with a sequence encoding a phage coat protein into the genome of a filamentous bacteriophage such as M13 or lambda phage (λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, CA) can also be used in this approach) or variants thereof.

[0156] Briefly, mRNA is isolated from a B cell population and used to generate heavy and light chain immunoglobulin cDNA expression libraries in λImmunoZap™(H) and λImmunoZap™(L) and similar vectors. These vectors can be screened individually or co-expressed to form Fab fragments or antibodies. Positive plaques can then be converted to non-lytic plasmids that allow high-level expression of monoclonal antibody fragments from E. coli.

[0157] In one embodiment, the variable regions of the genes expressing the monoclonal antibody of interest in a hybridoma are amplified using nucleotide primers. These primers can be synthesized by one skilled in the art or purchased from commercial sources, which also include, among others, V H area, V L area, C H Area, and C L Primers for mouse and human variable regions, including the V region, are commercially available. These primers can be used to amplify the heavy or light chain variable region, which can then be inserted into a vector. These vectors can then be introduced into E. coli, yeast, or mammalian systems for expression. H and V L Large amounts of single-chain proteins containing fusions of domains can be produced using these methods.

[0158] After cells producing the antigen-binding molecules provided herein are obtained using any of the above immunization and other techniques, the specific antibody genes can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein. The antibodies produced therefrom can be sequenced, the CDRs identified, and the DNA encoding the CDRs can be manipulated as previously described to generate other antibodies according to the invention.

[0159] Those skilled in the art will understand that some proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (e.g., lysine or arginine) by the action of carboxypeptidase (e.g., as described in Harris, (1995) J. Chromatog. 705:129-34).

[0160] An alternative method for producing murine monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, e.g., mice that have been treated (e.g., primed with pristane) to promote the formation of ascites containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Baines and Thorpe, (1992) in Methods in Molecular Biology, 10:79-104 (The Humana Press)). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on particular properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light or heavy chain) antibodies, and anti-idiotypic antibodies.

[0161] Although the disclosed antigen-binding molecules were produced in a murine system, human, partially human, or humanized antibodies may be suitable for many applications, particularly those involving administration of the antibodies to human subjects, and other types of antigen-binding molecules may be suitable for particular applications. Such antibodies can be prepared as described herein and form an aspect of the present disclosure.

[0162] The present disclosure provides antigen-binding molecules that specifically bind to the anti-CD20 scFv-14 binding domain and subsequences thereof, molecules comprising these sequences, and cells that present such molecules. Antigen-binding molecules that cross-compete with the antigen-binding molecules disclosed herein form another aspect of the present disclosure.

[0163] In some embodiments, an antibody or antigen-binding molecule that specifically binds to the anti-CD20 scFv-14 binding domain binds to the same or overlapping epitope as a reference antibody disclosed herein. In certain embodiments, the antibody or antigen-binding molecule binds to the same or overlapping epitope as the reference antibody. IV. Antigen-binding molecules: specific for GALV gp70 protein

[0164] The present disclosure further relates to antigen-binding molecules, including antibodies that specifically bind to GALV gp70 protein, as well as molecules and cells that display such molecules and / or that cross-compete with one or more of the antigen-binding molecules described herein. A heavy chain antigen-binding molecule may comprise a unique set of CDR sequences as defined in Tables 7A, 7B, and 7C, and is exemplified by the light chain CDR1, CDR2, and CDR3 sequences provided in Tables 8A, 8B, and 8C. Related clones can be found in Tables 9 and 10. In various embodiments, an scFv form of an antigen-binding molecule may comprise a heavy chain binding molecule linked to a light chain binding molecule by a linker amino acid sequence (e.g., a "Whitlow" linker). Exemplary linker sequences are described herein.

[0165] In various embodiments, the antigen binding molecules described herein may be used in one or more methods (eg, methods described herein and methods in the art).

[0166] In various embodiments, an antigen-binding molecule may comprise one or more CDRs. In various embodiments, an antigen-binding molecule may comprise one or more framework regions. In various embodiments, an antigen-binding molecule may comprise three CDRs spaced apart between four framework regions.

[0167] In various embodiments, an antigen-binding molecule may comprise one or more CDRs incorporated into a variable heavy chain. In various embodiments, an antigen-binding molecule may comprise one or more CDRs incorporated into a variable light chain. In various embodiments, an antigen-binding molecule may comprise a variable heavy chain connected to a variable light chain by a linker.

[0168] In various embodiments, an antigen-binding molecule may comprise a light chain. In various embodiments, an antigen-binding molecule may comprise a heavy chain. In various embodiments, an antigen-binding molecule may comprise a light chain and a heavy chain connected by a disulfide bond. In various embodiments, an antigen-binding molecule may comprise a first heavy chain connected to a second heavy chain by a disulfide bond. In various embodiments, an antigen-binding molecule may comprise two light chains and two heavy chains.

[0169] The encoded antibody or antigen-binding molecule of the present disclosure can be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule may be single-chain. In certain embodiments, the antigen-binding molecule may be selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof. In one particular embodiment, the antibody or antigen-binding molecule may comprise an scFv.

[0170] In certain embodiments, an antigen-binding molecule such as an antibody may comprise a single chain, and the heavy chain variable region and the light chain variable region may be connected by a linker. Hmay be located at the N-terminus of the linker, and V L may be located at the C-terminus of the linker. L may be located at the N-terminus of the linker, and V H may be located at the C-terminus of the linker.

[0171] TIFF2026507879000012.tif146170

[0172] TIFF2026507879000013.tif139170

[0173] TIFF2026507879000014.tif137170

[0174] TIFF2026507879000015.tif119170

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[0178] TIFF2026507879000019.tif157170

[0179] TIFF2026507879000020.tif89128

[0180] In one embodiment, the antigen-binding molecules of the present disclosure are antibodies and antigen-binding fragments thereof. In certain embodiments, the antibodies of the present disclosure comprise at least one CDR set forth in Tables 7A-7C and 8A-8C. In certain embodiments, the antibodies of the present disclosure comprise at least one heavy chain variable region from Table 9. In certain embodiments, the antibodies of the present disclosure comprise at least one light chain variable region from Table 10. In certain embodiments, the antibodies of the present disclosure comprise a combination of heavy and light chain variable regions set forth in Table 11. In another aspect, the present disclosure provides hybridomas capable of producing the antibodies disclosed herein, as well as methods of producing antibodies from hybridomas, as described herein and known in the art.

[0181] Humanized antibodies are described herein and can be prepared by known techniques. In one embodiment, a humanized monoclonal antibody comprises a variable domain (or all or part of its antigen-binding site) of a mouse or rabbit antibody and a constant domain derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen-binding site of a mouse or rabbit monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for producing engineered monoclonal antibodies include those described in Riechmann et al. (1988) Nature 332:323, Liu et al. (1987) Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al. (1989) Bio / Technology 7:934, and Winter et al. (1993) TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are described, for example, in U.S. Patent Nos. 5,869,619; 5,225,539, 5,821,337, 5,859,205, 6,881,557, Padlan et al., (1995) FASEB J. 9:133-39; Tamura et al., (2000) J. Immunol. 164:1432-41; Zhang et al., (2005) Mol. Immunol. 42(12):1445-1451; Hwang et al., Methods. (2005) 36(1):35-42; Dall'Acqua et al., (2005) Methods 36(1):43-60; and Clark, (2000) Immunology Today. 21(8):397-402.

[0182] The antigen-binding molecules of the present invention may also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any number of techniques familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (including, for example, B lymphocytes), in vitro immunization of human B cells, fusion of splenocytes from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other procedures known in the art and based on the disclosure herein.

[0183] Procedures for producing human monoclonal antibodies in non-human animals have been developed. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into mice to replace the inactivated mouse genes. In this technique, elements of human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci (see also Bruggemann et al., 1997) Curr. Opin. Biotechnol. 8:455-58).

[0184] Examples of techniques for the production and use of transgenic animals for the production of human or partially human antibodies are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al., Antibody Engineering: Methods and Protocols, (Lo, ed.) Humana Press, NJ, 191-200 (2003); Kellermann et al., (2002) Curr Opin Biotechnol. 13:593-97; Russell et al., (2000) Infect Immun. 68:1820-26; Gallo et al., (2000) Eur J. Immun. 30:534-40; Davis et al., (1999) Cancer Metastasis Rev. 18:421-25; Green, (1999) J Immunol Methods 231:11-23;Jakobovits,(1998)Advanced Drug Delivery Reviews 31:33-42;Green et al.,(1998)J Exp Med.188:483-95;Jakobovits,(1998)Exp.Opin.Invest.Drugs.7:607-14;Tsuda et al. al.,(1997)Genomics,42:413-21;Mendez et al.,(1997)Nat.Genet.15:146-56;Jakobovits,(1994)Curr Biol.4:761-63;Arbones et al.,(1994)Immunity 1:247-60;Green et al. al.,(1994)Nat.Genet.7:13-21;Jakobovits et al.,(1993)Nature 362:255-58;Jakobovits et al.,(1993)Proc Natl Acad Sci USA 90:2551-55;Chen et al.,(1993)Intl Immunol 5:647-656;Choi et al.,(1993)Nature Genetics 4:117-23;Fishwild et al.,(1996)Nature Biotechnology 14:845-51;Lonberg et al.,(1994)Nature 368:856-59;Lonberg,(1994)Handbook of Experimental Pharmacology 113:49-101;Neuberger,(1996)Nature Biotech 14:826;Taylor et al.,(1992)Nucleic Acids Research 20:6287-95;Taylor et al Proc Nat Acad Sci USA 97:722-27;Tomizuka et al.,(1997)Nature Genetics 16:133-43; 90:3720-24;Tuaillon et al.,(1994)J Immunol 152:2912-20;Lonberg et al.,(1994)Nature 368:856;Taylor et al.,(1994)Intl Immunol 6:579;Environment 5,877,397;Bruggemann et al.,(1997)Curr.Opin.Biotechnol.8:455-58;Jakobovits et al al.,(1995)Ann.NYacad.Sci.764:525-35.

[0185] Another method for obtaining the antigen-binding molecules of the present invention is by using phage display, which has been well established for this purpose. See, for example, Winter et al., (1994) Ann. Rev. Immunol. 12:433-55; Burton et al., (1994) Adv. Immunol 57:191-280. A combinatorial library of human or mouse immunoglobulin variable region genes can be generated in a phage vector that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind to scFv-14, as well as molecules containing this sequence and cells that display such molecules. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., (1989) Science 246:1275-81; Sastry et al., (1989) Proc. Natl. Acad. Sci. USA 86:5728-32; Alting-Mees et al., (1990) Strategies in Molecular Biology 3:1-9; Kang et al., (1991) Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., (1992) J. Mol. Biol. 227:381-388; Schlebusch et al., (1997) Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments can be inserted in frame with a sequence encoding a phage coat protein into the genome of a filamentous bacteriophage such as M13 or lambda phage (λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, CA) can also be used in this approach) or variants thereof.

[0186] Briefly, mRNA is isolated from a B cell population and used to generate heavy and light chain immunoglobulin cDNA expression libraries in λImmunoZap™(H) and λImmunoZap™(L) and similar vectors. These vectors can be screened individually or co-expressed to form Fab fragments or antibodies. Positive plaques can then be converted to non-lytic plasmids that allow high-level expression of monoclonal antibody fragments from E. coli.

[0187] In one embodiment, the variable regions of the genes expressing the monoclonal antibody of interest in a hybridoma are amplified using nucleotide primers. These primers can be synthesized by one skilled in the art or purchased from commercial sources, which also include, among others, V H area, V L area, C H Area, and C L Primers for mouse and human variable regions, including the V region, are commercially available. These primers can be used to amplify the heavy or light chain variable region, which can then be inserted into a vector. These vectors can then be introduced into E. coli, yeast, or mammalian systems for expression. H and V L Large amounts of single-chain proteins containing fusions of domains can be produced using these methods.

[0188] After cells producing the antigen-binding molecules provided herein are obtained using any of the above immunization and other techniques, the specific antibody genes can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein. The antibodies produced therefrom can be sequenced, the CDRs identified, and the DNA encoding the CDRs can be manipulated as previously described to generate other antibodies according to the invention.

[0189] Those skilled in the art will understand that some proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (e.g., lysine or arginine) by the action of carboxypeptidase (e.g., as described in Harris, (1995) J. Chromatog. 705:129-34).

[0190] An alternative method for producing murine monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, e.g., mice that have been treated (e.g., primed with pristane) to promote the formation of ascites containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Baines and Thorpe, (1992) in Methods in Molecular Biology, 10:79-104 (The Humana Press)). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on particular properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light or heavy chain) antibodies, and anti-idiotypic antibodies.

[0191] Although the disclosed antigen-binding molecules were produced in a murine system, human, partially human, or humanized antibodies may be suitable for many applications, particularly those involving administration of the antibodies to human subjects, and other types of antigen-binding molecules may be suitable for particular applications. Such antibodies can be prepared as described herein and form an aspect of the present disclosure.

[0192] The present disclosure provides antigen-binding molecules that specifically bind to the GALV gp70 protein and subsequences thereof, molecules comprising these sequences, and cells that present such molecules. Antigen-binding molecules that cross-compete with the antigen-binding molecules disclosed herein form another aspect of the present disclosure.

[0193] In some embodiments, an antibody or antigen-binding molecule that specifically binds to a GALV gp70 protein binding domain binds to the same or overlapping epitope as a reference antibody disclosed herein. In certain embodiments, the antibody or antigen-binding molecule binds to the same or overlapping epitope as the reference antibody. a) Antibodies

[0194] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy chains (HC) and two light chains (LC), which may be interconnected by disulfide bonds or antigen-binding molecules. Each HC chain contains a heavy chain variable region (V H ) and a heavy chain constant region (CH). The heavy chain constant region may contain three constant domains, CH1, CH2, and CH3. Each LC chain contains a light chain variable region (V L ) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. H Area and V L The regions can be further subdivided into hypervariable regions called "complementarity determining regions" (CDRs), separated by more conserved regions called "framework regions" (FRs). H and V Lcontains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0195] In some embodiments, the antigen-binding molecules of the present disclosure specifically bind to at least a portion of scFv-14, molecules comprising the same or a related sequence, and cells presenting such molecules. In certain embodiments, the antigen-binding molecules of the present disclosure specifically bind to at least a portion of scFv-14, molecules comprising the same or a similar sequence, and cells presenting such molecules at a concentration of 1x10 -6 Under M, 1x10 -7 Under M, 1x10 -8 Less than M or 1x10 -9 K less than M D In one specific embodiment, the antigen-binding molecule specifically binds to at least a portion of scFv-14, as well as molecules comprising these sequences and cells presenting such molecules, at a concentration of 1x10 -7 K less than M D In another embodiment, the antigen-binding molecule specifically binds to scFv-14, as well as molecules having the same or similar sequences, and cells presenting such molecules, at a concentration of 1x10 -8 K less than M D In some embodiments, the antigen-binding molecule specifically binds to scFv-14, as well as molecules comprising the same or similar sequence, and cells presenting such molecules, at a concentration of about 1 x 10 -7 M, approx. 2x10 -7 M, about 3x10 -7 M, approx. 4x10 -7 M, about 5x10 -7 M, about 6x10 -7 M, about 7x10 -7 M, approx. 8x10 -7 M, approx. 9x10 -7 M, about 1x10 -8 M, approx. 2x10-8 M, about 3x10 -8 M, approx. 4x10 -8 M, about 5x10 -8 M, about 6x10 -8 M, about 7x10 -8 M, approx. 8x10 -8 M, approx. 9x10 -8 M, about 1x10 -9 M, approx. 2x10 -9 M, about 3x10 -9 M, approx. 4x10 -9 M, about 5x10 -9 M, about 6x10 -9 M, about 7x10 -9 M, approx. 8x10 -9 M, approx. 9x10 -9 M, about 1x10 -10 M, or approximately 5x10 -10 K of M D It binds specifically at K D can be calculated using standard methodologies as described herein and elsewhere in the art.

[0196] In specific embodiments, the antigen-binding molecules of the present disclosure are identified in Tables 3-4 and comprise the identified heavy and light chain amino acid sequences set forth in those tables, respectively. b) ScFv

[0197] In various embodiments, the scFv is a V of an immunoglobulin or analog. H and V L In various embodiments, the V H and V L may be connected by a linker. In various embodiments, the scFv does not contain the constant regions normally present in an antibody.

[0198] In various embodiments, scFvs facilitate phage display, which allows convenient expression of the antigen-binding domain as a single peptide. In other embodiments, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas. scFvs can be used for a variety of different purposes. In various embodiments, scFvs can be incorporated into flow cytometry and immunohistochemistry diagnostic assays. In other embodiments, scFvs can be used as the antigen-binding domain of artificial T cell receptors (chimeric antigen receptors). c) Linker

[0199] When used in biotechnological and biotherapeutic applications, linker sequences can be peptide-based and can serve a range of scientifically relevant purposes. For example, linkers can be used simply as spacer moieties to impart desired structural and / or functional properties to a larger molecule. In another example, linkers can impart little or no structural or functional properties to a larger molecule, but can simply be used as a distinguishing feature (e.g., a "marker" or "biomarker" or "tag") that uniquely identifies the larger molecule. In yet another example, linkers can be used to impart a recognizable feature that can serve as a binding site for an antibody to the larger molecule containing the linker sequence.

[0200] In various embodiments, the linker can comprise a sequence of amino acids that form a peptide. For example, the linker can comprise a peptide sequence of about 20 to about 30 amino acids. In some embodiments, the linker can comprise a peptide sequence of about 25 amino acids. In some embodiments, the linker peptide comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids. In some embodiments, the linker comprises about 8 amino acids to about 18 amino acids (e.g., 10 amino acids).

[0201] In various embodiments, the linker can include a flexible portion. For example, the flexible portion can be rich in glycine residues. In various embodiments, the linker can be soluble or partially soluble. For example, the linker can include one or more serine and / or threonine residues. In various embodiments, the linker can be V H N-terminus of V L In other embodiments, the linker may be connected to the C-terminus of V H The C-terminus of V L It can be connected to the N-terminus of

[0202] When a linker sequence is used as a distinguishing, detectable, or identifiable feature of a larger molecule, an antibody that specifically binds to the linker sequence to the exclusion of other sequences present in the larger molecule can serve as a detection agent. Such antibodies can be labeled with a moiety that is detectable under certain conditions. Additional uses for such antibodies include purifying and isolating molecules containing the linker, characterizing molecules in specific settings, enriching a population of molecules containing and / or displaying the linker, and therapeutic applications.

[0203] In 1993, Whitlow et al. disclosed a synthetic linker peptide containing the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 121) (Whitlow et al., 1993) Prot. Eng. 6(8):989-95). The disclosed peptide was studied as a component of scFv and was designed to remove proteolytic sites identified in previous linker peptides. Whitlow et al. concluded that this newly designed synthetic linker peptide was more stable to proteolysis in vitro compared to the previous linker peptide on which its sequence was based and also showed less aggregation compared to the same previous linker. Whitlow et al. did not disclose antigen-binding molecules based on second-generation linker peptides.

[0204] In various embodiments, a "Whitlow" linker sequence may be included in the scFv antigen-binding molecules described herein. In various embodiments, the linker sequence may be included in a larger amino acid sequence that also includes heavy and light chain amino acid sequences.

[0205] There are various other linkers available in the art that may be suitable. One example is the "G4S linker" having the sequence: (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 126). In various embodiments, the linker may include SEQ ID NO: 126 and one or more additional Gly residues. In various embodiments, the linker may include SEQ ID NO: 126 and one or less Gly residues. The linker may vary in length based on the number of Gly residues. V. Polynucleotides Encoding Antibodies and Antigen-Binding Molecules Anti-CD20 scFv-14 binding molecule

[0206] In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 1 to 120). In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 1 to 10). In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 11 to 20).

[0207] In some embodiments, the polynucleotides of the present invention encode antigen-binding molecules comprising a heavy chain variable region amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10.

[0208] In some embodiments, a polynucleotide of the present invention encodes an antigen-binding molecule, and the antigen-binding molecule comprises a light chain variable amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 20.

[0209] It will be understood by those skilled in the art that due to the degeneracy of the genetic code, variations in the disclosed polynucleotide sequences are possible, and therefore, such variants of the disclosed polynucleotide sequences form an aspect of the present disclosure. GALV gp70 binding molecule

[0210] In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 127-324). In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 303-314). In various embodiments, the polynucleotide may encode an antigen-binding molecule (SEQ ID NOs: 315-324).

[0211] In some embodiments, the polynucleotides of the present invention encode antigen-binding molecules comprising a heavy chain variable region amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 303 to 314.

[0212] In some embodiments, a polynucleotide of the present invention encodes an antigen-binding molecule, and the antigen-binding molecule comprises a light chain variable amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 315 to 324.

[0213] It will be understood by those skilled in the art that due to the degeneracy of the genetic code, variations in the disclosed polynucleotide sequences are possible, and therefore, such variants of the disclosed polynucleotide sequences form an aspect of the present disclosure.

[0214] TIFF2026507879000021.tif218170TIFF2026507879000022.tif210170

[0215] TIFF2026507879000023.tif201170TIFF2026507879000024.tif140170VI.Vector, Cell, and Pharmaceutical Composition

[0216] In certain aspects, provided herein are vectors comprising the polynucleotides of the present disclosure. In some embodiments, the present invention relates to a vector or a set of vectors comprising a polynucleotide encoding an antibody or antigen-binding molecule that specifically binds to anti-CD20 scFv-14 (SEQ ID NOs: 1-120). In some embodiments, the present invention relates to a vector or a set of vectors comprising a polynucleotide encoding an antibody or antigen-binding molecule that specifically binds to GALV gp70 (SEQ ID NOs: 127-324).

[0217] Any vector known in the art may be suitable for expressing the antibodies and antigen-binding molecules of the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0218] In another aspect, provided herein are cells comprising the polynucleotides or vectors of the invention. In some embodiments, the present invention relates to cells, including in vitro cells, comprising polynucleotides encoding the antigen-binding molecules described herein. In some embodiments, the present invention relates to cells, e.g., in vitro cells, comprising polynucleotides encoding antibodies or antigen-binding molecules thereof that specifically bind to anti-CD20 scFv-14 or GALV gp70 protein, as disclosed herein, molecules comprising these sequences, and cells that present such molecules.

[0219] Any cell can be used as a host cell for polynucleotides and vectors encoding all or fragments of the antibodies and antigen-binding molecules of the present invention. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Escherichia, e.g., Escherichia, e.g., E. coli; Bacilli, e.g., B. subtilis and B. licheniformis; Pseudomonas, e.g., P. aeruginosa; and Streptomyces. In some embodiments, the host cell is a mammalian cell, e.g., a human cell. In some embodiments, the host cell is a CHO cell, and in other embodiments, the host cell is an sP2 / 0 or other mouse cell. The host cells of the present invention can be obtained from any source known in the art.

[0220] Other aspects of the present disclosure may be directed to compositions comprising the polynucleotides described herein, the vectors described herein, the antibodies, antigen-binding molecules described herein, and / or the in vitro cells described herein. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the compositions comprise an excipient.

[0221] In one embodiment, the composition comprises polynucleotides encoding antibodies or antigen-binding molecules that specifically bind to anti-CD20 scFV-14 or GALV gp70 protein, molecules comprising these sequences, and cells that present such molecules. In another embodiment, the composition comprises in vitro cells comprising polynucleotides encoding antibodies or antigen-binding molecules thereof encoded by the polynucleotides disclosed herein.

[0222] In some embodiments, a composition comprises two or more different antibodies or antigen-binding molecules that specifically bind to anti-CD20 scFv-14 or GALV gp70 protein, molecules comprising these sequences, and cells presenting such molecules. In some embodiments, a composition comprises two or more antibodies or antigen-binding molecules that specifically bind to anti-CD20 scFv-14 or GALV gp70 protein, molecules comprising these sequences, and cells presenting such molecules, wherein the antibodies or antigen-binding molecules bind to two or more epitopes. In some embodiments, the antibodies or antigen-binding molecules do not compete with each other for binding to their epitopes. In some embodiments, two or more of the antibodies or antigen-binding molecules provided herein are combined together in a pharmaceutical composition. Preferably, such compositions are suitable for administration to subjects, including humans. VII. Exemplary Methods

[0223] The following sections describe various exemplary methods of using the antigen-binding molecules disclosed herein. Any of the antigen-binding molecules and fragments thereof disclosed herein, including those set forth in the tables, figures, and accompanying sequence listing, can be used in the disclosed methods.

[0224] T cells can be used in some of the disclosed methods. Such T cells can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0225] In various embodiments, the antigen-binding molecule specifically binds to at least a portion of molecules comprising scFv-14 and / or similar sequences and cells displaying such sequences. In various further embodiments, the antigen-binding molecule specifically binds to at least a portion of molecules comprising GALV gp70 protein and / or similar sequences and virus particles displaying such sequences. In various embodiments, the antigen-binding molecule may comprise one or more of: (a) light chain CDR1, (b) light chain CDR2, (c) light chain CDR3, (d) heavy chain CDR1, (e) heavy chain CDR2, and (f) heavy chain CDR3. In various embodiments, the light chain and heavy chain may be connected by a linker.

[0226] In various embodiments, the antigen-binding molecule may comprise a variable heavy chain. In various embodiments of the anti-CD20 scFv-14 binding molecule, the variable heavy chain variable region may comprise one of SEQ ID NOs: 1-10. In various embodiments, the claimed antigen-binding molecules disclosed herein (e.g., V sequences comprising SEQ ID NOs: 1-10) may be used. H V of an antigen-binding molecule containing the sequence HV is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to H Any antigen-binding molecule comprising the amino acid sequence can be used.

[0227] In various embodiments, the antigen-binding molecule may comprise a variable heavy chain. In various embodiments of the GALV gp70-binding molecule, the variable heavy chain variable region may comprise one of SEQ ID NOs: 303-314. In various embodiments, the claimed antigen-binding molecules disclosed herein (e.g., V sequences comprising SEQ ID NOs: 303-314) may be used. H V of an antigen-binding molecule containing the sequence H V is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to H Any antigen-binding molecule comprising the amino acid sequence can be used.

[0228] In various embodiments of the anti-CD20 scFv-14 binding molecule, the variable heavy chain may comprise one of CDR1, wherein CDR1 comprises one of SEQ ID NOs: 21-41. In various embodiments, the heavy chain may comprise one of CDR2, wherein CDR2 comprises one of SEQ ID NOs: 42-65. In various embodiments, the heavy chain may comprise one of CDR3, wherein CDR3 comprises one of SEQ ID NOs: 66-85.

[0229] In various embodiments of the GALV gp70 binding molecule, the variable heavy chain can comprise one of CDR1, wherein CDR1 comprises one of SEQ ID NOs: 127-138, 157-168, and 187-198. In various embodiments, the heavy chain can comprise one of CDR2, wherein CDR2 comprises one of SEQ ID NOs: 139-150, 169-180, and 199-210. In various embodiments, the heavy chain can comprise one of CDR3, wherein CDR3 comprises one of SEQ ID NOs: 151-156, 181-186, 211-222, and DYY.

[0230] In various embodiments of the anti-CD20 scFv-14 binding molecule, the light chain may comprise one of SEQ ID NOs: 11-20. In various embodiments, the claimed antigen-binding molecules disclosed herein (e.g., Vs comprising SEQ ID NOs: 11-20) may be used. L V of an antigen-binding molecule containing the sequence L V is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to L Any antigen-binding molecule comprising the amino acid sequence can be used.

[0231] In various embodiments of the GALV gp70 binding molecule, the light chain may comprise one of SEQ ID NOs: 315-324. In various embodiments, the claimed antigen binding molecules disclosed herein (e.g., V comprising SEQ ID NOs: 315-324) may be used. L V of an antigen-binding molecule containing the sequence L V is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to L Any antigen-binding molecule comprising the amino acid sequence can be used.

[0232] In various embodiments of the anti-CD20 scFv-14 binding molecule, the variable light chain may comprise one of CDR1, wherein CDR1 comprises one of SEQ ID NOs: 86-99. In various embodiments, the light chain may comprise one of CDR2, wherein CDR2 comprises one of SEQ ID NOs: 100-111. In various embodiments, the light chain may comprise one of CDR3, wherein CDR3 comprises one of SEQ ID NOs: 112-120.

[0233] In various embodiments of the GALV gp70 binding molecule, the variable light chain can comprise one of CDR1, wherein CDR1 comprises one of SEQ ID NOs: 223-232, 253-262, and 283-292. In various embodiments, the light chain can comprise one of CDR2, wherein CDR2 comprises one of SEQ ID NOs: 233-242, 263-272, SGS, GTN, RAS, DTS, and KVS. In various embodiments, the light chain can comprise one of CDR3, wherein CDR3 comprises one of SEQ ID NOs: 243-252, 273-282, and 293-302.

[0234] In various embodiments, the variable light chain may be linked to the variable heavy chain by a linker (e.g., a Whitlow linker).

[0235] In further embodiments of the disclosed methods, the antigen-binding molecule comprises one or more of: (a) light chain CDR1, (b) light chain CDR2, (c) light chain CDR3, (d) heavy chain CDR1, (e) heavy chain CDR2, and (f) heavy chain CDR3 (SEQ ID NOs: 21-120).

[0236] In various embodiments of the disclosed methods, the antigen-binding molecule of anti-CD20 scFv-14 comprises a heavy chain (HC), and the HC can comprise a heavy chain variable region (VH) sequence comprising one of SEQ ID NOs: 1 to 10. Furthermore, embodiments of the disclosed methods can use antigen-binding molecules comprising a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VH) sequence comprising one of SEQ ID NOs: 1 to 10).

[0237] In various embodiments of the disclosed methods, the antigen-binding molecule of GALV gp70 comprises a heavy chain (HC), and the HC can comprise a heavy chain variable region (VH) sequence comprising one of SEQ ID NOs: 303 to 314. Furthermore, embodiments of the disclosed methods can use antigen-binding molecules comprising a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VH) sequence comprising one of SEQ ID NOs: 303 to 314).

[0238] In various embodiments of the disclosed methods, the antigen-binding molecule of anti-CD20 scFv-14 comprises a light chain (LC), and the LC can comprise a light chain variable region (LH) sequence comprising one of SEQ ID NOs: 11 to 20. In various embodiments of the disclosed methods, the light chain comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3. Furthermore, embodiments of the disclosed methods can employ antigen-binding molecules comprising a VL amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VL) of one of the sequences comprising SEQ ID NOs: 11 to 20).

[0239] In various embodiments of the disclosed methods, the antigen-binding molecule of GALV gp70 comprises a light chain (LC), and the LC can comprise a light chain variable region (LH) sequence comprising one of SEQ ID NOs: 315 to 324. In various embodiments of the disclosed methods, the light chain comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3. Furthermore, embodiments of the disclosed methods can employ antigen-binding molecules comprising a VL amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VL) of one of the sequences comprising SEQ ID NOs: 315 to 324).

[0240] In certain embodiments of the disclosed methods, the variable heavy chain comprises SEQ ID NO:5.

[0241] In certain embodiments of the disclosed methods, the variable light chain comprises SEQ ID NO:15.

[0242] In a specific embodiment of the disclosed methods, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0243] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0244] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0245] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; or an HCDR according to any one of SEQ ID NOs: 129, 159, and 189. R1; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; HCDR3 according to SEQ ID NO: 214 or DYY; HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR3 according to SEQ ID NO: 218 or DYY; HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY;HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197; HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209; HCDR3 according to SEQ ID NO: 221 or DYY; or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198; HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210; HCDR3 according to SEQ ID NO: 222 or DYY.

[0246] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0247] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0248] Given the above description of antigen-binding molecules that can be used in the disclosed methods, representative methods will now be discussed in more detail. a) Methods for determining the number of cells or viral particles displaying a molecule of interest

[0249] There are situations in which it may be desirable to determine the number of cells present in a sample that express a molecule of interest. For example, it may be desirable to determine the number of immune cells present in a sample obtained from a subject that express a molecule of interest. Alternatively, it may be desirable to determine the number of transfected cells that express a molecule of interest, which can be used as a measure of the level of transfection efficiency. The disclosed method can be used in these and other applications in which it is desirable to determine the number of cells present in a sample that express a molecule of interest.

[0250] Accordingly, there is provided a method for determining the number of cells in a sample that present a molecule, wherein the molecule comprises an amino acid sequence selected from the group consisting of any one or more of the amino acid sequences set forth in Tables 3A-3C, 4A-4C or 7A-7C, 8A-8C, 9-10.

[0251] In one embodiment, a sample is provided that contains cells known to express or suspected to express a molecule of interest comprising an amino acid sequence selected from the group consisting of any of the amino acid sequences described herein.

[0252] In certain embodiments, the selected amino acid sequence is QVQLQQSGAELMKPGASVKLSCKATGHTFTGYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTTEDSAIYYCAREGFAYWGQGTLVTVSA (SEQ ID NO: 5). In other embodiments, the selected amino acid sequence is DIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK (SEQ ID NO: 15). In other embodiments, the selected amino acid sequence is selected from (SEQ ID NOs: 25, 46, 70, 32, 54, 39, 62, 80, 90, 98, 104, 116), or any combination.

[0253] In a particular embodiment, the selected amino acid sequence is (SEQ ID NO: 325).

[0254] The cells can be of any type and can be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In a preferred embodiment, the cells are immune cells. The immune cells of the present method can be any type of immune cell (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). T cells (including T cytotoxic, T helper, and Treg cells) are particularly preferred. In a specific embodiment, the cells are T cells, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be used in this embodiment of the disclosed method. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the disclosed methods, the T cells can be in vivo T cells or in vitro T cells. Furthermore, the cells can be placed in or isolated from any environment in which the cells can be maintained in viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, tissue grown ex vivo, a suitable buffer, etc.

[0255] In a specific embodiment, the molecule of interest is a CAR. When the molecule is a CAR, it can be any of CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD49g (ITGA7), CD49h (ITGA8), CD49i (ITGA9), CD49m (ITGA10), CD49m (ITGA11), CD49m (ITGA12), CD49m (ITGA13), CD49m (ITGA14), CD49m (ITGA15), CD49m (ITGA16), CD49m (ITGA17), CD49m (ITGA18), CD49m (ITGA19 ... ), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR 2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2 ), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF1) 4), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6) , CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta,The antibody may comprise a molecule or fragment thereof selected from the group consisting of IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a combination thereof.

[0256] The sample is then contacted with an antigen-binding molecule that specifically binds to a molecule of interest and comprises a detectable label under conditions that allow the formation of a binding complex comprising cells present in the sample and the antigen-binding molecule. The antigen-binding molecule is preferably an antigen-binding molecule (or a fragment thereof) disclosed herein, for example, in the figures, sequence listing, or this section of the disclosure. Any antigen-binding molecule that specifically binds to all or a portion of an anti-CD20 scFv-14 molecule can be used in the disclosed methods. Several examples of suitable antigen-binding molecules are provided herein, including, for example, those having one or more CDRs set forth in any of the tables presented herein, such as Tables 3A-3C, 4A-4C, 7A-7C, and 8A-8C.

[0257] Any detectable label can be used in the present methods, and an appropriate label can be selected using a desired set of criteria. Examples of types of detectable labels include Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarin, aminocouramin, methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, and the like. Red), allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTruquoise 2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira OrangeOrange), mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, peridinin chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Other types of detectable labels include optical dyes, radioactive labels (e.g., as described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies (2010)). 3 H, 11 C. 14 C. 15 N, 18 F, 35 S, 64 CU, 90 Y, 99 Tc, 111 In, 124 I, 125 I, 131 I), photochromic compounds, magnetic labels (e.g., DYNABEADS), and the like, which are expressly incorporated herein by reference. Strategies for labeling proteins are known in the art and can be used in the disclosed methods.

[0258] The label can be associated with the antigen-binding molecule at any position in the molecule, but it is preferable to associate the label with the molecule at a position (or multiple positions, if multiple labels are used) that does not alter the binding properties of the molecule (unless such altered binding activity is desired). Any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of a molecule of interest, including an anti-CD20 scFv-14 molecule, can be used in the disclosed methods. In another aspect, any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of a molecule of interest, including a GALV gp70 molecule, can be used in the disclosed methods.

[0259] In a specific embodiment of the disclosed methods, with respect to an anti-CD20 scFv-14 molecule, the antigen-binding molecule comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0260] In a specific embodiment of the disclosed methods, with respect to an anti-CD20 scFv-14 molecule, the antigen-binding molecule comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0261] In a specific embodiment of the disclosed methods, with respect to an anti-CD20 scFv-14 molecule, the antigen-binding molecule comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0262] In certain embodiments of the disclosed methods, GALV With respect to the gp70 binding molecule, HCDR1 has a sequence according to any one of SEQ ID NOs: 127-138, 157-168, and 187-198, HCDR2 has a sequence according to any one of SEQ ID NOs: 139-150, 169-180, and 199-210, HCDR3 has a sequence according to any one of SEQ ID NOs: 151-156, 181-186, 211-222, and DYY, LCDR1 has a sequence according to any one of SEQ ID NOs: 223-232, 253-262, and 283-292, LCDR2 has a sequence according to any one of SEQ ID NOs: 233-242, 263-272, SGS, GTN, RAS, DTS, and KVS, and LCDR3 has a sequence according to any one of SEQ ID NOs: 243-252, 273-282, and 293-302.

[0263] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212, an HCDR4 according to any one of SEQ ID NOs: 129, 159, and 189, R1, HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and any one of SEQ ID NOs: 153, 183, and 215 an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206, HCDR3 according to SEQ ID NO: 218 or DYY, HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219, HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196, HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208, HCDR3 according to SEQ ID NO: 220 or DYY,HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, HCDR3 according to SEQ ID NO: 221 or DYY, or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, HCDR3 according to SEQ ID NO: 222 or DYY.

[0264] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0265] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0266] The antigen-binding molecule can be placed on any surface, or no surface at all. For example, the antigen-binding molecule can be present in a buffer solution, and the buffer solution and antigen-binding molecule can be contacted with the sample. Alternatively, the antigen-binding molecule can be associated with a surface. Suitable surfaces include agarose beads, magnetic beads such as DYNABEADS™, or plastic, glass, or ceramic plates such as well-equipped plates, bags such as cell culture bags, etc. The surface itself can be placed in another structure, such as a column.

[0267] Conditions that allow for the formation of binding complexes will depend on various factors, but generally, aqueous buffers of physiological pH and ionic strength, such as phosphate-buffered saline (PBS), favor the formation of binding complexes and are preferred in the disclosed methods.

[0268] The number of cells present in the binding complex in the sample is then determined. The specific method used to determine the number of cells present in the binding complex depends on the nature of the label selected. For example, if a fluorescent label is selected, FACS can be used. If an isotopic label is selected, mass spectrometry, NMR, or other techniques can be used. If a magnetic label is selected, magnetic-based cell sorting can be used. Microscopy can also be used. The output of these detection methods can be in the form of cell number, or the output can be in a form that allows for the calculation of the number of cells based on the output. b) A method for determining the presence or absence of a molecule

[0269] It is valuable to have the ability to separate populations of different molecules, particularly biologically related molecules, from one another. The antigen-binding molecules provided herein can be used to achieve such separation and can be used in a range of biotechnological, biopharmaceutical, and therapeutic applications.

[0270] In one embodiment of the present disclosure, a method is provided for isolating a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule. In another embodiment of the present disclosure, a method is provided for isolating a molecule comprising one or more of the amino acid sequences set forth in Tables 3A-3C and 4A-4C.

[0271] In a further aspect of the disclosure, methods are provided for isolating molecules comprising all or a portion of a GALV gp70 molecule. In another aspect of the disclosure, methods are provided for isolating molecules comprising a molecule comprising one or more of the amino acid sequences set forth in Tables 7A-7C and 8A-8C.

[0272] In one embodiment, the method comprises providing a sample known or suspected to contain all or a portion of an anti-CD20 scFv-14 molecule, hi one embodiment, the method comprises providing a sample known or suspected to contain all or a portion of a GALV gp70 molecule.

[0273] In one embodiment, the method involves providing a sample known or suspected to contain one or more amino acid sequences of interest.

[0274] In certain embodiments, the selected amino acid sequence is QVQLQQSGAELMKPGASVKLSCKATGHTFTGYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTTEDSAIYYCAREGFAYWGQGTLVTVSA (SEQ ID NO: 5). In other embodiments, the selected amino acid sequence is DIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK (SEQ ID NO: 15). In other embodiments, the selected amino acid sequence is selected from (SEQ ID NOs: 25, 46, 70, 32, 54, 39, 62, 80, 90, 98, 104, 116), or any combination.

[0275] In a particular embodiment, the selected amino acid sequence is (SEQ ID NO: 325).

[0276] In a specific embodiment, the molecule of interest is a CAR. When the molecule is a CAR, it can be any of CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD49g (ITGA7), CD49h (ITGA8), CD49i (ITGA9), CD49m (ITGA10), CD49m (ITGA11), CD49m (ITGA12), CD49m (ITGA13), CD49m (ITGA14), CD49m (ITGA15), CD49m (ITGA16), CD49m (ITGA17), CD49m (ITGA18), CD49m (ITGA19 ... ), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR 2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2 ), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF1) 4), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6) , CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta,The antibody may comprise a molecule or fragment thereof selected from the group consisting of IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a combination thereof.

[0277] Antigen-binding molecules are provided that specifically bind to all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein, and optionally contain a detectable label. If it is decided to use a detectable label, any detectable label can be used in this method, as described herein, and an appropriate label can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green), Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, and the like. Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Suitable optical dyes, including fluorophores, are described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is expressly incorporated herein by reference, and include radiolabels (e.g., 3 H, 11 C. 14 C.15 N, 18 F, 35 S, 64 CU, 90 Y, 99 Tc, 111 In, 124 I, 125 I, 131 I), photochromic compounds, Halo tags, Atto dyes, Tracy dyes, proteinaceous fluorescent labels (e.g., proteinaceous fluorescent labels include, but are not limited to, green fluorescent proteins including Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession Number U55762), blue fluorescent proteins (BFP, Quantum Biotechnologies, Inc. Stauber, 1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent proteins (Clontech Labs., Inc.), luciferase (Ichiki et al., (1996) Curr. Biol. 6:178-182), and the like). al., (1993) J. Immunol. 150:5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be employed in the disclosed methods.

[0278] The label can be associated with the antigen-binding molecule at any position within the molecule; however, it is preferable to associate the label with the molecule at a position (or at multiple positions, if multiple labels are used) that does not alter the binding properties of the molecule (unless such altered binding activity is desired). With regard to the anti-CD20 scFv-14 molecule, any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of the anti-CD20 scFv-14 molecule can be used, for example, those disclosed herein, e.g., those having one or more CDRs shown in Tables 3A-3C and 4A-4C. With regard to the GALV gp70 protein, any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of the GALV gp70 protein can be used, for example, those disclosed herein, e.g., those having one or more CDRs shown in Tables 7A-7C and 8A-8C.

[0279] In a specific embodiment of the disclosed methods, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0280] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0281] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0282] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212, an HCDR4 according to any one of SEQ ID NOs: 129, 159, and 189, R1, HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and any one of SEQ ID NOs: 153, 183, and 215 an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206, HCDR3 according to SEQ ID NO: 218 or DYY, HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219, HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196, HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208, HCDR3 according to SEQ ID NO: 220 or DYY,HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, HCDR3 according to SEQ ID NO: 221 or DYY, or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, HCDR3 according to SEQ ID NO: 222 or DYY.

[0283] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0284] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0285] The antigen-binding molecule can be placed on any surface, or no surface at all. For example, the antigen-binding molecule can be present in a buffer solution, and the buffer solution and antigen-binding molecule can be contacted with the sample. Alternatively, the antigen-binding molecule can be associated with a surface. Suitable surfaces include agarose beads, magnetic beads such as DYNABEADS™, or plastic, glass, or ceramic plates such as well-equipped plates, bags such as cell culture bags, etc. The surface itself can be placed in another structure, such as a column.

[0286] The sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex comprising a molecule containing a selected amino acid sequence and the antigen-binding molecule. The conditions that allow the formation of the binding complex depend on various factors, but generally, an aqueous buffer solution of physiological pH and ionic strength, such as phosphate-buffered saline (PBS), favors the formation of the binding complex and is preferred in the disclosed method. Since the components of the binding complex can be arranged on a surface as described herein, the formed binding complex can also be arranged on a surface.

[0287] At this stage, no binding complexes may be formed, or multiple binding complexes may be formed, each containing one or more antigen-binding molecules bound to an anti-CD20 scFv-14 molecule or a molecule containing all or a portion of the GALV gp70 protein. Unbound molecules and / or unbound antigen-binding molecules containing the selected amino acid sequence may be present in the local environment of any formed binding complexes.

[0288] Any molecules that are not part of the binding complex are then separated from any formed binding complex. The method of removal depends on the structure and / or local environment of the binding complex. For example, if the antigen-binding molecules are placed on beads, plates, or bags, unbound components of the reaction mixture can be washed away using a solution that leaves the formed binding complex intact. If the binding complex is placed on beads, the beads themselves may be placed in a column or other structure, and the same approach can be used.

[0289] The solution used to induce the formation of the binding complex can be used, for example, as a wash solution to remove unbound components. Any suitable buffer or solution can be used that does not disrupt the formed binding complex. Typically, buffers with high salt concentrations and non-physiological pHs that contain chaotropic agents or denaturing agents are preferably avoided when performing this step of the method.

[0290] The formed binding complex is then separated into (a) molecules containing all or a portion of the anti-CD20 scFv-14 molecule or GALV gp70 protein, and (b) antigen-binding molecules (e.g., one or more of SEQ ID NOS: 21-120) or molecules containing a sequence according to one or more of SEQ ID NOS: 127-324. Separation can be achieved using standard methodologies known to those skilled in the art. For example, a solution of appropriate pH and composition can be washed over the complex. A commonly used solution for this purpose is 0.1 M glycine HCl, pH 2.5-3.0, and separation can be achieved using this solution. Other solutions that can be used include 100 mM citric acid, pH 3.0, 50-100 mM triethylamine or triethanolamine, pH 11.5; 150 mM ammonium hydroxide, pH 10.5; 0.1 M glycine·NaOH, pH 10.0; 0.1 M Tris-acetate, pH 7.7, containing 5 M lithium chloride, 3.5 M magnesium chloride or potassium chloride, 3.0 M potassium chloride, 2.5 M sodium iodide or potassium iodide, 0.2-3.0 M sodium thiocyanate, and 2.0 M NaCl; 2-6 M guanidine HCl, 2-8 M urea, 1.0 M ammonium thiocyanate, 1% sodium deoxycholate, 1% SDS; and 10% dioxane, 50% ethylene glycol, pH 8-11.5.

[0291] After separation, if the anti-CD20 scFv-14 molecule or a molecule containing all or a portion of the GALV gp70 protein is the primary target, it can be recovered. Alternatively, if the antigen-binding molecule is the primary target, it can be recovered. c) A method for determining the presence or absence of a molecule

[0292] As disclosed herein, it may be desirable to isolate molecules comprising all or a portion of an anti-CD20 scFv-14 molecule. In other cases, simply knowing whether a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule is present or absent in a sample is sufficient information. For example, it may be useful to know that such molecules are expressed, regardless of expression level. In other cases, it may be desirable to know whether a purification process or step designed to remove such molecules was effective. Thus, qualitative determination of the presence or absence of a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule may be useful in multiple applications. The amino acid sequences set forth in Tables 3A-3C and 4A-4C can be used in any of the quantitative or qualitative methods described herein to determine the presence, absence, and / or amount of all or a portion of an anti-CD20 scFv-14 molecule.

[0293] As disclosed herein, it may be desirable to isolate molecules comprising all or a portion of the GALV gp70 protein. In other cases, simply knowing whether a molecule comprising all or a portion of the GALV gp70 protein molecule is present or absent in a sample is sufficient information. For example, it may be useful to know that such a molecule is expressed, regardless of expression level. In other cases, it may be desirable to know whether a purification process or step designed to remove such a molecule was effective. Thus, qualitative determination of the presence or absence of a molecule comprising all or a portion of the GALV gp70 protein may be useful in several applications. The amino acid sequences set forth in Tables 7A-7C and 8A-8C may be used in any of the quantitative or qualitative methods described herein to determine the presence, absence, and / or amount of all or a portion of the GALV gp70 protein.

[0294] With this in mind, methods are provided for determining the presence or absence in a sample of an anti-CD20 scFv-14 molecule or a molecule comprising all or a portion of a GALV gp70 protein in the sample.

[0295] In one embodiment, the method involves providing a sample known or suspected to contain all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein.

[0296] In a specific embodiment, all or a portion of the anti-CD20 scFv-14 molecule is a CAR. When a molecule is a CAR, it may be any of the following: CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1) , CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), C D158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-Zeta), CD258(LIGHT), CD268(BAF FR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK -p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1,The antibody may comprise a molecule or fragment thereof selected from the group consisting of NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a combination thereof.

[0297] Provided are antigen-binding molecules (e.g., SEQ ID NOS: 20 to 120) comprising a detectable label, to which the antigen-binding molecule (e.g., all or a portion of an anti-CD20 scFv-14 molecule) specifically binds. In another aspect, provided are antigen-binding molecules (e.g., SEQ ID NOS: 127 to 324) comprising a detectable label, to which the antigen-binding molecule (e.g., all or a portion of a GALV gp70 protein) specifically binds. An appropriate label can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green), Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, and the like. Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Suitable optical dyes, including fluorophores, are described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is expressly incorporated herein by reference, and include radioactive labels (e.g.,3 H, 11 C. 14 C. 15 N, 18 F, 35 S, 64 CU, 90 Y, 99 Tc, 111 In, 124 I, 125 I, 131 I), photochromic compounds, Halo tags, Atto dyes, Tracy dyes, proteinaceous fluorescent labels (e.g., proteinaceous fluorescent labels include, but are not limited to, green fluorescent proteins including Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession Number U55762), blue fluorescent proteins (BFP, Quantum Biotechnologies, Inc. Stauber, 1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent proteins (Clontech Labs., Inc.), luciferase (Ichiki et al., (1996) Curr. Biol. 6:178-182), and the like). al., (1993) J. Immunol. 150:5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be employed in the disclosed methods.

[0298] In a specific embodiment of the disclosed methods, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0299] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0300] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0301] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212, an HCDR4 according to any one of SEQ ID NOs: 129, 159, and 189, R1, HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and any one of SEQ ID NOs: 153, 183, and 215 an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206, HCDR3 according to SEQ ID NO: 218 or DYY, HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219, HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196, HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208, HCDR3 according to SEQ ID NO: 220 or DYY,HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, HCDR3 according to SEQ ID NO: 221 or DYY, or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, HCDR3 according to SEQ ID NO: 222 or DYY.

[0302] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0303] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0304] The label can be associated with the antigen-binding molecule at any position in the molecule, but is preferably associated with the molecule at a position (or at multiple positions when multiple labels are used) that does not alter the binding properties of the molecule (unless such altered binding activity is desired). Any antigen-binding molecule that specifically binds to all or a portion of the anti-CD20 scFv-14 molecule or GALV gp70 protein can be used, for example, those disclosed herein, e.g., those having one or more CDRs shown in Tables 3A to 3C, 4A to 4C, 7A to 7C, and 8A to 8C.

[0305] The sample is then contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex containing the cells present in the sample and the antigen-binding molecule. The antigen-binding molecule can be placed on any surface, or not placed on a surface at all. For example, the antigen-binding molecule can be present in a buffer solution, and the buffer solution-antigen-binding molecule can be contacted with the sample. Alternatively, the antigen-binding molecule can be associated with a surface. Suitable surfaces include agarose beads, magnetic beads such as DYNABEADS™, or plastic, glass, or ceramic plates such as well-equipped plates, bags such as cell culture bags, etc. The surface itself can be placed in a separate structure, such as a column.

[0306] The sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex containing the anti-CD20 scFv-14 molecule or a molecule containing all or a portion of the GALV gp70 protein and the antigen-binding molecule. The conditions that allow the formation of the binding complex depend on various factors, but generally, an aqueous buffer solution of physiological pH and ionic strength, such as phosphate-buffered saline (PBS), favors the formation of the binding complex and is preferred in the disclosed method. Because the components of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on a surface.

[0307] At this stage, no binding complexes may be formed, or multiple binding complexes may be formed, each comprising one or more antigen-binding molecules bound to a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule (or one or more molecules comprising all or a portion of an anti-CD20 scFv-14 molecule bound to an antigen-binding molecule [e.g., SEQ ID NOs: 1-120]). Non-binding molecules comprising all or a portion of an anti-CD20 scFv-14 molecule and / or non-binding antigen-binding molecules may also be present in the local environment of any formed binding complexes.

[0308] In other embodiments, at this stage, no binding complexes may be formed, or multiple binding complexes containing one or more antigen-binding molecules bound to molecules containing all or a portion of the GALV gp70 protein may be formed. Non-binding molecules containing all or a portion of the GALV gp70 protein and / or non-binding antigen-binding molecules may be present in the local environment of the formed binding complexes.

[0309] Any molecules that are not part of the binding complex are then separated from any formed binding complex. The method of removal depends on the structure and / or local environment of the binding complex. For example, if the antigen-binding molecules are placed on beads, plates, or bags, unbound components of the reaction mixture can be washed away using a solution that leaves the formed binding complex intact. If the binding complex is placed on beads, the beads themselves may be placed in a column or other structure, and the same approach can be used.

[0310] The solution used to induce the formation of the binding complex can be used, for example, as a wash solution to remove unbound components. Any suitable buffer or solution that does not disrupt the formed binding complex can also be used. Typically, buffers with high salt concentrations and non-physiological pHs that contain chaotropes or denaturants should be avoided when performing this step of the method.

[0311] Finally, the presence or absence of a binding complex containing a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein-binding molecule (e.g., one or more of Tables 3A-3C, 4A-4C, 7A-7C, 8A-8C, and 9-10) is detected. The specific method used to detect the presence or absence of a binding complex depends on the nature of the label selected. For example, if a fluorescent label is selected, FACS can be used. If an isotopic label is selected, mass spectrometry, NMR, or other techniques can be used. If a magnetic label is selected, magnetic-based cell sorting can be used. Microscopy can also be used. The end result of this method is a qualitative assessment of the presence or absence of an antigen-binding molecule comprising a detectable label, and thus the presence or absence of its binding partner, which molecule comprises all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein.

[0312] As with all of the disclosed methods, the anti-CD20 scFv-14 molecule or a molecule comprising all or a portion of the GALV gp70 protein can be placed in any environment. In a preferred embodiment, the anti-CD20 scFv-14 molecule or a molecule comprising all or a portion of the GALV gp70 protein is expressed on the surface of a cell. In this embodiment, the cell can be of any type, human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In a preferred embodiment, the cell is an immune cell. The immune cell of this method can be any type of immune cell (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). T cells (including T cytotoxic, T helper, and Treg cells) are particularly preferred. In a specific embodiment, the cell is a T cell, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be used in this embodiment of the disclosed method, and the cell can be a human cell or a non-human cell. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. In additional embodiments, the cells are T cells that present a CAR. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the disclosed method, the T cells can be in vivo T cells or in vitro T cells.

[0313] In additional embodiments, the cells can be placed in or isolated from any environment in which the cells can be maintained in viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, tissue grown ex vivo, a suitable buffer, etc. d) How to increase the concentration of a molecule

[0314] Very often, the molecule of interest is present in a sample at a lower level than desired. For example, when cells are transfected with an exogenous gene, the expression level of the protein(s) encoded by the exogenous gene is low. The same is true for molecules secreted from cells. Although such molecules are often present in small amounts, they can still be detected using the methods provided herein if they contain all or part of the anti-CD20 scFv-14 molecule and / or one or more of the molecules listed in Tables 3A-3C and 4A-4C. The same is true for viral particles. Although such viral particles are often present in small amounts, they can still be detected using the methods provided herein if they contain all or part of the GALV gp70 protein. One solution to the problem of low expression levels is to increase the concentration of the molecule of interest, which may be free in solution or expressed on the surface of cells. The concentration of a molecule of interest expressed intracellularly can also be increased, but the cells must first be lysed to release the molecule.

[0315] To address this issue, there are methods for increasing the concentration of cells presenting molecules comprising all or a portion of an anti-CD20 scFv-14 molecule and / or one or more molecules described in Tables 3A-3C and 4A-4C. In a further aspect, described herein are methods for increasing the concentration of viral particles presenting molecules and / or one or more molecules comprising all or a portion of a GALV gp70 protein.

[0316] In one embodiment, the method comprises providing a sample comprising cells known or suspected to contain all or a portion of an anti-CD20 scFv-14 molecule and / or a molecule comprising an amino acid sequence selected from the group consisting of one or more molecules listed in Tables 3A-3C and 4A-4C. In a further embodiment, the method comprises providing a sample comprising cells known or suspected to contain all or a portion of a GALV gp70 protein molecule and / or a molecule comprising an amino acid sequence selected from the group consisting of one or more molecules listed in Tables 7A-7C and 8A-8C.

[0317] In a specific embodiment of the disclosed methods, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0318] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0319] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0320] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212, an HCDR4 according to any one of SEQ ID NOs: 129, 159, and 189, R1, HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and any one of SEQ ID NOs: 153, 183, and 215 an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206, HCDR3 according to SEQ ID NO: 218 or DYY, HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219, HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196, HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208, HCDR3 according to SEQ ID NO: 220 or DYY,HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, HCDR3 according to SEQ ID NO: 221 or DYY, or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, HCDR3 according to SEQ ID NO: 222 or DYY.

[0321] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0322] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0323] In specific embodiments, the anti-CD20 scFv-14 molecule and / or a molecule comprising all or a portion of one or more of the molecules described in Tables 3A-3C and 4A-4C is a CAR. When a molecule is a CAR, it may be any of the following: CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITG A4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4 -1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2D L5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD 3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD 335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS),The antibody may comprise a molecule or fragment thereof selected from the group consisting of LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a combination thereof.

[0324] Antigen-binding molecules that specifically bind to all or a portion of an anti-CD20 scFv-14 molecule (e.g., one or more of the molecules described in Tables 3A-3C and 4A-4C) or specifically bind to a GALV gp70 protein and optionally include a detectable label are provided. If it is preferred to use a detectable label, any detectable label can be used in this method as described herein, and an appropriate label can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green), Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, and the like. Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Suitable optical dyes, including fluorophores, are described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is expressly incorporated herein by reference, and include radiolabels (e.g.,3 H, 11 C. 14 C. 15 N, 18 F, 35 S, 64 CU, 90 Y, 99 Tc, 111 In, 124 I, 125 I, 131 I), photochromic compounds, Halo tags, Atto dyes, Tracy dyes, proteinaceous fluorescent labels (e.g., proteinaceous fluorescent labels include, but are not limited to, green fluorescent proteins including Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession Number U55762), blue fluorescent proteins (BFP, Quantum Biotechnologies, Inc. Stauber, 1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent proteins (Clontech Labs., Inc.), luciferase (Ichiki et al., (1996) Curr. Biol. 6:178-182), and the like). al., (1993) J. Immunol. 150:5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be employed in the disclosed methods.

[0325] The label can be associated with the antigen-binding molecule at any position in the molecule, although it is preferable to associate the label with the molecule at a position (or multiple positions, if multiple labels are used) that does not alter the binding properties of the molecule (unless such altered binding activity is desired). With respect to the anti-CD20 scFv-14 molecule, any antigen-binding molecule that specifically binds to a molecule comprising all or a portion of the anti-CD20 scFv-14 molecule (e.g., one or more of the molecules described in Tables 3A-3C and 4A-4C); or one or more molecules comprising all or a portion of the anti-CD20 scFv-14 molecule bound to an antigen-binding molecule or a fragment thereof) can be used, for example, those disclosed herein, e.g., those having one or more CDRs described in Tables 3A-3C and 4A-4C. With respect to the GALV gp70 protein, any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of the GALV gp70 protein can be used, for example, those disclosed herein, e.g., those having one or more of the CDRs shown in Tables 7A to 7C and 8A to 8C.

[0326] In a specific embodiment of the disclosed methods, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0327] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0328] In a specific embodiment of the disclosed method, the antigen binding molecule of anti-CD20 scFv-14 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116.

[0329] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, an HCDR3 according to SEQ ID NO: 211 or DYY, an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188, an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200, an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212, an HCDR4 according to any one of SEQ ID NOs: 129, 159, and 189, R1, HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201, HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191, HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203, and any one of SEQ ID NOs: 153, 183, and 215 an HCDR3 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206, HCDR3 according to SEQ ID NO: 218 or DYY, HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219, HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196, HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208, HCDR3 according to SEQ ID NO: 220 or DYY,HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, HCDR3 according to SEQ ID NO: 221 or DYY, or HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, HCDR3 according to SEQ ID NO: 222 or DYY.

[0330] In certain embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 288; 7, an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN, an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285, an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS LCDR2 according to any one of SEQ ID NOs: 245, 275, and 295; LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289, LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN, LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299, LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, SEQ ID NOs: 240, 270,and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

[0331] In a specific embodiment of the disclosed methods, with respect to a GALV gp70 binding molecule, the light chain variable region and the heavy chain variable region of the binding molecule comprise an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; HCDR1 according to any one of SEQ ID NOs: 140, 170, and 200; HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201 , HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190, HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202, HCDR3 according to SEQ ID NO: 214 or DYY, any one of SEQ ID NOs: 227, 257, and 287 an LCDR1 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN;LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297, HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192, HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204, HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216, LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286, LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS, LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296, SEQ ID NOs: 133, 163, and HCDR1 according to any one of SEQ ID NOs: 145, 175, and 205; HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; HCDR2, HCDR3 according to SEQ ID NO: 218 or DYY, LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284, LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN, LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294, HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195, HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207, HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219, any one of SEQ ID NOs: 223, 253, and 283 LCDR1 according to any one of SEQ ID NOs: 233, 263, and SGS; LCDR2 according to any one of SEQ ID NOs: 243, 273, and 293; HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; HCDR3 according to SEQ ID NO: 220 or DYY; LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; SEQ ID NOs: 249, 279,and 299, an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, an HCDR3 according to SEQ ID NOs: 221 or DYY, an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291, an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN, an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301, or an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NOs: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300. ,

[0332] The antigen-binding molecule (e.g., one or more molecules described in Tables 3A-3C, 4A-4C, 7A-7C, and 8A-8C) can be disposed on any surface, or no surface at all. For example, the antigen-binding molecule can be present in a buffer solution, and the buffer-antigen-binding molecule can be contacted with the sample. Alternatively, the antigen-binding molecule can be associated with a surface. Suitable surfaces include agarose beads, magnetic beads such as DYNABEADS™, or plastic, glass, or ceramic plates such as well-equipped plates, bags such as cell culture bags, etc. The surface itself can be disposed in another structure, such as a column.

[0333] The cells expressing a molecule comprising all or a portion of the anti-CD20 scFv-14 molecule can be of any type and can be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In a preferred embodiment, the cells are immune cells. The immune cells of the present method can be any type of immune cell (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). T cells (including T cytotoxic, T helper, and Treg cells) are particularly preferred. In a specific embodiment, the cells are T cells, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be used, and the cells can be human or non-human. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. The T cells can be autologous, allogeneic, or xenogeneic. In additional embodiments, the cells are T cells that present a CAR. The T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the disclosed methods, the T cells can be in vivo T cells or in vitro T cells. Furthermore, the cells can be placed in or isolated from any environment that can maintain the cells in viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, a suitable buffer, etc.

[0334] A sample containing cells and / or viral particles is contacted with an antigen-binding molecule under conditions that allow the formation of a binding complex comprising all or a portion of an anti-CD20 scFv-14 molecule or GALV gp70 protein and the antigen-binding molecule (e.g., one or more molecules described in Tables 3A-3C, 4A-4C, 7A-7C, or 8A-8C). The conditions that allow the formation of a binding complex depend on various factors, but generally, an aqueous buffer solution at physiological pH and ionic strength, such as phosphate-buffered saline (PBS), favors the formation of a binding complex and is preferred in the disclosed methods. Because components of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on a surface.

[0335] At this stage, no binding complexes may have formed, or multiple binding complexes may have formed, each containing one or more antigen-binding molecules (e.g., one or more molecules described in Tables 3A-3C, 4A-4C, 7A-7C, and 8A-8C) bound to all or a portion of the anti-CD20 scFv-14 molecule or GALV gp70 protein. Non-binding molecules (e.g., one or more molecules described in Tables 3A-3C, 4A-4C, 7A-7C, and 8A-8C) containing all or a portion of the anti-CD20 scFv-14 molecule, GALV gp70 protein, and / or non-binding antigen-binding molecule may also be present in the local environment of any formed binding complexes.

[0336] Any molecules or cells that are not part of the binding complex are then separated from any formed binding complex. The method of removal depends on the structure and / or local environment of the binding complex. For example, if the antigen-binding molecule is bound to beads, a plate, or a bag, unbound components of the reaction mixture can be washed away with a solution that leaves the formed binding complex intact. If the binding complex is bound to beads, the beads themselves can be placed in a column or other structure, and the same approach can be used.

[0337] The solution used to induce the formation of the binding complex can be used, for example, as a wash solution to remove unbound components. Any suitable buffer or solution that does not disrupt the formed binding complex can also be used. Typically, buffers with high salt concentrations and non-physiological pHs that contain chaotropes or denaturants should be avoided when performing this step of the method.

[0338] At this stage of the method, a population of cells presenting a molecule comprising all or a portion of the anti-CD20 scFv-14 molecule or viral particles presenting GALV gp70 protein is present. If a detectable label is used, the concentration of the cells or viral particles can be easily determined in accordance with the nature of the label. There are no cells or viral particles that do not express the anti-CD20 scFv-14 molecule or a molecule comprising all or a portion of the GALV gp70 protein, and therefore the population (or concentration) of cells presenting a molecule comprising all or a portion of the anti-CD20 scFv-14 molecule or viral particles presenting GALV gp70 protein is increased compared to the level before the method was performed.

[0339] If the concentration of virus particles displaying anti-CD20 scFv-14 molecules and / or molecules comprising all or part of one or more molecules set forth in Tables 3A-3C, 4A-4C, or GALV gp70 protein and / or one or more molecules set forth in Tables 7A-7C and 8A-8C is not at the desired level, the above steps can be repeated as many times as desired. In the context of this step of the method, the desired number of times may be zero if the desired cell concentration is already present. [Example]

[0340] Hyperimmunized mice were immunized with scFv14 fused to the crystallizable fragment (Fc) domain derived from mouse immunoglobulin protein, isotype G2a. These culture supernatants were screened for the presence of antibodies that exhibited specific binding to scFv14. Antibody heavy and light chain gene sequences (e.g., SEQ ID NOS: 1-20) from hybridomas selected from this screening were used to generate antibodies that were again checked for specificity for scFv14. Antibodies selected from this screening were conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC) fluorophores and characterized by flow cytometry.

[0341] Of the 25 hybridoma supernatants, 14 showed specific binding to human CAR T cells expressing scFv14, an anti-CD20-targeting scFv, but not to an irrelevant anti-CD20 CAR scFv, FMC63-bearing anti-CD19 CAR, or untransduced T cells from the same healthy donor. Ten of the 14 clones were found suitable for antibody production. After antibody production, all 10 clones were retested for selectivity and sensitivity against scFv14 and an irrelevant anti-CD20 CAR and an anti-CD19 CAR bearing FMC63. Of these, five were conjugated to fluorophores for characterization by flow cytometry. One of these, clone 24C12, showed robust, sensitive, and specific binding to scFv14, but not to an irrelevant anti-CD20 CAR or an anti-CD19 CAR with FMC63, and was selected for use as a flow cytometry reagent to characterize scFv14 expression in anti-CD20 samples.

[0342] An antibody generation and characterization campaign yielded antibody clone 24C12, which strongly binds to the anti-CD20 scFv contained in the anti-CD20 CAR but does not bind to the irrelevant anti-CD20 CAR or anti-CD19 CAR scFv, regardless of conjugation to either PE or FITC. The Examples section below details the relevant methods.

[0343] The reagents used in this study included FACS staining buffer, goat anti-mouse IgG AF-488, mouse IgG isotype control, Whitlow linker control (LC) PE, LC AF-647, Live / Dead fixable violet stain, Live / Dead fixable aqua stain, 24C12 PE, and 24C12 FITC. FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; PE, phycoerythrin. LC is a custom-designed antibody that binds to the peptide linker between the light and heavy chains of chimeric antigen receptor (CAR) single-chain variable fragments (scFv), allowing for assessment of overall CAR transduction efficiency. Abbreviations: AF, Alexa Fluor; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; PE, phycoerythrin. KIP-1 is a custom-made antibody that binds to the peptide linker between the light and heavy chains of chimeric antigen receptor (CAR) single-chain variable fragments (scFv), allowing for assessment of overall CAR transduction efficiency. Instruments used in this study included a Sorvall Legend XTR Centrifuge, a Vi-Cell XR, and a FACS Fortessa X-20 II. Abbreviations: FACS, fluorescence-activated cell sorter.

[0344] These studies used an anti-CD19 CAR and three anti-CD20 CARs (Table 14). The anti-CD19 CAR, FMC63-28z, contained the FMC63 anti-CD19-targeting scFv. The three anti-CD20 CARs differed only in the scFv used to target CD20. The scFvs included scFv2, scFv14, and Leu16. scFv14 and scFv2 were fully human anti-CD20 scFvs that differed in their complementarity-determining regions. Leu16 is a mouse anti-human CD20 scFv. See, e.g., Wu et al., (2001) Protein Eng. 2001;14(12):1025-33, which is incorporated herein by reference in its entirety for any purpose. Sequence analysis was completed using IgBLAST (Ig Basic Local Alignment Search Tool) to identify the Leu16 and FMC63 germline genes. TIFF2026507879000025.tif45170

[0345] Abbreviations: CAR, chimeric antigen receptor; scFv, single-chain variable fragment; Vh, variable heavy chain; Vl, variable light chain; Vk, variable region gene segment. I. Example 1: Hybridoma Generation

[0346] Abveris DiversimAb™ Hyperimmune Mice (Canton, Massachusetts) were immunized with scFv-Fc proteins derived from scFv14 and mouse IgG2a Fc. Hybridoma supernatants were first tested in a dilution series for sensitivity to CARs bearing scFv14 and, as a negative control, to CARs bearing FMC63. They were then tested in a selectivity screen against CARs containing scFv2, scFv14, and Leu16. Selected binders for scFv14 underwent antibody sequencing and recombinant protein production, but not for FMC63, scFv2, or Leu16. The purified recombinant antibodies were tested again to confirm their specificity for scFv14 and then conjugated to a fluorophore. The bound antibodies were rescreened for specificity and sensitivity, and one clone was selected for use in detecting scFv14. II. Example 2: Hybridoma Susceptibility Screening

[0347] To test the sensitivity of the hybridoma supernatants, a dilution series screening was performed. Healthy donor T cells, either untransduced (NTD) or transduced with scFv14- or FMC63-carrying CAR, were incubated with the hybridoma supernatants and serially diluted in staining buffer. Negative controls consisted of an immunoglobulin (Ig) isotype control, conditioned medium from an irrelevant hybridoma, and pooled serum from pre-immunized mice. Polyclonal serum from mouse immunization was used as a positive control.

[0348] Cells were incubated with the diluted supernatant for 45 minutes at room temperature (RT), then harvested and washed twice with staining buffer. Samples were stained. Supernatants and control samples were stained with goat anti-mouse IgG conjugated to Alexa Fluor (AF)-488 (1:4,000). Controls for determining overall CAR expression were stained with anti-linker-specific antibody LC conjugated to phycoerythrin (PE) (1:1,000). All samples were incubated in staining buffer containing the viability dye Live-Dead Fixable Violet (1:2,000). Cells were stained for 45 minutes at room temperature, harvested, washed twice with staining buffer, and then immediately read on a BD Fortessa™ flow cytometer. Data were analyzed using FlowJo™ software (BD, version 10.6), and events were systematically gated for cells (using area by forward scatter [FSC]-side scatter [SSC]-area plots), single cells (using FSC-area by FSC-height plots), live cells (viability dye), and either phycoerythrin (PE) (for CAR control antibodies) or AF-488 (for supernatant samples), with gating thresholds set based on NTD control cells. III. Example 3: Hybridoma Specificity Testing

[0349] For specificity testing, human T cells transduced with CAR carrying either scFv2, scFv14, or Leu16 scFv were used. To ensure robust staining in the specificity screening, supernatants showing binding to scFv14 were tested at the highest concentration used in the dilution screening. Cells were stained and analyzed. IV. Example 4: Characterization of Recombinant Antibodies

[0350] After sequencing the antibody variable heavy (VH) and variable light (VL) domains from hybridomas selected from dilution series and specificity screening, antibodies were produced by Genscript (Piscataway, NJ) according to standard procedures. Briefly, proteins were expressed using Expi293F cells and affinity-purified in one step using MabSelect SuRe LX (GE Healthcare, Cat. No. 17-5474-02). Purity was assessed by SDS-PAGE and SEC-HPLC. Antibodies were sterile-filtered through a 0.22 μm filter, packaged aseptically, and stored at -80°C.

[0351] For recombinant antibody specificity and selection testing, healthy human donor T cells transduced with CARs bearing either scFv2, scFv14, or FMC63 were harvested, washed twice with staining buffer, and then incubated with either anti-scFv14 antibody or a mouse IgG isotype control, both at 300 ng / mL, or in the absence of primary antibody as a negative control. An NTD healthy donor T cell control was also included. Cells were stained and analyzed. V. Example 5: Fluorophore conjugation to recombinant antibodies

[0352] The anti-scFv14 antibody, 24C12, was sent to BD Pharmingen™ for custom conjugation to the fluorophores PE and fluorescein isothiocyanate (FITC). Briefly, the heterobifunctional crosslinking reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was conjugated to PE, and SMCC-PE was covalently conjugated to the reduced antibody. The 1:1 PE-conjugated antibody was then purified and buffer exchanged into PBS using size-exclusion chromatography. A reactive FITC molecule was conjugated to EV-aFMC63 according to standard protocols, allowing the fluorophore to be attached to primary amines on the antibody. Unbound fluorophores were removed, and the buffer exchanged into PBS, pH 7.4, using standard size-exclusion chromatography methods. VI. Example 6: Characterization of Fluorophore-Conjugated Antibodies

[0353] To characterize the fluorophore-conjugated 24C12 antibody, donor T cells transduced with CAR bearing either scFv2, scFv14, or FMC63 were harvested, washed twice with staining buffer, and then incubated with serial dilutions of 24C12 labeled with either PE or FITC. NTD T cells from the same healthy donor, an isotype control, and an unstained control were included. All samples were incubated in staining buffer containing the viability dye Live-Dead Fixable Aqua (1:1,000). Cells were stained for 15 minutes at 4°C, harvested, washed twice with staining buffer, and then fixed in 0.6% (v / v) paraformaldehyde for 10 minutes at room temperature and stored at 4°C until read on a BD Fortessa™ flow cytometer. Data were analyzed using FlowJo™ software (BD, version 10.6), and events were systematically gated for cells (using area by forward scatter [FSC]-side scatter [SSC]-area plots), single cells (using FSC-area by FSC-height plots), live cells (viability dye), and either PE, FITC, or AF-647, with gating thresholds set based on NTD control cells. VII. Example 7: Characterization of Hybridoma Supernatants

[0354] A panel of 25 cryopreserved hybridomas and associated culture supernatants (test report KIT19025-1) was sent to Kite for screening (Table 15). The supernatants were characterized according to the methods described herein. Based on the selection criteria described herein, hybridomas (Table 15) were sequenced, produced as recombinant antibodies, and characterized. TIFF2026507879000026.tif175166Abbreviations: NB, no join; NOTE: This table lists the hybridomas, the corresponding total protein concentrations in the supernatants, and the affinity coefficients (KD(M)) estimated by the Octet equilibrium binding assay (see ELN EXP-20-BD1634). VIII. Example 8: Hybridoma Sensitivity and Dilution Serial Screening

[0355] To assess the relative binding sensitivity of hybridoma culture supernatants to scFv-14, the supernatants were serially diluted and screened against NTD T cells, scFv-14-containing CAR T cells, or FMC63-containing CAR T cells. Polyclonal serum from immunized mice (Abveris™ KIT19025-1) was used as a positive control. Pooled normal serum from unimmunized mice, conditioned medium (CM) from an unrelated hybridoma, and a mouse IgG isotype control were used as negative controls.

[0356] Panel A of Figure 1 shows that both the scFv14-bearing CAR and the FMC63-bearing CAR are expressed by transduced T cells as determined by staining with the control anti-linker antibody LC (73.3% and 83.7% LC, respectively). + ). LC is a custom-designed antibody, scFv, that binds to a peptide linker between the light and heavy chains of the CAR scFv, allowing for assessment of overall CAR transduction efficiency. CM, isotype control, and normal mouse serum control showed no binding to scFv-14 (Figure 1, panels B-D). No binding of these controls to FMC63-28z CAR cells was observed (data not shown). As expected, CAR T cells bearing scFv14 stained positive with the polyclonal antiserum (Figure 1, panel E). None of the supernatants showed binding to NTD T cells (data not shown) or T cells expressing FMC63-bearing CAR (data not shown).

[0357] Because relatively weak binding may be caused by low antibody concentrations in the supernatants, a range-finding preliminary screen was performed to determine the appropriate range for testing relative sensitivity in serial dilution assays. Based on the results of the preliminary screen (data not shown), 25 hybridoma culture supernatants were divided into two groups. Nine of the 25 supernatants in group 1 were tested in a two-fold dilution series starting at a 1:500 (v / v) dilution using fluorescence-activated cell sorting (FACS) staining buffer. Binding to scFv-14 was determined by staining with goat anti-mouse IgG conjugated to AF-488 (data not shown). The remaining 16 were tested in a two-fold dilution series starting at a 1:125 (v / v) dilution using FACS staining buffer. Binding to scFv-14 was determined by staining with goat anti-mouse IgG conjugated to AF-488 (data not shown).

[0358] As a final check, the supernatant that showed no binding in the first dilution series (29C7) and 12 samples that showed no binding in the second dilution series (23A9, 23G3, 24D9, 24H1, 26G4, 27B9, 12F8, 13G4, 18D7, 19E5, 20C4, and 21B5) were tested at a single dilution of 1:2 with staining buffer. This final dilution revealed two supernatants that showed binding to scFv14 bearing CAR but not to the NTD control (see Figure 2) or CAR bearing FMC63 (Figure 16). Hybridomas that showed binding to scFv14 at the lowest dilution in each of the respective series but not to NTD T cells or FMC63-28z were selected for specificity screening.

[0359] Results from the dilution series indicate that a total of 14 hybridoma supernatants bind to scFv-14 but not to the NTD control or anti-CD19 CAR T cells. The following hybridomas were selected for further specificity screening: 13C2, 22H5, 23C11, 24C12, 25B2, 25F10, 29F8, 29F1, 23D4, 23E1, 24C7, 26E1, 18D7, and 24D9.

[0360] There was a large shift in mean fluorescence intensity (MFI) among the selected hybridomas. Figure 3 summarizes the binding at the highest concentration tested for each of the 14 hybridomas selected for specificity screening compared to the MFI of the sample. AF-488 + The fraction of cells that are AF-488 (see Figure 3A) and AF-488 in each sample + The MFI for the populations (see Figure 3B) is shown. IX. Example 9: Hybridoma specificity screening

[0361] To assess specificity for scFv14, supernatants were screened for binding to T cells expressing CARs bearing scFv14, CARs bearing irrelevant anti-CD20 scFv2, or CARs bearing Leu16, or NTD T cell controls. Pooled polyclonal post-immunization antisera were used as a positive control, while negative controls consisted of pooled pre-immunization mouse sera, CM from an irrelevant hybridoma, and a mouse IgG isotype control (see Figure 4). As determined by LC PE staining, overall CAR expression for CAR T cells bearing scFv2, scFv14, and Leu16 was 77.7%, 88.0%, and 84.5%, respectively (see Figure 4). All three CARs showed some binding to the polyclonal antisera. Because the immunogen was an anti-CD20 CAR, the polyclonal antisera are expected to react to various shared domains in the CARs tested in this assay. The 14 hybridomas that showed binding to scFv14 in the dilution series were then screened at the highest concentration used in this series (see Figure 5). + Controls were included as a reference to determine the expression percentage of each CAR. The results show that all 14 hybridoma supernatants specifically bind to scFv-14, but not to any of the negative controls. X. Example 10: Selection of Hybridomas for Sequencing and Antibody Production

[0362] The purpose of the dilution series and selection screening described in the Examples herein was to select candidate hybridomas for production. Based on these results, 10 of the 14 hybridomas tested for specificity were selected for sequencing and antibody production. Some clones were not selected if their sequences were very similar to other clones or if they appeared to be weak binders in the hybridoma supernatant screening. The 10 hybridomas included 18D7, 22H5, 23C11, 23E1, 24C12, 24C7, 25B2, 25F10, 29F1, and 29F8. XI. Example 11: Confirmation and Selection of Antibody Bindings for Characterization

[0363] To confirm the selectivity of antibody clones for scFv-14, they were screened against NTD T cells or T cells expressing CAR with either scFv-14, scFv2, or FMC63. CAR expression was determined by LC staining and found to be 68.4%, 67.5%, and 67.7%, respectively. In flow cytometry experiments, antibody clones 24C12, 29F1, 24C7, 23E1, 23C11, 18D7, 25B2, and 29F8 demonstrated specific binding to scFv-14. Based on these results, all eight clones were sent to BD Biosciences™ (San Diego, CA) for conjugation to PE and FITC. XII. Example 12: Post-Conjugation Characterization of 24C12

[0364] Of the eight recombinant antibodies sent to BD™ for conjugation to fluorescent dyes, only five arrived at Kite Pharma™ in time for inclusion in this report. While all five of the anti-scFv-14 antibodies were found to be selective for the anti-CD20 scFv component of KITE-363 based on their overall excellent binding properties (data not shown), one of the antibodies, 24C12, was selected for final characterization.

[0365] Antibody clone 24C12 conjugated to either PE or FITC was screened against healthy donor T cells that were either NTD or transduced to express CAR with either scFv2, scFv14, or FMC63. Antibody 24C12 shows specific binding to scFv14 regardless of fluorophore conjugate (see Figure 6). PE at the highest concentration of antibody tested (256 ng / mL) was significantly higher than that of FITC. + Cells (see Figure 6A) or FITC + A fraction of cells (see Figure 6B) showed selective binding to CAR bearing scFv14 and lacked binding to irrelevant CAR bearing scFv2 or FMC63. For reference, overall CAR expression in CAR-transduced T cells bearing scFv2, scFv14, and FMC63, as determined by staining with LC-AF647, was 80.6%, 78.7%, and 80.7%, respectively (see Figure 6).

[0366] Serial dilutions of 24C12 PE (see Figure 7A) and 24C12 FITC (see Figure 7B) show that both are selective for scFv14. XIII. Example 13: Generation of Recombinant Protein Immunogens and Screening Reagents

[0367] To facilitate the development and screening of antibodies that recognize the envelope protein, gibbon ape leukemia virus (GALV) gp70 (Uniprot P21415), several recombinant proteins were designed, expressed, and purified from human Expi293 cells containing mouse Fc, human monoFc, or His tags and used as potential immunogens and screening reagents. These proteins contained the entire predicted viral surface-exposed portion of GALV gp70, encompassing the receptor-binding domain (RBD) of GALV gp70 (residues 42–474), the coiled-coil (CC) domain of GALV gp70 (residues 505–616), or the RBD + CC domain (residues 42–616) (Figure 8). The CC domain is composed of heptad repeats that associate to form a noncovalent trimer. Analytical size-exclusion chromatography demonstrated that the CC domain-containing recombinant proteins formed trimers.

[0368] K562 cells expressing the endogenous SLC20A1 (PIT1) receptor or a negative control cell line (CHO) were stained with a dilution series of each recombinant protein containing a human monoFc tag and a fluorophore-conjugated anti-human Fc secondary antibody. An anti-SLC20A1 antibody (Proteintech, catalog no. 12423-1-AP) was included as a control. Recombinant GALV gp70 proteins containing the RBD bound to K562 cells in a dose-dependent manner, whereas proteins consisting of only the CC domain did not. No binding to the negative control cells was observed (Figures 9A-9B). Taken together, these data suggest appropriate structure and function, providing confidence in our reagents for antibody generation and screening. XIV. Example 14: Hybridoma Campaign and Screening

[0369] Abveris DiversimAb™ and DiverGimab™ hyperimmune mice (Canton, MA) were immunized with replication-incompetent empty retroviral (RVV) particles (payload-free) containing the envelope protein GALV gp70. Mice were boosted with recombinant mouse IgG2a Fc-tagged GALV gp70 protein (residues 42-616) or empty RVV particles. Mouse sera were titered by serial dilution in an indirect ELISA coated with recombinant soluble huIgG1 monoFc-tagged gibbon GALV gp70 protein (residues 42-616, 42-474, or 505-616). An unrelated huIgG1 monoFc-tagged protein was used as a negative control. Mouse sera were also tested for sensitivity to a PG 13-based stable packaging cell line constitutively producing viral particles containing the GALV gp70 envelope protein by flow cytometry in serial dilutions. PG13 cells were stained with normal mouse serum (NMS), no stain (NS), or an isotype control as a negative control. Based on positive ELISA and flow cytometry data, two mice were selected for hybridoma fusion (Figures 10A-10B). XV. Example 15: Hybridoma Supernatant Screening

[0370] Thousands of hybridoma fusion supernatants were screened using an indirect ELISA coated with recombinant soluble huIgG1 monoFc-tagged gibbon GALV gp70 protein (residues 42-616). An unrelated huIgG1 monoFc-tagged protein was used as a negative control. Only 60 positive binders were identified, and these were then subjected to a secondary ELISA screen coated with recombinant soluble huIgG1 monoFc-tagged gibbon GALV gp70 protein (residues 42-616, 505-616, or 42-474). An unrelated huIgG1 monoFc-tagged protein was used as a negative control. This further narrowed the list of positive antibodies to 17 clones (Figure 11). After this analysis, it was discovered that one of the hybridoma cell lines had died, reducing the number of positive hybridomas to 16.

[0371] The supernatants of 16 viable hybridoma lines tested in the ELISA-based screen were screened by flow cytometry in a dilution series for sensitivity to a PG 13-based stable packaging cell line that constitutively produces viral particles containing the GALV gp70 envelope protein (Figure 12). NIH-3T3 parental cells served as negative controls and were incubated with the same hybridoma supernatants and serially diluted in staining buffer. Negative controls consisted of an immunoglobulin (Ig) isotype control and normal mouse serum from two different mice (non-immunized). Cells were incubated with the diluted supernatants for 45 minutes at room temperature (RT), then harvested and washed twice with staining buffer. Supernatants and control samples were stained with PE-conjugated secondary antibodies. Data were analyzed using FlowJo™ software (BD, version 10.6), and events were systematically gated for cells (using a forward scatter [FSC] area × side scatter [SSC] area plot), single cells (using an FSC area × FSC height plot), and phycoerythrin (PE), with gating thresholds set based on negative control cells. Eight of the 16 viable hybridoma supernatants that were positive by ELISA were also confirmed to be positive by flow cytometry. Positive clones in this experiment were 35C11, 40A3, 8G8, 9A1, 9G11, 4F1, 2D3, and 40A6. XVI. Example 16: Sensitivity and specificity testing of recombinant antibodies

[0372] All hybridoma clones positive for GALV gp70 binding by ELISA were sequenced using next-generation sequencing (NGS). After sequencing the antibody variable heavy (VH) and variable light (VL) domains from the hybridomas, the antibodies were cloned into standard mammalian expression vectors in a mouse IgG2a format and produced at small scale. All unique VH and VL sequences resulted in a panel of 12 recombinant clonal antibodies being produced, which, if the purified yield was high enough for testing, were functionally tested at small scale. To test the sensitivity and specificity of the purified antibodies, a single 10-fold dilution of each antibody was incubated with PG13 cells or NIH-3T3 cells as a negative control (Figure 13). The following clones had sufficient purified yields for testing and were positive for PG13 staining: 8G8, 9A1, 40A3, 4F1, 35C11, and 9G11. XVII. Example 17: Sensitivity and dilution series screening of purified antibodies

[0373] To confirm the results of small-scale production and screening, and to retest clones with low small-scale purification yields, antibodies were produced on a larger scale. Briefly, antibodies were expressed in ExpiCHO cells using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, Cat. A29133). Antibodies were affinity purified on a HiTrap MabSelect SuRe column (Cytiva, Product No. 11003493) followed by size-exclusion chromatography on a HiLoad Superdex 200 16 / 600 (Cytiva, Product No. 28989335). A panel of nine antibodies was successfully produced, and the purity of each antibody was determined to be >95% by SDS-PAGE gel and analytical SEC (SEC-UPLC). Antibodies were sterile filtered through a 0.22 μm filter and stored at -80°C. The following clones were selected for further analysis: 35C11, 3C8, 40A3, 40A6, 8G8, 9A1, 9G11, 2D3, and 4F1.

[0374] To test the sensitivity and specificity of the purified antibodies, a titration / 3-fold dilution series was performed (from a top concentration of 10 μg / mL down to 0.005 μg / mL), and PG13 cells were incubated with each of the antibodies or the highest concentration of the mouse IgG isotype control (Figures 14A-14I). NIH-3T3 cells were used as a negative control and were incubated with the same purified antibody dilution series containing staining buffer. Both cell lines were also stained with secondary antibody alone (in the absence of primary antibody) as a negative control. Cells were stained and analyzed. XVIII. Example 18: Affinity Ranking and Specificity Testing of Purified Antibodies Using Biolayer Interferometry

[0375] The relative affinity ranking, epitope binning, and specificity of purified antibodies were measured by biolayer interferometry using Octet Red 96 (Sartorius). Briefly, purified antibodies were loaded onto an AMC biosensor (product number 18-5088) at 2 μg / mL and tested for binding to 100 nM recombinant soluble huIgG1 monoFc-tagged gibbon GALV gp70 protein analyte (residues 42-616, 42-474, or 505-616) (Figures 15A-15H). Antibody specificity was confirmed using a negative control analyte, soluble VSV-G (residues 17-467) huIgG1 monoFc-tagged protein. Competition assays were performed to identify clones with unique or non-overlapping epitopes. These experiments identified clones 35C11, 8G8, and 40A3 as having non-overlapping or non-competing epitopes within residues 42-474 of GALV-gp70. XIX. Example 19: Characterization of Purified Antibodies

[0376] A panel of nine recombinant antibodies was successfully produced. The antibodies were characterized and screened according to the methods described in Example 18. The affinity values ​​in Table 16 are for antibody clones against huIgG1 monoFc-tagged gibbon GALV gp70 protein (residues 42-474). TIFF2026507879000027.tif99164 Abbreviations: NB, no join; NOTE: This table lists antibody clones and purification yields in mg / L when produced in ExpiCHO cells and purified in two steps. Affinity values ​​(KD(M)) were estimated by Octet binding assay. XX. Example 20: Fluorophore conjugation to recombinant antibodies

[0377] Three anti-GALV gp70 antibodies, clones 8G8, 40A3, and 35C11, were selected as lead antibody candidates due to their binding properties and conjugated to Dylight™ 650 (Thermo Fisher Scientific) according to standard protocols for coupling fluorophores to primary amines on antibodies. Unbound fluorophores were removed using size-exclusion chromatography. The purity of each conjugated antibody was determined to be >95% analytical SEC (SEC-UPLC), and the degree of labeling was determined for each using spectrophotometry. XXI. Example 21: Characterization of Fluorophore-Conjugated Antibodies

[0378] For characterization of the fluorophore-conjugated 8G8, 40A3, and 35C11 antibodies, PG13 cells were incubated with 200 ng of each fluorophore-conjugated antibody. NIH-3T3 cells were used as a positive control. Both cell lines were also stained with secondary antibody alone (in the absence of primary antibody) as a negative control. Cells were stained and analyzed (Figures 15A-15H). Incorporation by Reference

[0379] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference in this specification should not be construed as an admission that such reference is prior art to the present disclosure. In the event that any definitions or terms provided in a reference incorporated by reference differ from the terms and explanations provided herein, the terms and definitions shall control.

[0380] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. It will be understood, however, that no matter how detailed the above appears in text, the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. An isolated antigen-binding molecule that binds to an anti-CD20 binding region, comprising: a heavy chain variable (VH) sequence having at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-10; a light chain variable (VL) sequence having at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 11-20; and a linker connecting the VH to the VL.

2. The isolated antigen-binding molecule of claim 1, comprising a VH amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 1 to 10.

3. The isolated antigen-binding molecule of claim 1, comprising a VL amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 11 to 20.

4. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 21-41.

5. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 42-65.

6. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CD3 selected from the group consisting of SEQ ID NOs: 66-85.

7. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a light chain CDR1 selected from the group consisting of SEQ ID NOs: 86-99.

8. 10. The isolated antigen binding molecule of any one of the preceding claims, comprising a light chain CDR2 selected from the group consisting of SEQ ID NOs: 100-111.

9. 10. The isolated antigen binding molecule of any one of the preceding claims, comprising a light chain CDR3 selected from the group consisting of SEQ ID NOs: 112-120.

10. 10. The isolated antigen-binding molecule of claim 1, wherein the linker comprises an amino acid sequence.

11. The isolated antigen-binding molecule of claim 10, wherein the amino acid sequence of the linker comprises a sequence having at least about 80% sequence identity to SEQ ID NO:

121.

12. The isolated antigen-binding molecule of claim 10, wherein the amino acid sequence of the linker comprises a sequence having at least about 80% sequence identity to SEQ ID NO:

126.

13. The isolated antigen-binding molecule of claim 11, wherein the amino acid sequence of the linker comprises a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

121.

14. The isolated antigen-binding molecule of claim 12, wherein the amino acid sequence of the linker comprises a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

126.

15. The isolated antigen-binding molecule of any one of the preceding claims, further comprising a detectable label selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

16. 20. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 25, 32, and 39.

17. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 46, 54, and 62.

18. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 70 and 80.

19. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 90 and 98.

20. 10. The isolated antigen-binding molecule of any one of the preceding claims, comprising a light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 104 and 110.

21. 10. The isolated antigen binding molecule of any one of the preceding claims, comprising a light chain CDR3 sequence comprising SEQ ID NO:

116.

22. 1. An antigen-binding system, antibody, or antigen-binding fragment thereof, comprising a GALV gp70-binding motif, wherein the GALV gp70-binding motif comprises three heavy chain complementarity-determining regions (HCDRs) of any one of heavy chain variable regions (HCVRs) selected from the group consisting of SEQ ID NOs: 303-314, and three light chain CDRs (LCDRs) of a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 315-324.

23. the GALV gp70-binding motif comprises a first domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a second domain comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3); (i) the HCDR1 has a sequence according to any one of SEQ ID NOs: 127-138, 157-168, and 187-198; (ii) the HCDR2 has a sequence according to any one of SEQ ID NOs: 139-150, 169-180, and 199-210; (iii) the HCDR3 has a sequence according to any one of SEQ ID NOs: 151-156, 181-186, 211-222, and DYY; (iv) the LCDR1 has a sequence according to any one of SEQ ID NOs: 223-232, 253-262, and 283-292; (v) the LCDR2 has a sequence according to any one of SEQ ID NOs: 233-242, 263-272, SGS, GTN, RAS, DTS, and KVS; (vi) The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 22, wherein the LCDR3 has a sequence according to any one of SEQ ID NOs: 243-252, 273-282, and 293-302.

24. The HCDR is (i) an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187, an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199, and an HCDR3 according to SEQ ID NO: 211 or DYY; (ii) an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; and an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; (iii) an HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; an HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; and an HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; (iv) an HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; an HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; and an HCDR3 according to SEQ ID NO: 214 or DYY; (v) an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; and an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; (vi) an HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; and an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; (vii) an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; and an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; (viii) an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194, an HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206, and an HCDR3 according to SEQ ID NO: 218 or DYY; (ix) an HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; an HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; and an HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; (x) an HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; an HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; and an HCDR3 according to SEQ ID NO: 220 or DYY; (xi) an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197, an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209, and an HCDR3 according to SEQ ID NO: 221 or DYY; or (xii) comprises an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, and an HCDR3 according to SEQ ID NO: 222 or DYY; and The LCDR is (i) an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; (ii) an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; and an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; (iii) an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (iv) an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; and an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (v) an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; and an LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; (vi) an LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; an LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; (vii) an LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; an LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; and an LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; (viii) an LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; an LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299; (ix) an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291; an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301; or (x) an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN; and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300, according to any one of SEQ ID NOs: 250, 280, and 300;

25. the antigen-binding system, the antibody, or the antigen-binding fragment thereof comprises a first domain comprising three heavy chain complementarity-determining regions (HCDRs) and a second domain comprising three light chain complementarity-determining regions (LCDRs); the HCDR and LCDR are (i) an HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; an HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; an HCDR3 according to SEQ ID NO: 211 or DYY; an LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; an LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; an LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; (ii) an HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; an HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; an HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; an LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; an LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; and an LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; (iii) an HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; an HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; an HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; and an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (iv) an HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; an HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; an HCDR3 according to SEQ ID NO: 214 or DYY; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (v) an HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; an HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; an HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; an LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; an LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; an LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; (vi) an HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192; an HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; an HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; an LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; an LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; an LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; (vii) an HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; an HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; an HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; an LCDR1 according to any one of SEQ ID NOs: 225, 255, and 285; an LCDR2 according to any one of SEQ ID NOs: 235, 265, and RAS; an LCDR3 according to any one of SEQ ID NOs: 245, 275, and 295; (viii) an HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; an HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; an HCDR3 according to SEQ ID NO: 218 or DYY; an LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; an LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; an LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294; (ix) an HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; an HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; an HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; an LCDR1 according to any one of SEQ ID NOs: 223, 253, and 283; an LCDR2 according to any one of SEQ ID NOs: 233, 263, and SGS; an LCDR3 according to any one of SEQ ID NOs: 243, 273, and 293; (x) an HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196; an HCDR2 according to any one of SEQ ID NOs: 148, 178, and 208; an HCDR3 according to SEQ ID NO: 220 or DYY; an LCDR1 according to any one of SEQ ID NOs: 229, 259, and 289; an LCDR2 according to any one of SEQ ID NOs: 239, 269, and GTN; an LCDR3 according to any one of SEQ ID NOs: 249, 279, and 299; (xi) an HCDR1 according to any one of SEQ ID NOs: 137, 167, and 197; an HCDR2 according to any one of SEQ ID NOs: 149, 179, and 209; an HCDR3 according to SEQ ID NO: 221 or DYY; an LCDR1 according to any one of SEQ ID NOs: 231, 261, and 291; an LCDR2 according to any one of SEQ ID NOs: 241, 271, and GTN; an LCDR3 according to any one of SEQ ID NOs: 251, 281, and 301; or (xii) The antigen-binding system, antibody, or antigen-binding fragment thereof according to any one of claims 22 to 24, comprising an HCDR1 according to any one of SEQ ID NOs: 138, 168, and 198, an HCDR2 according to any one of SEQ ID NOs: 150, 180, and 210, an HCDR3 according to SEQ ID NO: 222 or DYY, an LCDR1 according to any one of SEQ ID NOs: 230, 260, 290, an LCDR2 according to any one of SEQ ID NOs: 240, 270, and GTN, and an LCDR3 according to any one of SEQ ID NOs: 250, 280, and 300.

26. the antigen-binding system, the antibody, or the antigen-binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs; (i) the heavy chain variable domain is at least 80% identical to one of SEQ ID NOs: 303-314; (ii) The antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 22 to 25, wherein the light chain variable domain is at least 80% identical to one of SEQ ID NOs: 315 to 324.

27. the antigen-binding system, the antibody, or the antigen-binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs; (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 303 and the light chain variable domain is at least 80% identical to SEQ ID NO: 324; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 304 and the light chain variable domain is at least 80% identical to SEQ ID NO: 320; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 305 and the light chain variable domain is at least 80% identical to SEQ ID NO: 318; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 306 and the light chain variable domain is at least 80% identical to SEQ ID NO: 319; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 307 and the light chain variable domain is at least 80% identical to SEQ ID NO: 319; or (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 308 and the light chain variable domain is at least 80% identical to SEQ ID NO: 318; or (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 309 and the light chain variable domain is at least 80% identical to SEQ ID NO: 317; or (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 310 and the light chain variable domain is at least 80% identical to SEQ ID NO:

316. (ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 311 and the light chain variable domain is at least 80% identical to SEQ ID NO:

315. (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 312 and the light chain variable domain is at least 80% identical to SEQ ID NO: 321; (xi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 313 and the light chain variable domain is at least 80% identical to SEQ ID NO: 323; or (xii) The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 26, wherein the heavy chain variable domain is at least 80% identical to SEQ ID NO: 314 and the light chain variable domain is at least 80% identical to SEQ ID NO:

322.

28. 28. The antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 22 to 27, wherein the three HCDRs and the three LCDRs are comprised in a single polypeptide.

29. 28. The antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 22 to 27, wherein the three HCDRs are comprised in a first polypeptide and the three LCDRs are comprised in a second polypeptide.

30. 30. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 29, wherein the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

31. A nucleic acid encoding at least one polypeptide according to any one of claims 22 to 30.

32. A vector comprising the nucleic acid of claim 31.

33. 33. A method of producing an engineered cell comprising transfecting or transducing a cell with the nucleic acid of claim 31 or the vector of claim 32.

34. A cell encoding or expressing the antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 22 to 30, optionally wherein the cell is an immune cell.

35. 31. The antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 22 to 30, further comprising a detectable label.

36. 36. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 35, wherein the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

37. The fluorescent label may be selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarin, aminocouramin, methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, and Lissamine Rhodamine B. B), Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTrquoise 2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan), wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange37. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 36, wherein the antigen-binding protein is selected from the group consisting of: 1) Fluorescent Protein IgE (Fluorescent Protein IgE), 2) Fluorescent Protein IIgE, 3) Fluorescent Protein IIIgE, 4) Fluorescent Protein IgE, 5) Fluorescent Protein IIgE, 6) Fluorescent Protein IIIgE, 7) Fluorescent Protein IgE, 8) Fluorescent Protein IIgE, 9) Fluorescent Protein IIIgE, 10) Fluorescent Protein IIgE, 11) Fluorescent Protein IIgE, 12) Fluorescent Protein IIIgE, 13) Fluorescent Protein IIIgE, 14) Fluorescent Protein IIIgE, 15) Fluorescent Protein IIIgE, 16) Fluorescent Protein IIIgE, 17) Fluorescent Protein IIIgE, 18) Fluorescent Protein IIIgE, 19) Fluorescent Protein IIIgE, 12) Fluorescent Protein IIIgE, 19 ...

38. 1. A method for determining the number of viral particles expressing a gibbon ape leukemia virus (GALV) gp70 protein having the amino acid sequence of SEQ ID NO: 325, comprising: (a) providing a sample known or suspected to contain viral particles expressing the GALV gp70 protein; (b) contacting the sample with an antigen-binding system, antibody, or antigen-binding fragment thereof under conditions that allow the formation of one or more binding complexes comprising virus particles and the antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 21 to 29, wherein the antigen-binding system, antibody, or antigen-binding fragment thereof further comprises a detectable label; (c) detecting one or more binding complexes by detection of the detectable label; (d) determining the number of virus particles present in the sample based on the detecting of step (c).

39. 1. A method for determining the presence or absence of viral particles expressing a gibbon ape leukemia virus (GALV) gp70 protein having the amino acid sequence of SEQ ID NO: 325, comprising: (a) providing a sample known or suspected to contain viral particles expressing the GALV gp70 protein; (b) providing an antigen-binding molecule that specifically binds to the GALV gp70 protein, wherein the antigen-binding molecule further comprises a detectable label; (c) contacting the sample with the antigen-binding molecule under conditions that allow the formation of a binding complex between the GALV gp70 and the antigen-binding protein; (d) separating any molecules from the binding complex that are not part of the binding complex; and (e) detecting the presence or absence of the binding complex.

40. 40. The method of claim 38 or 39, wherein the antigen-binding molecule is disposed on a surface selected from the group consisting of agarose beads, magnetic beads, plastic well plates, glass well plates, ceramic well plates, and cell culture bags.

41. 40. The method of claim 38 or 39, wherein the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a proteinaceous fluorescent label, a magnetic label, a radioactive label, and a hapten.

42. The fluorescent label may be selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarin, aminocouramin, methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red, and the like. Red), allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTrquoise 2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan), wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange42. The method of claim 41 , wherein the RFP is selected from the group consisting of: 1) Phycoerythrin (Petroleum Orange), 2) mOrange, 3) allophycocyanin (APC), 4) mKO, 5) TurboRFP, tdTomato, 6) TagRFP, 7) DsRed monomer, 8) DsRed2 ("RFP"), 9) mStrawberry, 10) TurboFP602, 11) AsRed2, 12) mRFP1, 13) J-Red, 14) R-phycoerythrin (RPE), 15) B-phycoerythrin (BPE), 16) mCherry, 17) HcRed1, 18) Katusha, 19) P3, 20) Peridinin Chlorophyll (PerCP), 21) mKate (TagFP635), 22) TurboFP635, 23) mPlum, and 24) mRaspberry.

43. The method of any one of claims 38 to 42, wherein the detecting is a flow-based detection method.

44. 44. The method of claim 43, wherein the flow-based detection method is flow virometry.

45. 43. The method of any one of claims 38 to 42, wherein said detecting is performed by ELISA, biolayer interferometry (BLI), Western blot, or any combination thereof.

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