Antigen binding molecules and methods of use

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 489,373, filed on March 9, 2023, and U.S. Provisional Patent Application No. 63 / 620,111, filed on January 11, 2024, each of which is hereby incorporated in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on February 8, 2024, is named K-1137-WO-PCT_SL.xml, and is 312,108 bytes in size. Technical Field

[0005] The present disclosure relates to antigen-binding molecules, such as antibodies, that specifically bind to targets, including the anti-CD20 scFv-14 binding domain or the gibbon ape leukemia virus (GALV) protein gp70, as well as molecules comprising these sequences and cells presenting such molecules, polynucleotides encoding such antigen-binding molecules, and humanized forms of the antigen-binding molecules, and also discloses methods of using the antigen-binding molecules. Background Art

[0006] Antigen binding molecules, including antibodies and fragments such as Fab, F(ab')2, scFv, etc., are used in immunotherapy and solid-phase based applications such as biosensors, affinity chromatography and immunoassays. These antibodies and other antigen binding molecules derive their utility from their ability to specifically bind to their targets.

[0007] Anti-idiotypic antibodies are a subset of antibodies and are antibodies raised against immune antibodies. These anti-idiotypic antibodies exhibit specific binding to the idiotype (unique antigenic determinants on the antibody surface) of the immune antibody. Anti-idiotypic antibodies can generally be classified into three different groups: (1) antibodies that recognize an idiotype that is different from the antigen binding site (ABS) on the immune antibody; (2) antibodies that recognize epitopes within the ABS and mimic the structure and form 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, e.g., Pan et al., (1995) FASEB J 9:43-49).

[0008] There is a further need to detect and quantify viral particles. In particular, viral envelope proteins such as the gibbon ape leukemia virus (GALV) protein gp70 provide excellent targets for viral detection. Antigen-binding molecules specific for GALV gp70 would have many uses, for example in assays such as flow-based viral detection methods.

[0009] Disclosed herein are antigen-binding molecules, including 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 also form an aspect of the present disclosure. Applications and uses of these antigen-binding molecules are also disclosed. Summary of the Invention

[0010] 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 the 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.

[0011] In various aspects, disclosed is an isolated antigen binding molecule 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 a VH of an antigen binding molecule described herein.

[0012] In various aspects, disclosed is an isolated antigen binding molecule 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.

[0013] In various embodiments, the antigen binding molecules of separation include a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 21-41. In various embodiments, the antigen binding molecules of separation include a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 42-65. In various embodiments, the antigen binding molecules of separation include a heavy chain CD3 selected from the group consisting of SEQ ID NO: 66-85. In various embodiments, the antigen binding molecules of separation include a light chain CDR1 selected from the group consisting of SEQ ID NO: 86-99. In various embodiments, the antigen binding molecules of separation include a light chain CDR2 selected from the group consisting of SEQ ID NO: 100-111. In various embodiments, the antigen binding molecules include a light chain CDR3 selected from the group consisting of SEQ ID NO: 112-120.

[0014] 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 molecules comprise 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 the antigen binding molecules described herein.

[0015] 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.

[0016] 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.

[0017] In certain aspects, the antigen binding system, antibody, or antigen binding fragment thereof comprises a GALV gp70 binding motif, wherein the GALV gp70 binding motif comprises the sequence 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 sequence of three light chain CDRs (LCDRs) of a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 315-324.

[0018] In certain aspects, 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), wherein

[0019] (i) HCDR1 has a sequence according to any one of SEQ ID NOs: 127-138, 157-168 and 187-198;

[0020] (ii) HCDR2 has a sequence according to any one of SEQ ID NOs: 139-150, 169-180, and 199-210;

[0021] (iii) HCDR3 has a sequence according to any one of SEQ ID NOs: 151-156, 181-186, 211-222, and DYY;

[0022] (iv) LCDR1 has a sequence according to any one of SEQ ID NOs: 223-232, 253-262, and 283-292;

[0023] (v) LCDR2 has a sequence according to any one of SEQ ID NOs: 233-242, 263-272, SGS, GTN, RAS, DTS, and KVS; and

[0024] (vi) LCDR3 has a sequence according to any one of SEQ ID Nos: 243-252, 273-282 and 293-302.

[0025] In certain aspects, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises HCDRs comprising:

[0026] (i) a HCDR1 according to any one of SEQ ID Nos: 127, 157 and 187; a HCDR2 according to any one of SEQ ID Nos: 139, 169 and 199; a HCDR3 according to SEQ ID NO: 211 or DYY;

[0027] (ii) a HCDR1 according to any one of SEQ ID Nos: 128, 158 and 188; a HCDR2 according to any one of SEQ ID Nos: 140, 170 and 200; a HCDR3 according to any one of SEQ ID Nos: 151, 181 and 212;

[0028] (iii) a HCDR1 according to any one of SEQ ID Nos: 129, 159 and 189; a HCDR2 according to any one of SEQ ID Nos: 141, 171 and 201; a HCDR3 according to any one of SEQ ID Nos: 152, 182 and 213;

[0029] (iv) a HCDR1 according to any one of SEQ ID Nos: 130, 160 and 190; a HCDR2 according to any one of SEQ ID Nos: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY;

[0030] (v) a HCDR1 according to any one of SEQ ID Nos: 131, 161 and 191; a HCDR2 according to any one of SEQ ID Nos: 143, 173 and 203; a HCDR3 according to any one of SEQ ID Nos: 153, 183 and 215;

[0031] (vi) a HCDR1 according to any one of SEQ ID Nos: 132, 162 and 192; a HCDR2 according to any one of SEQ ID Nos: 144, 174 and 204; a HCDR3 according to any one of SEQ ID Nos: 154, 184 and 216;

[0032] (vii) a HCDR1 according to any one of SEQ ID Nos: 133, 163 and 193; a HCDR2 according to any one of SEQ ID Nos: 145, 175 and 205; a HCDR3 according to any one of SEQ ID Nos: 155, 185 and 217;

[0033] (viii) a HCDR1 according to any one of SEQ ID Nos: 134, 164 and 194; a HCDR2 according to any one of SEQ ID Nos: 146, 176 and 206; a HCDR3 according to SEQ ID NO: 218 or DYY;

[0034] (ix) a HCDR1 according to any one of SEQ ID Nos: 135, 165 and 195; a HCDR2 according to any one of SEQ ID Nos: 147, 177 and 207; a HCDR3 according to any one of SEQ ID Nos: 156, 186 and 219;

[0035] (x) a HCDR1 according to any one of SEQ ID Nos: 136, 166 and 196; a HCDR2 according to any one of SEQ ID Nos: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY;

[0036] (xi) a HCDR1 according to any one of SEQ ID Nos: 137, 167 and 197; a HCDR2 according to any one of SEQ ID Nos: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or

[0037] (xii) a HCDR1 according to any one of SEQ ID Nos: 138, 168 and 198; a HCDR2 according to any one of SEQ ID Nos: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY, and

[0038] and LCDRs, which include:

[0039] (i) LCDR1 according to any one of SEQ ID Nos: 232, 262 and 292; LCDR2 according to any one of SEQ ID Nos: 242, 272 and GTN; LCDR3 according to any one of SEQ ID Nos: 252, 282 and 302;

[0040] (ii) 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;

[0041] (iii) LCDR1 according to any one of SEQ ID Nos: 227, 257 and 287; 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;

[0042] (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;

[0043] (v) 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;

[0044] (vi) 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;

[0045] (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;

[0046] (viii) 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;

[0047] (ix) 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

[0048] (x) LCDR1 according to any one of SEQ ID Nos: 230, 260, 290; LCDR2 according to any one of SEQ ID Nos: 240, 270 and GTN; LCDR3 according to any one of SEQ ID Nos: 250, 280 and 300.

[0049] In certain aspects, 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), wherein:

[0050] The HCDR and the LCDR comprise:

[0051] (i) a HCDR1 according to any one of SEQ ID Nos: 127, 157 and 187; a HCDR2 according to any one of SEQ ID Nos: 139, 169 and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID Nos: 232, 262 and 292; a LCDR2 according to any one of SEQ ID Nos: 242, 272 and GTN; a LCDR3 according to any one of SEQ ID Nos: 252, 282 and 302;

[0052] (ii) a HCDR1 according to any one of SEQ ID Nos: 128, 158 and 188; a HCDR2 according to any one of SEQ ID Nos: 140, 170 and 200; a HCDR3 according to any one of SEQ ID Nos: 151, 181 and 212; a LCDR1 according to any one of SEQ ID Nos: 228, 258 and 288; a LCDR2 according to any one of SEQ ID Nos: 238, 268 and KVS; a LCDR3 according to any one of SEQ ID Nos: 248, 278 and 298;

[0053] (iii) a HCDR1 according to any one of SEQ ID Nos: 129, 159 and 189; a HCDR2 according to any one of SEQ ID Nos: 141, 171 and 201; a HCDR3 according to any one of SEQ ID Nos: 152, 182 and 213; a LCDR1 according to any one of SEQ ID Nos: 226, 256 and 286; a LCDR2 according to any one of SEQ ID Nos: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID Nos: 246, 276 and 296

[0054] (iv) a HCDR1 according to any one of SEQ ID Nos: 130, 160 and 190; a HCDR2 according to any one of SEQ ID Nos: 142, 172 and 202; a HCDR3 according to SEQ ID NOs: 214 or DYY; a LCDR1 according to any one of SEQ ID Nos: 227, 257 and 287; a LCDR2 according to any one of SEQ ID Nos: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID Nos: 247, 277 and 297;

[0055] (v) a HCDR1 according to any one of SEQ ID Nos: 131, 161 and 191; a HCDR2 according to any one of SEQ ID Nos: 143, 173 and 203; a HCDR3 according to any one of SEQ ID Nos: 153, 183 and 215; a LCDR1 according to any one of SEQ ID Nos: 227, 257 and 287; a LCDR2 according to any one of SEQ ID Nos: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID Nos: 247, 277 and 297;

[0056] (vi) a HCDR1 according to any one of SEQ ID Nos: 132, 162 and 192; a HCDR2 according to any one of SEQ ID Nos: 144, 174 and 204; a HCDR3 according to any one of SEQ ID Nos: 154, 184 and 216; a LCDR1 according to any one of SEQ ID Nos: 226, 256 and 286; a LCDR2 according to any one of SEQ ID Nos: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID Nos: 246, 276 and 296;

[0057] (vii) a HCDR1 according to any one of SEQ ID Nos: 133, 163 and 193; a HCDR2 according to any one of SEQ ID Nos: 145, 175 and 205; a HCDR3 according to any one of SEQ ID Nos: 155, 185 and 217; a LCDR1 according to any one of SEQ ID Nos: 225, 255 and 285; a LCDR2 according to any one of SEQ ID Nos: 235, 265 and RAS; a LCDR3 according to any one of SEQ ID Nos: 245, 275 and 295;

[0058] (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; HCDR3 according to SEQ ID NOs: 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

[0059] (ix) a HCDR1 according to any one of SEQ ID Nos: 135, 165 and 195; a HCDR2 according to any one of SEQ ID Nos: 147, 177 and 207; a HCDR3 according to any one of SEQ ID Nos: 156, 186 and 219; a LCDR1 according to any one of SEQ ID Nos: 223, 253 and 283; a LCDR2 according to any one of SEQ ID Nos: 233, 263 and SGS; a LCDR3 according to any one of SEQ ID Nos: 243, 273 and 293;

[0060] (x) a HCDR1 according to any one of SEQ ID Nos: 136, 166 and 196; a HCDR2 according to any one of SEQ ID Nos: 148, 178 and 208; a HCDR3 according to SEQ ID Nos: 220 or DYY; a LCDR1 according to any one of SEQ ID Nos: 229, 259 and 289; a LCDR2 according to any one of SEQ ID Nos: 239, 269 and GTN; a LCDR3 according to any one of SEQ ID Nos: 249, 279 and 299;

[0061] (xi) a HCDR1 according to any one of SEQ ID Nos: 137, 167 and 197; a HCDR2 according to any one of SEQ ID Nos: 149, 179 and 209; a HCDR3 according to SEQ ID Nos: 221 or DYY; a LCDR1 according to any one of SEQ ID Nos: 231, 261 and 291; a LCDR2 according to any one of SEQ ID Nos: 241, 271 and GTN; a LCDR3 according to any one of SEQ ID Nos: 251, 281 and 301; or

[0062] (xii) a HCDR1 according to any one of SEQ ID Nos: 138, 168 and 198; a HCDR2 according to any one of SEQ ID Nos: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY; a LCDR1 according to any one of SEQ ID Nos: 230, 260, 290; a LCDR2 according to any one of SEQ ID Nos: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID Nos: 250, 280 and 300.

[0063] 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, wherein:

[0064] (i) the heavy chain variable domain is at least 80% identical to any one of SEQ ID Nos: 303-314; and

[0065] (ii) the light chain variable domain is at least 80% identical to any one of SEQ ID Nos: 315-324.

[0066] 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, wherein:

[0067] (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;

[0068] (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;

[0069] (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;

[0070] (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;

[0071] (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;

[0072] (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;

[0073] (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;

[0074] (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;

[0075] (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;

[0076] (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;

[0077] (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

[0078] (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.

[0079] In certain aspects, a GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is featured, wherein the three HCDRs and the three LCDRs are comprised by a single polypeptide.

[0080] In certain aspects, a GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is featured, wherein the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide.

[0081] In certain aspects, a GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof is featured, wherein the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

[0082] In certain aspects, nucleic acids encoding at least one GALV gp70 binding polypeptide as described above are disclosed.

[0083] In certain aspects, vectors comprising such nucleic acids are disclosed.

[0084] In certain aspects, a method of producing an engineered cell is disclosed, wherein the method comprises transfecting or transducing the cell with a nucleic acid or vector as described immediately above.

[0085] In certain aspects, disclosed is a cell encoding or expressing a GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof, optionally wherein the cell is an immune cell.

[0086] In certain aspects, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof further comprises a detectable label.

[0087] In certain aspects, 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.

[0088] In certain aspects, the GALV gp70 antigen binding system, antibody, or antigen-binding fragment thereof comprises a detectable label that is a fluorescent label selected from the group consisting of: Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5. Cy7, TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan Cyan), wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green Green), ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, Ypet, TurboYFP, ZsYellow1, KusabiraOrange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinocyanin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

[0089] In certain aspects, 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:

[0090] (a) providing a sample known or suspected to contain viral particles expressing GALV gp70 protein;

[0091] (b) contacting the sample with an antigen binding system, antibody, or antigen binding fragment thereof according to any one of claims 21 to 29, under conditions that allow for the formation of one or more bound complexes comprising viral particles and the antigen binding system, antibody, or antigen binding fragment thereof, wherein the antigen binding system, antibody, or antigen binding fragment thereof further comprises a detectable label;

[0092] (c) detecting the one or more bound complexes by detecting the detectable label, and

[0093] (d) determining the number of viral particles present in the sample based on the detection in step (c).

[0094] In certain aspects, disclosed is a method of 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, the method comprising:

[0095] (a) providing a sample known or suspected to contain viral particles expressing GALV gp70 protein;

[0096] (b) providing an antigen-binding molecule that specifically binds to GALV gp70 protein, wherein the antigen-binding molecule further comprises a detectable label;

[0097] (c) contacting the sample with the antigen-binding molecule under conditions that allow formation of a binding complex between GALV gp70 and the antigen-binding protein;

[0098] (d) separating from the binding complex any molecules that are not part of the binding complex; and

[0099] (e) Detecting the presence or absence of the bound complex.

[0100] In certain aspects, a method for determining the number of viral particles or a method for determining the presence or absence of viral particles is featured, wherein the antigen binding molecules are 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.

[0101] In certain aspects, a method for determining the number of viral particles or a method for determining the presence or absence of viral particles is featured, 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.

[0102] In certain aspects, a method for determining the number of viral particles or a method for determining the presence or absence of viral particles is featured, wherein the fluorescent label is selected from the group consisting of: Atto dye, Alexafluor dye, quantum dot, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation) , GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midurish Blue, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGF P, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azomei Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, Ypet, TurboYFP, ZsYellow1, Kusabila 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, Peridin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

[0103] In certain aspects, methods for determining the number of viral particles or methods for determining the presence or absence of viral particles are featured, wherein the detection is performed using a flow-based detection method.

[0104] In certain aspects, a method for determining the number of viral particles or a method for determining the presence or absence of viral particles is featured, wherein the flow-based detection method is a flow cytometric viral assay.

[0105] In certain aspects, methods for determining the number of viral particles or methods for determining the presence or absence of viral particles are featured, wherein detection is performed by ELISA, biolayer interferometry (BLI), Western blot, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 Panels A to E in the figure are experimental data comparing the expression data of transduced T cells.

[0107] Figure 2 Shown are experimental data demonstrating binding of CAR carrying scFv 14 compared to controls.

[0108] Figure 3 Panel A in shows experimental data on the binding of supernatants of potential hybridoma candidates to scFv14.

[0109] Figure 3 Panel B in Figure 2 shows experimental data on the binding of supernatants of potential hybridoma candidates to scFv14.

[0110] Figure 4 Data from experiments evaluating specificity for scFv 14 are shown. Supernatants were screened for binding to T cells expressing CAR carrying scFv 14, carrying an irrelevant anti-CD20 scFv2, or carrying a Leu16-carrying CAR, or NTD T cell controls.

[0111] Figure 5 Data from experiments evaluating the specificity of scFv14 are shown.

[0112] Figure 6 Panel A in Figure 3 shows experimental data for clone 24C12 conjugated to PE screened against healthy donor T cells that either retained the NTD or were transduced to express CARs carrying scFv2, scFv14, or FMC63.

[0113] Figure 6 Panel B in Figure 3 shows experimental data for clone 24C12 conjugated to FITC screened against healthy donor T cells that either retained the NTD or were transduced to express CARs carrying scFv2, scFv14, or FMC63.

[0114] Figure 7 Panel A in Figure 3 shows experimental data for clone 24C12 conjugated to PE screened against healthy donor T cells that either retained the NTD or were transduced to express CARs carrying scFv2, scFv14, or FMC63.

[0115] Figure 7Panel B in Figure 3 shows experimental data for clone 24C12 conjugated to FITC screened against healthy donor T cells that either retained the NTD or were transduced to express CARs carrying scFv2, scFv14, or FMC63.

[0116] Figure 8 A schematic diagram shows the design of the recombinant GALV gp70 viral envelope protein. A secretion signal (SS) was included on 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, including residues 42-616 (numbering according to Uniprot P21415), was prepared using an N-terminal affinity tag. Truncations were prepared using a C-terminal affinity tag, including the coiled-coil region (residues 505-616) or only the receptor-binding domain (residues 42-474).

[0117] Figure 9A Shown are K562 or CHO cells stained with an anti-SLC20A1 antibody (Proteintech catalog number 12423-1-AP) and a PE-conjugated secondary antibody. Cells stained with the secondary antibody alone served as a control. The percentage of cells positive for SLC20A1 surface expression is shown (94.7% for K562 and 0% for CHO). Figure 9B Shown are K562 or CHO cells stained with a dilution series of recombinant GALV gp70 protein, followed by staining with a PE-conjugated secondary antibody. When K562 cells were stained with gp70 (42-474) or (42-616), but not (505-616), the PE mean fluorescence intensity (MFI) increased in a dose-dependent manner. No staining was observed for CHO cells.

[0118] Figure 10A Indirect ELISA titers measured using immobilized GALV gp70 (42-616) protein against immunized mouse sera or normal mouse sera as a negative control are shown. The 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 are shown (left and center) stained with serum from immunized mice selected for hybridoma fusion and a dilution series of PE-conjugated secondary antibodies. Negative controls are shown, including no staining (NS), normal mouse serum (unimmunized mice: NMS1 and NMS2), or isotype control antibody staining of PG13 cells (right).

[0119] Figure 11Shown is a relative comparison of indirect ELISA titers measured using immobilized GALV gp70 proteins (42-616), (505-616), (42-616), or an affinity-tagged negative control protein after incubation with hybridoma supernatants. 17 positive hybridoma clones are shown.

[0120] Figure 12 PG13 cells stably producing viral particles containing GALV gp70 envelope protein were incubated with a dilution series of each hybridoma supernatant and stained with PE-conjugated secondary antibody. The relative PE signal for each hybridoma is shown.

[0121] Figure 13 PG13 cells stably producing viral particles containing the GALV gp70 envelope protein (top) 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.

[0122] 14A to 14I PG13 cells (left), which stably produce viral particles containing the GALV gp70 envelope protein, were incubated with a dilution series of each large-scale purified antibody clone (from a high of 10 μg / mL down to 0.005 μg / mL). NIH-3T3 cells (right) were stained with the same dilution series of the antibody clones as a negative control. Isotype controls were also included for both cell lines stained at a high concentration of 10 μg / mL.

[0123] Figures 15A to 15H Purified antibody clones loaded onto AMC biosensors (2 ug / mL) are shown. Octet sensorgrams (association and dissociation) of all clones are shown, demonstrating binding to 100 nM monoFc-tagged GALV gp70 protein residues (42-474).

[0124] Figure 16 Hybridoma supernatants that did not bind to FMC63-28z at the highest concentration tested are shown. Sample identity is indicated on the left side of each graph. Each graph shows a histogram of fluorescence intensity values ​​(x-axis) plotted against the frequency of their corresponding pattern (y-axis) normalized to that value. DETAILED DESCRIPTION

[0125] In certain aspects, embodiments of the present application relate to anti-idiotypic antigen binding molecules, including antibodies, that specifically bind to antigen binding molecules that specifically bind to anti-CD20 scFv 14 (see Kanyarat Thueng-in, Jeeraphong Thanongsaksrikul, Surasak Jittavisutthikul, Watee Seesuay, Monrat Chulanetra, Yuwaporn Sakolvaree, Potjanee Srimanote, and 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).

[0126] The anti-CD20 scFv-14 has the amino acid sequence:

[0127] VH DNA - CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAAAGAATATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAGTGGTCACACATACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTGCGCCAGAGGGCCTCACTACGACGACTGGAGCGGATTTATCATATGGTTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA (SEQ ID NO: 122)

[0128] VL DNA - GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCTTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGGTTTCCTCCTACCTTTGGCCAAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 123)

[0129] VH protein- QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS (SEQ ID NO: 124)

[0130] VL protein - DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK (SEQ ID NO: 125)

[0131] Also provided are humanized forms of the antigen-binding molecules, molecules comprising anti-CD20 scFv 14, and cells presenting molecules comprising anti-CD20 scFv 14. Also disclosed are polynucleotides encoding the antigen-binding molecules, vectors comprising the polynucleotides, and in vitro cells comprising the polynucleotides and the vectors.

[0132] Methods of using the disclosed antigen binding molecules are provided. The antigen binding molecules, polynucleotides, vectors, in vitro cells, and methods described herein are useful in a range of applications, for example as reagents to detect the presence of, quantify the amount of, screen for, purify, and biomarker research focused on portions comprising anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules. Therapeutic uses are also provided, for example applications in which the biological activity of portions comprising anti-CD20 scFv14 and cells presenting such molecules is modulated (enhanced or inhibited), and dose ranging studies involving therapeutic agents comprising anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules and cells presenting such molecules.

[0133] The antigen binding molecules (e.g., scFv, antibodies, etc.) disclosed herein were generated from hybridomas generated using mouse-derived B cells, but can be readily humanized using standard methods known to those of skill in the art, as well as those methods as described herein. Representative humanized versions of the disclosed antigen binding molecules can be generated as described herein.

[0134] In certain additional aspects, embodiments of the application relate to antigen binding molecules, including antibodies, that specifically bind to viral coat proteins. In certain aspects, the antigen binding molecules bind to the gibbon ape leukemia virus gp70 envelope protein (GALV gp70). GALV gp70 is a surface protein that attaches the virus to a host cell by binding to its receptor.

[0135] GALV gp70 has the amino acid sequence: (SEQ ID NO: 325).

[0136] Additionally, polynucleotides encoding GALV gp70 binding molecules, as well as vectors comprising the polynucleotides and in vitro cells comprising the polynucleotides and the vectors are disclosed.

[0137] Methods of 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 range of applications, such as as reagents to detect the presence of GALV gp70-containing portions, molecules containing this sequence, cells that present such molecules, and viral particles, quantifying the amount of GALV gp70-containing portions, molecules containing this sequence, and cells that present such molecules, screening for GALV gp70-containing portions, molecules containing this sequence, and cells that present such molecules, purifying GALV gp70-containing portions, molecules containing this sequence, and cells that present such molecules, and biomarker studies focused on GALV gp70-containing portions, molecules containing this sequence, and cells that present such molecules. Also provided are therapeutic uses, such as applications in which the biological activity of GALV gp70-containing portions and cells that present such molecules is modulated (enhanced or inhibited), and dose range studies of therapeutic agents involving GALV gp70-containing portions, molecules containing this sequence, and cells that present such molecules.

[0138] I. Definition

[0139] In order to make the present disclosure more easily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout this application. The headings provided herein do not limit the various aspects of the present disclosure, which should be understood by reference to this specification as a whole.

[0140] It should be understood that wherever herein aspects are described with the language "comprising," other similar aspects described as "consisting of" and / or "consisting essentially of" are also provided.

[0141] The units, prefixes, and symbols used herein are provided in their International System of Units (SI) accepted form.Numerical ranges are inclusive of the numbers defining the range.

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. 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, Cammack et al., eds., 2nd ed., (2006), Oxford University Press provides those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0143] As used herein, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g. Immunolo—y - A Synthesis (2nd ed.), Golub and Green, eds., Sinauer Assoc., Sunderland, Mass. (1991), which is incorporated herein by reference for any purpose. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as α-amino acids, α-disubstituted amino acids, N-alkyl amino acids), lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, eN-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, according to standard usage and convention, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction.

[0144] As used herein, the terms "a" and "an" are used according to standard convention and mean one or more, unless the context dictates otherwise.

[0145] As used herein, the term "about" refers to a value or composition 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, according to the practice in the art, "about" or "consisting essentially of..." can mean within one or more standard deviations. Alternatively, "about" or "consisting essentially of..." can mean a range of up to 10% (i.e., ±10%). For example, about 5 mg can include any number from 4.5 mg to 5.5 mg. In addition, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to 5 times of a certain value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "consisting essentially of..." should be assumed to be within an acceptable error range for that specific value or composition.

[0146] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and fractions thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise indicated.

[0147] As used herein, the term "and / or," is understood to mean either one or both of the listed options is present, along with the proviso that one or both alternatives are not excluded. Accordingly, as used herein, such terms as "A and / or B" are intended to encompass the following aspects: "A and B" "A or B" "A" (alone) and "B" (alone). Likewise, as used herein, such terms as "A, B, and / or C" are 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).

[0148] As used herein, the use of the term alternative (e.g., "or") should be understood to mean one of the alternatives is present, both alternatives are present, or neither alternative is present.

[0149] As used herein, the term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0150] The term "antibody" includes naturally occurring and non-naturally occurring (recombinantly produced) antibodies, human and non-human antibodies, mono-specific antibodies, multi-specific antibodies (including bi-specific antibodies), immunoglobulins, synthetic antibodies, four-chain antibodies comprising two heavy chains 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 (see, e.g., Stocks, (2004) Drug Discovery Today 9(22):960-66), antibody fusions (which term encompasses antibody-drug conjugates), and sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen binding fragments thereof. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0151] Unless otherwise indicated, the term "antibody" also encompasses intact immunoglobulins or antigen-binding portions thereof that compete with intact antibodies 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 particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments comprising complementary determining regions (CDRs), single-chain antibodies (scFvs), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0152] As disclosed herein, non-human antibodies can be humanized using recombinant methods to reduce their immunogenicity in humans relative to antibodies that specifically bind to anti-CD20 scFv14, molecules comprising this sequence, and cells presenting such molecules. Unless explicitly stated, and unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments of antigen-binding molecules of any of the aforementioned immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single-chain antibodies (i.e., scFv).

[0153] As used herein, the term "antigen" means any molecule that elicits an immune response or is capable of being bound by an antibody or other antigen-binding molecule. The immune response may involve the production of antibodies, the activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide (including anti-CD20 scFv14), as well as molecules comprising the same sequence and cells presenting such molecules, can serve as an antigen. Typically, the antigen can be endogenously expressed, i.e., expressed from genomic DNA, or it can be recombinantly expressed or chemically synthesized. In a specific embodiment, the antigen comprises all or a portion of anti-CD20 scFv14 and a molecule comprising the same sequence, optionally conjugated to an adjuvant (such as keyhole limpet hemocyanin (KLH)) or Fc to facilitate screening.

[0154] As used herein, the term "antigen binding molecule" means a protein comprising a portion that binds to an antigen or target protein and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding molecule to the antigen. Examples of representative types of antigen binding molecules include scFv, human, mouse, or rabbit antibodies; humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; diabodies; tria antibodies; 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.

[0155] Antigen binding molecules may comprise alternative protein scaffolds or artificial scaffolds, for example, with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing introduced mutations to, for example, stabilize the three-dimensional structure of the antigen binding molecule, and 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). In addition, peptide antibody mimics ("PAMs") and scaffolds based on antibody mimics that utilize various components (e.g., fibronectin) as scaffolds may be used. Antigen binding molecules may have, for example, the structure of naturally occurring immunoglobulins.

[0156] An antigen-binding molecule may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or they 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 are capable of specifically binding to two different antigens (e.g., anti-CD20 scFv14 and a cell surfactant molecule).

[0157] 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, which specifically binds to anti-CD20 scFv14, molecules comprising anti-CD20 scFv14, and cells presenting such molecules. In further embodiments, the antigen binding molecule binds to a CAR comprising anti-CD20 scFv14, molecules comprising anti-CD20 scFv14, and can be expressed on immune cells (such as T cells).

[0158] The term "autologous" refers to any material that originates from the same individual and is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein ™ ) method involves collecting lymphocytes from a patient, then engineering them to express a construct, such as a CAR construct, and then administering them back to the same patient.

[0159] As used herein, the term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). K D By k off / k on The quotient of K A By k on / k off Calculate 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 antibodies and antigens. on and k off It can be determined by techniques known to those skilled in the art, such as BIAcore ® Or KinExA or surface plasmon resonance.

[0160] As used herein, the term "complementarity determining region" or "CDR" refers to an amino acid sequence that contributes to antigen binding specificity and affinity. The framework region helps maintain the correct conformation of the CDR to promote binding between the antigen-binding molecule and the 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 analysis of available complex crystal structures.

[0161] Table 1

[0162]

[0163] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy chain variable region and light chain variable region of an antibody or antigen-binding molecule thereof. In certain aspects, the CDRs of an antibody can be identified according to the Kabat numbering system (see, for example, Kabat et al. Sequences of Proteins of lmmunological Interest t , 5th Edition, NIH Publication 91-3242, Bethesda MD 1991). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at the following amino acid positions: amino acid positions 31 to 35, which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1); amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at the following amino acid positions: amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some embodiments, the CDRs of the antibodies described herein may be described according to the Kabat numbering scheme (although they can be readily interpreted in other numbering systems using Table 1 above). In some embodiments, the CDRs of the antibodies described herein may be described according to the Clothia numbering scheme. In some embodiments, the CDRs of the antibodies described herein can be described according to the IGMT numbering scheme.

[0164] In certain aspects, the CDRs of an antibody can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and 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). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; 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 identified according to the Chothia numbering scheme.

[0165] As used herein, a "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or within the framework region of an antibody or antigen-binding molecule (or fragment thereof) provided herein may be replaced with an amino acid residue having a similar side chain.

[0166] Conservative amino acid substitutions encompassed by the present disclosure may encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These non-naturally occurring amino acid residues include peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues can be divided into several categories based on common side chain properties:

[0167] Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0168] Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0169] Acidic: Asp, Glu;

[0170] Basic: His, Lys, Arg;

[0171] Residues that affect chain orientation: Gly, Pro; and

[0172] Aromatic: Trp, Tyr, Phe.

[0173] Non-conservative substitutions can involve exchanging a member of one of these classes with a member of another class. Such substituted residues can be introduced into regions of human antibodies, such as those homologous to non-human antibodies, or into non-homologous regions of the molecule. Exemplary conservative amino acid substitutions are listed in Table 2 below.

[0174] Table 2

[0175]

[0176] As used herein, the terms "constant region" and "constant domain" are interchangeable and have common meanings in the art. The constant region is the portion of an antibody, e.g., the carboxyl terminal portion of a light chain and / or a heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has an amino acid sequence that is more conserved than the variable domain of an immunoglobulin.

[0177] As used herein, the term "cross competition" means 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, such as where the binding of a binding molecule to an antigen completely blocks the ability of a reference binding molecule to bind to an antigen, or cross competition can be partial, such as where the binding of a binding molecule to an antigen reduces the ability of a reference binding molecule to bind to an antigen. In certain embodiments, the antigen binding molecules that cross compete with the reference antigen binding molecule bind to an epitope that is identical or overlapping with the reference antigen binding molecule. In other embodiments, the antigen binding molecules that cross compete with the reference antigen binding molecule bind to an epitope that is different from the reference antigen binding molecule. Many types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another, for example: solid phase direct or indirect radioimmunoassays (RIA); solid phase direct or indirect enzyme immunoassays (EIA); sandwich competition assays (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 assays, solid phase direct label sandwich assays (Harlow and Lane, 1988) Antibodies, A Laboratory Manual , Cold Spring Harbor Press); use I 125 Labeled 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).

[0178] The term "derivative" refers to a molecule that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, derivatives include covalent modifications, including but not limited to chemical linkage to polymers, lipids, or other organic or inorganic moieties. In certain embodiments, chemically modified antigen binding molecules (derivatives) can have a greater circulating half-life than unchemically modified antigen binding molecules. In some embodiments, derivative antigen binding molecules are covalently modified to include one or more water-soluble polymer attachments, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0179] The term "diabody" or dAB, as used herein, means a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a VH and a VL domain connected by a linker that is too short to allow for pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on the other polypeptide chain (see, e.g., Holliger et al. (1993) Proc Natl Acad Sci U.S.A. 90:6444-48, Poljak et al. (1994) Structure 2:1121-23, and Perisic et al. (1994) Strucure 2(12): 1217-26). If the two polypeptide chains of a diabody are identical, then the diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains of different sequences can be used to make diabodies with two different antigen binding sites. Similarly, triabodies and tetrabodies are antibodies which comprise three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, which can be the same or different.

[0180] As used herein, "epitope" is a term of art and refers to a local region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a contiguous amino acid of a polypeptide (a linear epitope or a continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (a conformational epitope, a nonlinear epitope, a discontinuous epitope, or a noncontinuous 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 to mass spectrometry (e.g., liquid chromatography electrospray ionization mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (e.g., Giege et al., (1994) ActaCrystallogr 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 can be refined using computer software such as X-PLOR (Yale University, 1992, disseminated by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) Vol. 114 and Vol. 115, edited by Wyckoff et al.) and BUSTER (Bricogne, (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne, (1997) Meth Enzymol 276A: 361-423, edited by 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 skilled in the art. See, e.g., Champe et al., (1995) J Biol Chem 270: 1388-94 and Cunningham and Wells, (1989) Science 244: 1081-85 for descriptions of mutagenesis techniques, including alanine and arginine scanning mutagenesis techniques.

[0181] As used herein, the term "Fab fragment" means a monovalent fragment having VL, VH, CL, and CH domains; a "F(ab')2 fragment" is a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; a "Fv fragment" has the VH and VL domains of a single arm of an antibody; and a "dAb fragment" has an antigen-binding fragment of a VH domain, a VL domain, or either a VH or a VL domain.

[0182] As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms in the context of antigen-binding molecules and are used interchangeably, and refer to a given molecule that preferentially binds to an antigen (e.g., an epitope or immune complex), as such binding is understood by those skilled in the art. For example, an antigen-binding molecule that specifically binds to an antigen may bind to other peptides or polypeptides, but with relatively lower affinity, as determined by, for example, immunoassays, BIAcore, or other methods. ® In a specific embodiment, the molecule that specifically binds to the antigen is bound to a molecule with a certain K A When the molecule binds to another antigen, K A is at least 2 log, 2.5 log, 3 log, 4 log or greater.

[0183] In another embodiment, molecules that specifically bind to an antigen (e.g., anti-CD20 scFv14) and molecules comprising the same sequence and cells presenting such molecules) are expressed at about 1 × 10 -7 The dissociation constant (K d ) combination. In some embodiments, when K d About 1 × 10 -9 M to about 5 × 10 -9 M, the antigen binding molecule specifically binds the antigen with "high affinity" (e.g., anti-CD20 scFv14 and molecules comprising the same sequence and cells presenting such molecules). In some embodiments, when K d About 1 × 10 -10 M to about 5 × 10 -10 When M, the antigen-binding molecule specifically binds the antigen with "very high affinity" (e.g., anti-CD20 scFv14, as well as molecules comprising the same sequence and cells presenting such molecules).

[0184] In another embodiment, a molecule that specifically binds to an antigen (e.g., anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules) does not cross-react with other proteins under similar binding conditions. In some embodiments, a molecule that specifically binds to an antigen (e.g., anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules) does not cross-react with other proteins that do not comprise anti-CD20 scFv14, molecules comprising this sequence and cells presenting such molecules. In some embodiments, provided herein is an antibody or fragment thereof that binds to anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules with a higher affinity than to another, unrelated antigen. In certain embodiments, provided herein is an antigen-binding molecule (e.g., an antibody) or fragment thereof that binds to anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater affinity than to another, unrelated antigen, as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In some embodiments, the extent of binding of an antigen-binding molecule, antibody, or antigen-binding fragment thereof that specifically binds to anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules described herein is less than 10%, 15%, or 20% of the binding of the antibody to the linker fragment protein, as measured by, e.g., a radioimmunoassay, as compared to an unrelated protein that does not comprise anti-CD20 scFv14 and molecules comprising this sequence and cells presenting such molecules.

[0185] As used herein, the term "heavy chain" when used in reference to an antibody, can refer to any of the different types based on the amino acid sequence of the constant domain, e.g., alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), which produce the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3, and IgG4.

[0186] As used herein, the term "immunoglobulin" means an immunologic molecule 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 of skill in the art, including but not limited to human IgGl, IgG2, IgG3, and IgG4. Many of the molecules described herein are immunoglobulins. As used herein, "isotype" means the antibody class or subclass (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0187] Immunoglobulins are tetrameric molecules typically composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50 kDa to 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 130 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-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 both light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Berzofsky and Berkower, Ch. 7 Fundamental Immunology (Paul, W., ed., Lippincott Williams & Wilkins (2012); the chapters and volumes of which are incorporated by reference in their entirety for all purposes.) The variable regions of each light / heavy chain pair form the antibody combining site, such that an intact immunoglobulin has two major binding sites.

[0188] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or "CDRs"). From N-terminus to C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain can be based on the phylogenetic tree of Kabat (see, e.g., Kabat et al.). Sequences of Proteins of lmmunological Interest , 5th ed., NIH Publication 91-3242, Bethesda MD (1991)) or Chothia (see, e.g., Chothia and Lesk (1987), J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger and Pluckthun (2001), J Mol Biol 309:657-670) as used herein. The Kabat, Chothia, IGMT, and Abm (Oxford Molecular) numbering systems are more fully described herein.

[0189] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. The in vitro cell may include human cells, such as T cells or dendritic cells, or it may include CHO, sp2 / 0, rabbit and other non-human cells.

[0190] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types based on the amino acid sequence of the constant domain, e.g., kappa (K) or lambda (l). Light chain amino acid sequences are known in the art. In particular embodiments, the light chain is a human light chain.

[0191] The term "neutralize" refers to an antigen binding molecule, scFv, antibody, or fragment thereof that binds to a ligand (e.g., a moiety comprising anti-CD20 scFv14 and 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 a binding site on the ligand, or otherwise alters the binding ability of the ligand by indirect means, such as a structural or energetic change in the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents a protein to which it binds from performing a biological function.

[0192] As used herein, the term "patient" means any person who is being treated for an abnormal physiological condition, such as cancer, or who has been formally diagnosed with a disorder, those who do not have a formally recognized condition, those who are receiving medical care, those who are at risk of developing a condition, and the like. The terms "subject" and "patient" are used interchangeably herein and include human and non-human animal subjects.

[0193] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably herein and mean a compound comprising of amino acid residues covalently linked by peptide bonds. A protein or peptide comprises at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a sequence of a protein or peptide. The term polypeptide encompasses any peptide or protein comprising two or more amino acids linked to one another by peptide bonds. As used herein, the term refers to both short chains (which are also sometimes referred to as peptides, oligopeptides, and oligomers) and longer chains (which are also sometimes referred to as proteins). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The term "polypeptide" includes natural, recombinant, synthetic, or combinations thereof.

[0194] In some respects, polypeptide and / or protein have one or more amino acid whose disappearance, addition and / or replacement of antigen binding molecules.Useful polypeptide fragment can comprise the immunological function fragment of antigen binding molecules, includes but not limited to a part etc. of other parts of one or more CDR districts, variable domains of heavy chain and / or light chain, antibody chain.One or more amino acid whose parts that can replace antigen binding molecules comprise the amino acid whose for example D or L form, be different from the amino acid whose amino acid, disappearance, non-natural existence and amino acid whose chemical analogue that is usually found in the same position of antigen binding molecules.

[0195] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs that have properties similar to those of the template peptide and form an aspect of the present disclosure. These types of non-peptide compounds are referred to as "peptide mimetics" or "peptidomimetic compounds." See, for example, Fauchere, (1986) Adv. Drug Res. (Testa, ed.) 15:29-69; Veber and 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.

[0196] These terms specifically encompass polypeptides, peptides, proteins and similar molecules comprising anti-CD20 scFv14, as well as molecules comprising this sequence and cells presenting such molecules.

[0197] As used herein, the term "percent identity" refers to the percentage of identical residues between the amino acids or nucleotides in the molecules being compared. For these calculations, gaps in the alignment, if any, must be resolved by a specific 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 Computational Molecular Biology , (ed. Lesk), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects , (Ed. Smith), 1993, NewYork: Academic Press; Computer Analysis of Sequence Data, Part I , (eds. Griffin and Griffin), 1994, New Jersey: Humana Press; von Heinje, (1987) Sequence Analysis in Molecular Biology , New York: Academic Press; Sequence Analysis PrimerMethods such as those described in Gribskov and Devereux, eds. (1991) Sequence Analysis Primer, New York: M. Stockton Press; and Carillo et al. (1988) J. Applied Math. 48:1073.

[0198] In calculating percent identity, the sequences in question are optimally aligned by comparison of sequences to give the maximum matching between the sequences. Computer programs for determining percent identity can be, for example, MOE (Chemical Computing Group) or DNASTAR (University of Wisconsin, Madison, WI). The computer algorithm GAP can be used to align two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned by optimal matching of their respective amino acids or nucleotides ("span of matches," as determined by the algorithm). Gap open penalty (which is calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonal of the comparison matrix used; "diagonal" is the score or number assigned by a particular comparison matrix to each perfect amino acid match) and gap extension penalty (which is typically 1 / 10ththe gap open penalty) and a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (see, e.g., Dayhoff et al. (1978) Atlas of Protein Sequence and Structure 3: 101- 153; Dayhoff et al. (1982) in A. MacCorquodale and K. Schellman (eds.), Protein Sequence Atlas of Protein Sequence and Structure 5:345-352; Henikoff et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919).

[0199] Certain alignment schemes for aligning two amino acid sequences can result in matching of only a short region of the two sequences, and this small aligned region can have very high sequence identity even if there is no significant relationship between the two full-length sequences. Thus, if desired, the alignment method chosen (e.g., the GAP program) can be adjusted to yield an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

[0200] As used herein, the terms "single-chain antibody" and "single-chain variable fragment (scFv)" are used interchangeably and mean a V L and V HThe scFv regions are connected via a linker to form an antigen-binding molecule that is a continuous protein chain, wherein the linker is long enough to allow the protein chain to fold back on itself and 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 No. 980250, p. 15, 2012., which is incorporated herein by reference in its entirety for any purpose.) scFv14 is a specific example of scFv.

[0201] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., a portion 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 the onset of disease or promotes disease regression (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free disease periods, or the prevention of impairment or disability resulting from the disease). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0202] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into cells 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 papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0203] As used herein, the terms "variable region" or "variable domain" are used interchangeably and refer to a portion of an antibody, typically a portion of a light chain or a heavy chain, generally located approximately at the amino terminus of the antibody and comprising about 100 to 130 amino acids in the heavy chain and about 90 to 115 amino acids in the light chain, which vary widely in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in 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.

[0204] In certain embodiments, the variable region of the antigen-binding molecule is a human variable region. In other embodiments, the variable region comprises rodent, human, or mouse CDRs and human framework regions (FRs). In other embodiments, the variable region is a primate (e.g., non-human primate) variable region. In other 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).

[0205] The terms "VH," "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.

[0206] The terms "VL," "VL domain," and "VL chain" are used interchangeably and refer to the light chain variable region of an antigen binding molecule, antibody, or antigen binding fragment thereof.

[0207] As used herein, the term "virion" means one or more complete viral particles as well as any portion of one or more viral particles.

[0208] Various aspects of the disclosure are described in further detail in the following subsections.

[0209] II. Overview

[0210] Immunotherapy using T cells engineered to express chimeric antigen receptors (CARs) has shown significant promise in the clinic, with the potential to cure relapsed B-cell malignancies. However, data from multiple clinical studies have identified a key vulnerability of treatment with anti-CD19 CAR T cells: susceptibility of tumor cells to antigen escape (i.e., downregulation or loss of detectable antigen on tumor cells), leading to tumor relapse after treatment. For example, in a Phase 1 / 2 clinical study (ZUMA-1) of axicabtagene ciloleucel, an autologous anti-CD19 CAR T-cell product, 39 of 88 responders (44%) relapsed later after treatment, and among all patients with available post-relapse samples, 4 of 16 patients (25%) had CD19-positive disease at diagnosis and CD19-negative disease after treatment. See, for example, 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 herein by reference in their entirety for any purpose. Additionally, patients receiving tisagenlecleucel (another anti-CD19 CAR T cell therapy) suffered disease relapse driven by antigen loss. See, for example, 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 herein by reference in their entirety for any purpose.

[0211] Nonclinical data have demonstrated that dual targeting of CARs to two independent target cell surface antigens is more effective in vitro and in vivo compared to monovalent CAR T cells, see, for example, 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, the entire text of which is incorporated herein by reference for any purpose. Similar to CD19, CD20 is a cell surface antigen expressed on most healthy B cells (from pre-B cells to memory B cells) as well as leukemia and lymphoma cells. In the context of monoclonal antibody therapy and CAR T cell therapy, proof of concept targeting CD20 has been demonstrated in the clinic and, when combined with CD19 targeting, may represent an effective strategy to reduce the probability 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 entireties of which are incorporated herein by reference for any purpose.

[0212] Both single and dual antigens (such as those targeting anti-CD19 / CD20 CAR T cell therapies) for the treatment of patients with relapsed or refractory B-cell malignancies need to be fully understood and characterized during the development and manufacturing process. More specifically, the embodiments herein describe antibodies specific for scFv14 to characterize specific protein expression of anti-CD20 CARs.

[0213] There is a further need to detect and quantify viral particles. In certain aspects, detection of viral particles can be achieved by detecting 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, for example, in assays such as flow-based viral detection methods.

[0214] III. Antigen Binding Molecules: Specific for Anti-CD20 scFv14

[0215] The present disclosure relates to antigen binding molecules that specifically bind to anti-CD20 scFv14 and molecules comprising the same sequence and cells presenting such molecules, including antibodies, and / or those antigen binding molecules that cross-compete with one or more antigen binding molecules described herein. The heavy chain antigen binding molecules may comprise a set of unique CDR sequences as defined in Table 3A, Table 3B, and Table 3C, and are exemplified by the light chain CDR1, CDR2, and CDR3 sequences provided as listed in Table 4A, Table 4B, and Table 4C. Related clones are found in Tables 5 and 6. In various embodiments, the antigen binding molecules in the form of scFv may include a heavy chain binding molecule connected to a light chain binding molecule via a linker amino acid sequence (e.g., a "Whitlow" linker). Examples of linker sequences are described herein.

[0216] In various embodiments, the antigen binding molecules described herein can be used in one or more methods (eg, those described herein and in the art).

[0217] In various embodiments, the antigen binding molecules may comprise one or more CDRs. In various embodiments, the antigen binding molecules may comprise one or more framework regions. In various embodiments, the antigen binding molecules may comprise three CDRs spaced apart and between four framework regions.

[0218] In various embodiments, the antigen binding molecules may comprise one or more CDRs incorporated into the variable heavy chain. In various embodiments, the antigen binding molecules may comprise one or more CDRs incorporated into the variable light chain. In various embodiments, the antigen binding molecules may comprise a variable heavy chain connected to the variable light chain via a linker.

[0219] In various embodiments, the antigen binding molecules may comprise a light chain. In various embodiments, the antigen binding molecules may comprise a heavy chain. In various embodiments, the antigen binding molecules may comprise a light chain and a heavy chain connected by a disulfide bond. In various embodiments, the antigen binding molecules may comprise a first heavy chain connected to a second heavy chain by a disulfide bond. In various embodiments, the antigen binding molecules may comprise two light chains and two heavy chains.

[0220] The antibodies or antigen-binding molecules encoded by the present disclosure can be single-chain or double-chain. In some embodiments, the antibodies or antigen-binding molecules can be single-chain. In certain embodiments, the antigen-binding molecules can be selected from the group consisting of: scFv, Fab, Fab', Fv, F(ab')2, dAb and any combination thereof. In a specific embodiment, the antibody or antigen-binding molecule can comprise scFv.

[0221] In certain embodiments, an antigen binding molecule such as an antibody may comprise a single chain in which the heavy chain variable region and the light chain variable region may be connected by a linker. H Can be located at the N-terminus of the linker, and V L Can be located at the C-terminus of the linker. In other embodiments, V L Can be located at the N-terminus of the linker, and V H Can be located at the C-terminus of the linker.

[0222] Table 3A: Anti-CD20 Heavy Chain Antigen Binding Molecule CDR1, CDR2 and CDR3 Amino Acid Sequences (Chothia Format) .

[0223]

[0224] Table 3B: Anti-CD20 Heavy Chain Antigen Binding Molecule CDR1, CDR2 and CDR3 Amino Acid Sequences (Kabat Format) .

[0225]

[0226] Table 3C: Anti-CD20 Heavy Chain Antigen Binding Molecule CDR1, CDR2 and CDR3 Amino Acid Sequences (IMGT Format) .

[0227]

[0228] Table 4A: Anti-CD20 Light Chain Antigen Binding Molecule CDR1, CDR2, and CDR3 Sequences (Chothia Format) .

[0229]

[0230] Table 4B: Anti-CD20 Light Chain Antigen Binding Molecule CDR1, CDR2 and CDR3 Sequences (Kabat Format) .

[0231]

[0232] Table 4C: Anti-CD20 Light Chain Antigen Binding Molecule CDR1, CDR2, and CDR3 Sequences (IMGT Format) .

[0233]

[0234] Table 5: Anti-CD20 heavy chain antigen binding molecule variable domain amino acid sequence .

[0235]

[0236] Table 6: Anti-CD20 light chain antigen binding molecule variable domain amino acid sequence .

[0237]

[0238] In one embodiment, the antigen-binding molecules of the present disclosure are antibodies and antigen-binding fragments thereof. In one embodiment, the antibodies of the present disclosure that are specific for anti-CD20 scFv comprise at least one CDR listed in Tables 3A to 3C and Tables 4A to 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 as known in the art.

[0239] Humanized antibodies are described herein and can be prepared by known techniques. In one embodiment, humanized monoclonal antibodies comprise the variable domains (or all or part of its antigen binding site) of mouse or rabbit antibodies and the constant domains derived from human antibodies. Alternatively, humanized antibody fragments can comprise the antigen binding site of mouse or rabbit monoclonal antibodies and the variable domain fragments (lacking antigen binding site) derived from human antibodies. The program for producing engineered monoclonal antibodies includes 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 described in those programs. In one embodiment, chimeric antibodies are CDR-transplanted antibodies. Techniques for humanizing antibodies are discussed, for example, in U.S. Pat. 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.

[0240] 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 of ordinary skill in the art. Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro Immunization, fusion of splenocytes from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V region phage library, or other procedures as known in the art and based on the disclosure herein.

[0241] Methods 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 are introduced into mice to replace the inactivated mouse genes. In this technology, elements of the human heavy chain and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted destruction of the endogenous heavy chain and light chain loci (see also Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58).

[0242] Examples of techniques for the production and use of transgenic animals that produce human or partially human antibodies are described in U.S. Patent 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; Russel 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. (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. (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 Pharmacology113: 49-101; Neuberger, (1996) Nature Biotech 14:826; Taylor et al., (1992) Nucleic Acids Research 20:6287-95; Taylor et al., (1994) Intl Immunol 6:579-91; Tomizuka et al., (1997) Nature Genetics 16:133-43; Tomizuka et al., (2000) Proc Nat Acad Sci USA 97:722-27; Tuaillon et al., (1993) Proc Nat Acad Sci USA 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; U.S. Patent No. 5,877,397; Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., (1995) Ann. N.Y. Acad. Sci. 764:525-35.

[0243] An additional method for obtaining the antigen binding molecules of the present invention is by using phage display, which is 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. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be produced in phage vectors, which can be screened to select Ig fragments (Fab, Fv, sFv or multimers thereof) that bind to scFv-14 and molecules containing this sequence and cells that present such molecules. See, e.g., U.S. Patent 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 multiple polynucleotide sequences encoding Ig variable region segments can be inserted in frame with sequences encoding phage coat proteins into a filamentous phage such as M13 or lambda phage (λImmunoZap). ™ (H) and λImmunoZap ™ (L) vector (Stratagene, La Jolla, Calif.) can also be used in this method) or its variants.

[0244] Briefly, mRNA was isolated from B cell populations and used for λ ImmunoZap ™ (H) and λImmunoZap ™ Heavy and light chain immunoglobulin cDNA expression libraries can be generated in IgG1 (IgE) and similar vectors. These vectors can be screened individually or co-expressed to produce Fab fragments or antibodies. Positive plaques can then be converted to non-lytic plasmids that allow high-level expression of monoclonal antibody fragments from Escherichia coli (E. coli).

[0245] In one embodiment, in a hybridoma, the variable region of the gene expressing the monoclonal antibody of interest is amplified using nucleotide primers. These primers can be synthesized by one of ordinary skill in the art or can be purchased from commercial sources, which also sell primers for mouse and human variable regions, including primers for V H , V L , C H , and C L regions, and the like). 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 mammal-based systems for expression. Using these methods, large amounts of single chain proteins containing fusions of V H , and V L domains can be produced.

[0246] Once cells producing the antigen binding molecules provided herein have been 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 thus produced can be sequenced, and the identified CDRs and DNA encoding CDRs can be manipulated as previously described to produce other antibodies according to the application.

[0247] Those of skill in the art will appreciate that some proteins, such as antibodies, can undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line used to express the protein as well as the culture conditions. Such modifications can include glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and changes in asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue such as lysine or arginine due to the action of carboxypeptidases (as described in, e.g., Harris, (1995) J Chromatog 705:129-34).

[0248] An alternative method for producing murine monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of a syngeneic mouse, e.g., a mouse that has been treated (e.g., pristane-primed) 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) Monoclonal Antibodies: Production, Engineering and Clinical Application, ed. R. L. Rodriguez, Plenum Press, New York, pp. 99- 156). Methods in Molecular BiologyMonoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on specific 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.

[0249] Although the disclosed antigen binding molecules are produced in mouse systems, human, partially human or humanized antibodies may be suitable for many applications, particularly those involving administration of antibodies to human subjects, and other types of antigen binding molecules will be suitable for certain applications. Such antibodies can be prepared as described herein and form one aspect of the present disclosure.

[0250] The present disclosure provides antigen binding molecules that specifically bind to the anti-CD20 scFv-14 binding domain and subsequences thereof, molecules comprising such sequences, and cells presenting such molecules. Antigen binding molecules that cross-compete with the antigen binding molecules disclosed herein form another aspect of the present disclosure.

[0251] 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 a reference antibody.

[0252] IV. Antigen Binding Molecules: Specific for GALV gp70 Protein

[0253] The present disclosure further relates to antigen binding molecules that specifically bind to GALV gp70 protein and molecules comprising the same sequence and cells that present such molecules, including antibodies, and / or those antigen binding molecules that cross-compete with one or more antigen binding molecules described herein. The heavy chain antigen binding molecules may comprise a set of unique CDR sequences as defined in Table 7A, Table 7B, and Table 7C, and are exemplified by the light chain CDR1, CDR2, and CDR3 sequences provided as listed in Table 8A, Table 8B, and Table 8C. Related clones are found in Tables 9 and 10. In various embodiments, the antigen binding molecules in the form of scFv may comprise a heavy chain binding molecule connected to a light chain binding molecule via a linker amino acid sequence (e.g., a "Whitlow" linker). Examples of linker sequences are described herein.

[0254] In various embodiments, the antigen binding molecules described herein can be used in one or more methods (eg, those described herein and in the art).

[0255] In various embodiments, the antigen binding molecules may comprise one or more CDRs. In various embodiments, the antigen binding molecules may comprise one or more framework regions. In various embodiments, the antigen binding molecules may comprise three CDRs spaced apart and between four framework regions.

[0256] In various embodiments, the antigen binding molecules may comprise one or more CDRs incorporated into the variable heavy chain. In various embodiments, the antigen binding molecules may comprise one or more CDRs incorporated into the variable light chain. In various embodiments, the antigen binding molecules may comprise a variable heavy chain connected to the variable light chain via a linker.

[0257] In various embodiments, the antigen binding molecules may comprise a light chain. In various embodiments, the antigen binding molecules may comprise a heavy chain. In various embodiments, the antigen binding molecules may comprise a light chain and a heavy chain connected by a disulfide bond. In various embodiments, the antigen binding molecules may comprise a first heavy chain connected to a second heavy chain by a disulfide bond. In various embodiments, the antigen binding molecules may comprise two light chains and two heavy chains.

[0258] The antibodies or antigen-binding molecules encoded by the present disclosure can be single-chain or double-chain. In some embodiments, the antibodies or antigen-binding molecules can be single-chain. In certain embodiments, the antigen-binding molecules can be selected from the group consisting of: scFv, Fab, Fab', Fv, F(ab')2, dAb and any combination thereof. In a specific embodiment, the antibody or antigen-binding molecule can comprise scFv.

[0259] In certain embodiments, an antigen binding molecule such as an antibody may comprise a single chain in which the heavy chain variable region and the light chain variable region may be connected by a linker. H Can be located at the N-terminus of the linker, and V L Can be located at the C-terminus of the linker. In other embodiments, V L Can be located at the N-terminus of the linker, and V H Can be located at the C-terminus of the linker.

[0260] Table 7A: GALV gp70 heavy chain antigen binding molecule CDR1, CDR2 and CDR3 amino acid sequences (Chothia format) .

[0261]

[0262] Table 7B: GALV gp70 heavy chain antigen binding molecule CDR1, CDR2 and CDR3 amino acid sequences (Kabat format) .

[0263]

[0264] Table 7C: GALV gp70 heavy chain antigen binding molecule CDR1, CDR2 and CDR3 amino acid sequences (IMGT format) .

[0265]

[0266] Table 8A: GALV gp70 light chain antigen binding molecule CDR1, CDR2 and CDR3 sequences (Chothia format) .

[0267]

[0268] Table 8B: GALV gp70 light chain antigen binding molecule CDR1, CDR2 and CDR3 sequences (Kabat format) .

[0269]

[0270] Table 8C: GALV gp70 light chain antigen binding molecule CDR1, CDR2 and CDR3 sequences (IMGT format) .

[0271]

[0272] Table 9: Amino acid sequences of the variable domains of the GALV gp70 heavy chain antigen-binding molecules .

[0273]

[0274] Table 10: Amino acid sequences of the variable domains of the GALV gp70 light chain antigen binding molecules .

[0275]

[0276] Table 11 GALV gp70 binding antibody clones .

[0277]

[0278] 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 shown 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 chain variable regions and light chain variable regions as shown in Table 11. In another aspect, the present disclosure provides hybridomas capable of producing antibodies disclosed herein and methods of producing antibodies from hybridomas, as described herein and as known in the art.

[0279] Humanized antibodies are described herein and can be produced by known techniques. In one embodiment, a humanized monoclonal antibody comprises the variable domain (or all or a portion of the antigen binding site) of a murine 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 murine or rabbit monoclonal antibody and a variable domain fragment (lacking the antigen binding site) derived from a human antibody. Procedures for generating 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 discussed in, e.g., 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.

[0280] The antigen binding molecules of the present application can also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any number of techniques familiar to those of ordinary skill in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein. in vitro

[0281] ​Methods 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 are introduced into mice to replace the inactivated mouse genes. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted disruption of the endogenous heavy and light chain loci (see also Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58).

[0282] Examples of techniques for the production and use of transgenic animals that produce human or partially human antibodies are described in U.S. Patent 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; Russel 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., (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., (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 Pharmacology113: 49-101; Neuberger, (1996) Nature Biotech 14:826; Taylor et al., (1992) Nucleic Acids Research 20:6287-95; Taylor et al., (1994) Intl Immunol 6:579-91; Tomizuka et al., (1997) Nature Genetics 16:133-43; Tomizuka et al., (2000) Proc Nat Acad Sci USA 97:722-27; Tuaillon et al., (1993) Proc Nat Acad Sci USA 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; U.S. Patent No. 5,877,397; Bruggemann et al. (1997) Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al. (1995) Ann. NY Acad. Sci. 764:525-35.

[0283] An additional method for obtaining the antigen binding molecules of the present invention is by using phage display, which is 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. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be produced in phage vectors, which can be screened to select Ig fragments (Fab, Fv, sFv or multimers thereof) that bind to scFv-14 and molecules containing this sequence and cells that present such molecules. See, e.g., U.S. Patent 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 multiple polynucleotide sequences encoding Ig variable region segments can be inserted in frame with sequences encoding phage coat proteins into a filamentous phage such as M13 or lambda phage (λImmunoZap). ™ (H) and λImmunoZap ™ (L) vector (Stratagene, La Jolla, Calif.) can also be used in this method) or its variants.

[0284] Briefly, mRNA was isolated from B cell populations and used for λ ImmunoZap ™ (H) and λImmunoZap ™ Heavy and light chain immunoglobulin cDNA expression libraries can be generated using IgG1 (IgE) and similar vectors. These vectors can be screened individually or co-expressed to generate 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.

[0285] In one embodiment, nucleotide primers are used to amplify the variable region of the gene expressing the monoclonal antibody of interest in a hybridoma. These primers can be synthesized by one of ordinary skill in the art or can be purchased from commercial sources, which also sell primers for mouse and human variable regions, including primers for V H 、V L 、C H and C L 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-based systems for expression. Using these methods, large quantities of V-containing proteins can be produced. H and V L A single-chain protein containing a fusion of the two domains.

[0286] In case any above-mentioned immunity and other technology are used to obtain the cell that produces the antigen binding molecules that this paper provides, just can be by cloning specific antibody gene from wherein separation and amplification DNA or mRNA according to standard procedure as described herein.Can order-check the antibody thus produced, and the DNA of the CDR identified and coding CDR can be manipulated to produce other antibodies according to the present invention as previously described.

[0287] Those skilled in the art will appreciate that some proteins (such as antibodies) can undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line and culture conditions used to express the protein. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are those that lose carboxyl terminal basic residues (such as lysine or arginine) due to the action of carboxypeptidases (as described in, for example, Harris, (1995) J Chromatog 705:129-34).

[0288] An alternative method for producing mouse monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, for example, mice that have been treated (e.g., pristane sensitization) to promote the formation of ascites containing the monoclonal antibodies. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein-A agarose, size exclusion chromatography, and ion exchange chromatography (see, for example, Baines and Thorpe, (1992)). Methods in Molecular Biology Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on specific 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.

[0289] Although the disclosed antigen binding molecules are produced in mouse systems, human, partially human or humanized antibodies may be suitable for many applications, particularly those involving administration of antibodies to human subjects, and other types of antigen binding molecules will be suitable for certain applications. Such antibodies can be prepared as described herein and form one aspect of the present disclosure.

[0290] The present disclosure provides antigen binding molecules that specifically bind to GALV gp70 protein and subsequences thereof, molecules comprising such sequences, and cells presenting such molecules.Antigen binding molecules that cross-compete with the antigen binding molecules disclosed herein form another aspect of the present disclosure.

[0291] 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 a reference antibody.

[0292] a) Antibodies

[0293] Antibodies (Abs) may include, but are not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Generally, antibodies 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 comprises a heavy chain variable region (V H ) and heavy chain constant region (CH). The heavy chain constant region may comprise three constant domains CH1, CH2 and CH3. Each LC chain may comprise a light chain variable region (V L ) and a light chain constant region. The light chain constant region may comprise a constant domain CL. V H and V L The V domain can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant region of Abs mediates the binding of immunoglobulins 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.

[0294] In some embodiments, the antigen-binding molecules of the present invention specifically bind to at least a portion of scFv-14, molecules comprising the same or related sequences, and cells presenting such molecules. In certain embodiments, the antigen-binding molecules of the present disclosure bind to at least a portion of scFv-14 at a concentration of less than 1 × 10 -6 M, less than 1 × 10 -7 M, less than 1 × 10 -8 M or less than 1 × 10 -9 M's K D Specifically binds to at least a portion of scFv-14 and molecules comprising the same or similar sequence and cells presenting such molecules. In a specific embodiment, the antigen binding molecule is expressed in an amount of less than 1 × 10 -7 M's K D Specifically binds to at least a portion of scFv-14, as well as molecules comprising these sequences and cells presenting such molecules. In another embodiment, the antigen binding molecule is expressed in an amount of less than 1 × 10 -8 M's K D Specifically binds to at least a portion of scFv-14 and molecules having the same or similar sequence and cells presenting such molecules. In some embodiments, the antigen binding molecule is present at about 1 × 10 -7 M, about 2 × 10 -7 M, about 3 × 10 -7 M, about 4 × 10 -7 M, about 5 × 10 -7 M, about 6 × 10 -7 M, about 7 × 10 -7 M, about 8 × 10 -7 M, about 9× 10 -7 M, about 1 × 10 -8 M, about 2 × 10 -8 M, about 3 × 10 -8 M, about 4 × 10 -8 M, about 5 × 10 -8 M, about 6 × 10 -8 M, about 7 × 10 -8 M, about 8 × 10 -8 M, about 9 × 10 -8 M, about 1 × 10 -9 M, about 2 × 10 -9 M, about 3 × 10 - 9 M, about 4 × 10 -9 M, about 5 × 10 -9 M, about 6 × 10 -9 M, about 7 × 10 -9M, about 8 × 10 -9 M, about 9 × 10 -9 M, about 1 × 10 -10 M or about 5 × 10 -10 M's K D Binds scFv-14 and molecules comprising the same or similar sequences and cells presenting such molecules. D Calculations can be made using standard methods, as described herein and elsewhere in the art.

[0295] In specific embodiments, the antigen binding molecules of the present disclosure are identified in Tables 3 to 4, and each comprises the identified heavy and light chain amino acid sequences described in those tables.

[0296] b) ScFv

[0297] In various embodiments, the scFv can be a V comprising an immunoglobulin or the like. H and V L In various embodiments, V H and V L The connection may be via a linker.In various embodiments, the scFv does not include the constant regions typically present in antibodies.

[0298] In various embodiments, scFv facilitates phage display, where it is convenient to express the antigen-binding domain as a single peptide. In other embodiments, scFv can be produced directly from subcloned heavy and light chains derived from hybridomas. scFv can be used for a variety of different purposes. In various embodiments, scFv can be incorporated into flow cytometry and immunohistochemical diagnostic assays. In other embodiments, scFv can be used as the antigen-binding domain of an artificial T cell receptor (chimeric antigen receptor).

[0299] c) Connectors

[0300] When used in biotechnology and biotherapy applications, the linker sequence can be peptide-based and can be used in a range of scientifically relevant applications. For example, the linker can simply serve as a spacer to impart desired structural and / or functional properties to a larger molecule. In another example, the linker can impart little or no structural or functional properties to the larger molecule, but can simply serve as a distinguishing feature (e.g., a "marker" or "biomarker" or "tag") to uniquely identify the larger molecule. In another example, the linker can be used to impart a recognizable feature that can serve as a binding site for an antibody against the larger molecule comprising the linker sequence.

[0301] In various embodiments, the joint can comprise a sequence of amino acids that form a peptide. For example, the joint can comprise a peptide sequence of about 20 to about 30 amino acids. In some embodiments, the joint can comprise a peptide sequence of about 25 amino acids. In some embodiments, the joint 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 joint comprises about 8 amino acids to about 18 amino acids (e.g., 10 amino acids).

[0302] In various embodiments, the linker may comprise a flexible portion. For example, the flexible portion may be rich in glycine residues. In various embodiments, the linker may be soluble or partially soluble. For example, the linker may comprise one or more serine and / or threonine residues. In various embodiments, the linker may be V H The N-terminus of V L In other embodiments, the linker can be connected to the C-terminus of H The C-terminus of V L N-terminal connection.

[0303] When the 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 applications of such antibodies include purification and isolation of molecules comprising the linker, characterization of molecules in a specific environment, enrichment of the concentration of a population of molecules comprising and / or presenting the linker, and therapeutic applications.

[0304] In 1993, Whitlow et al. disclosed a synthetic linker peptide comprising 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 an scFv and was designed to remove a proteolytic site identified in a previous linker peptide. Whitlow et al. concluded that this newly designed synthetic linker peptide was more stable to in vitro proteolysis when compared to the previous linker peptide on which its sequence was based, and also exhibited less aggregation than the same previous linker. Whitlow et al. did not disclose any antigen-binding molecules for their second-generation linker peptide.

[0305] 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 a heavy chain amino acid sequence and a light chain amino acid sequence.

[0306] Various other linkers available in the art may be suitable. One example is a "G4S linker" having the following sequence: (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 126). In various embodiments, the linker may comprise SEQ ID NO: 126 and one or more additional Gly residues. In various embodiments, the linker may comprise SEQ ID NO: 126 and one or fewer Gly residues. The length of the linker may vary based on the number of Gly residues.

[0307] V. Polynucleotides Encoding Antibodies and Antigen-Binding Molecules

[0308] Anti-CD20 scFv-14 binding molecule

[0309] In various embodiments, the polynucleotide may encode an antigen binding molecule (SEQ ID NOs: 1-120). In various embodiments, the polynucleotide may encode an antigen binding molecule (SEQ ID NOs: 1-10). In various embodiments, the polynucleotide may encode an antigen binding molecule (SEQ ID NOs: 11-20).

[0310] In some embodiments, the polynucleotides of the invention encode antigen binding molecules, wherein the antigen binding molecules comprise 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: 1-10.

[0311] In some embodiments, the polynucleotides of the invention encode antigen binding molecules, wherein the antigen binding molecules comprise 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: 11-20.

[0312] As will be appreciated by those skilled in the art, variations of the disclosed polynucleotide sequences are possible due to the degeneracy of the genetic code. Accordingly, such variants of the disclosed polynucleotide sequences form an aspect of the present disclosure.

[0313] GALV gp70 binding molecules

[0314] In various embodiments, the polynucleotides may encode antigen binding molecules (SEQ ID NOs: 127-324). In various embodiments, the polynucleotides may encode antigen binding molecules (SEQ ID NOs: 303-314). In various embodiments, the polynucleotides may encode antigen binding molecules (SEQ ID NOs: 315-324).

[0315] In some embodiments, the polynucleotides of the invention encode antigen binding molecules, wherein the antigen binding molecules comprise 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: 303-314.

[0316] In some embodiments, the polynucleotides of the invention encode antigen binding molecules, wherein the antigen binding molecules comprise 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: 315-324.

[0317] As will be appreciated by those skilled in the art, variations of the disclosed polynucleotide sequences are possible due to the degeneracy of the genetic code. Accordingly, such variants of the disclosed polynucleotide sequences form an aspect of the present disclosure.

[0318] Table 12: GALV gp70 heavy chain antigen binding molecule variable domain nucleic acid sequence .

[0319]

[0320]

[0321]

[0322] Table 13: GALV gp70 light chain antigen binding molecule variable domain nucleic acid sequence .

[0323]

[0324]

[0325] VI. Vectors, Cells, and Pharmaceutical Compositions

[0326] In certain aspects, provided herein are vectors comprising polynucleotides of the present disclosure. In some embodiments, the present invention relates to a vector or a set of vectors comprising polynucleotides encoding antibodies or antigen-binding molecules that specifically bind 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 polynucleotides encoding antibodies or antigen-binding molecules that specifically bind to GALV gp70 (SEQ ID NOs. 127-324).

[0327] 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, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0328] In other aspects, cells comprising polynucleotides or vectors of the invention are provided herein. In some embodiments, the invention relates to cells, in vitro cells, comprising polynucleotides encoding antigen binding molecules as described herein. In some embodiments, the invention relates to cells, such as in vitro cells, comprising polynucleotides encoding antibodies or antigen binding molecules thereof that specifically bind to anti-CD20 scFv-14 or GALV gp70 proteins, molecules comprising these sequences, and cells presenting such molecules, as disclosed herein.

[0329] Any cell can be used as a host cell for the 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, for example, Enterobacteriaceae, such as Escherichia, e.g., Escherichia coli; Bacilli, such as Bacilli subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the host cell is a mammalian cell, such as a human cell. In some embodiments, the host cell is a CHO cell, and in other embodiments, the host cell is sp2 / 0 or other murine cell. The host cells of the present invention can be obtained from any source known in the art.

[0330] Other aspects of the present disclosure may relate to compositions comprising polynucleotides as described herein, vectors as described herein, antibodies as described herein, antigen binding molecules and / or in vitro cells as 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.

[0331] In one embodiment, the composition comprises a polynucleotide encoding an antibody or antigen-binding molecule that specifically binds 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 an in vitro cell containing a polynucleotide encoding an antibody or antigen-binding molecule encoded by a polynucleotide disclosed herein.

[0332] In some embodiments, the composition comprises more than one different antibody or antigen binding molecule that specifically binds to anti-CD20 scFv-14 or GALV gp70 protein and molecules comprising these sequences and cells that present such molecules. In some embodiments, the composition comprises more than one antibody or antigen binding molecule that specifically binds to anti-CD20 scFv-14 or GALV gp70 protein and molecules comprising these sequences and cells that present such molecules, wherein the antibody or antigen binding molecule binds to more than one epitope. In some embodiments, the antibodies or antigen binding molecules will not compete with each other for binding to the epitope. In some embodiments, two or more antibodies or antigen binding molecules provided herein are combined together in a pharmaceutical composition. Preferably, such a composition will be suitable for use in subjects, including human administration.

[0333] VII. Exemplary Methods

[0334] The following sections describe various exemplary methods of using the antigen binding molecules disclosed herein. Any of the antigen binding molecules disclosed herein and fragments thereof, including those described in the tables, figures, and accompanying sequence listings, can be used in the disclosed methods.

[0335] In some disclosed methods, T cells may be used. Such T cells may be derived from any source known in the art. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population, or T cells may be obtained from a subject. T cells may 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. In addition, T cells may be derived from one or more T cell lines available in the art. Various techniques known to those skilled in the art (such as FICOLL) may also be used. ™ T cells are obtained from a unit of blood collected from a subject by separation and / or apheresis. Additional methods for isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0336] In various embodiments, antigen binding molecules specifically bind to at least a portion of scFv-14 and / or molecules comprising similar sequences and the cells presenting such sequences. In various other embodiments, antigen binding molecules specifically bind to at least a portion of GALV gp70 protein and / or molecules comprising similar sequences and the viral particles presenting such sequences. In various embodiments, antigen binding molecules can include one or more of the following: (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, light chain and heavy chain can be connected by a joint.

[0337] 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, an antigen binding molecule may be employed that comprises a variable heavy chain variable region that is consistent with an antigen binding molecule of the claims disclosed herein (e.g., a variable heavy chain variable region comprising SEQ ID NOs: 1-10). H sequence of the antigen-binding molecule) V 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 H Amino acid sequence.

[0338] 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, an antigen binding molecule may be employed that comprises a variable heavy chain variable region that is consistent with an antigen binding molecule of the claims disclosed herein (e.g., a variable heavy chain variable region comprising SEQ ID NOs: 303-314). H sequence of the antigen-binding molecule) V 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 H Amino acid sequence.

[0339] In various embodiments of the anti-CD20 scFv-14 binding molecules, the variable heavy chain may comprise one CDR1 in CDR1, wherein CDR1 comprises one of SEQ ID NOs: 21 to 41. In various embodiments, the heavy chain may comprise one CDR2 in CDR2, wherein CDR2 comprises one of SEQ ID NOs: 42 to 65. In various embodiments, the heavy chain may comprise one CDR3 in CDR3, wherein CDR3 comprises one of SEQ ID NOs: 66 to 85.

[0340] In various embodiments of the GALV gp70 binding molecules, the variable heavy chain can comprise one CDR1 in 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 CDR2 in 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 CDR3 in CDR3, wherein CDR3 comprises one of SEQ ID NOs: 151-156, 181-186, 211-222, and DYY.

[0341] 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, an antigen binding molecule comprising a light chain that is identical to an antigen binding molecule of the claims disclosed herein (e.g., comprising a light chain of SEQ ID NOs: 11-20) may be employed. L sequence of the antigen-binding molecule) V 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 L Amino acid sequence.

[0342] In various embodiments of the GALV gp70 binding molecule, the light chain can comprise one of SEQ ID NOs: 315-324. In various embodiments, an antigen binding molecule can be employed that comprises a light chain that is identical to an antigen binding molecule of the claims disclosed herein (e.g., a light chain comprising SEQ ID NOs: 315-324). L sequence of the antigen-binding molecule) V LV 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 L Amino acid sequence.

[0343] In various embodiments of the anti-CD20 scFv-14 binding molecules, the variable light chain can comprise one CDR1 in CDR1, wherein CDR1 comprises one of SEQ ID NOs: 86-99. In various embodiments, the light chain can comprise one CDR2 in CDR2, wherein CDR2 comprises one of SEQ ID NOs: 100-111. In various embodiments, the light chain can comprise one CDR3 in CDR3, wherein CDR3 comprises one of SEQ ID NOs: 112-120.

[0344] In various embodiments of the GALV gp70 binding molecules, the variable light chain can comprise one CDR1 in 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 CDR2 in 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 CDR3 in CDR3, wherein CDR3 comprises one of SEQ ID NOs: 243-252, 273-282, and 293-302.

[0345] In various embodiments, the variable light chain can be linked to the variable heavy chain via a linker (eg, a Whitlow linker).

[0346] In additional embodiments of the disclosed methods, the antigen binding molecule comprises one or more of the following: (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).

[0347] In various embodiments of the disclosed methods, the antigen binding molecule of the anti-CD20 scFv-14 comprises a heavy chain (HC), and the HC may comprise a heavy chain variable region (VH) sequence comprising one of SEQ ID NOs: 1 to 10. In addition, in embodiments of the disclosed methods, an antigen binding molecule 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 a VH of an antigen binding molecule of the claims disclosed herein (e.g., an antigen binding molecule comprising a variable region (VH) sequence of one of SEQ ID NOs: 1 to 10).

[0348] In various embodiments of the disclosed methods, the antigen-binding molecule of GALV gp70 comprises a heavy chain (HC), and the HC may comprise a heavy chain variable region (VH) sequence comprising one of SEQ ID NOs: 303-314. In addition, in embodiments of the disclosed methods, an antigen-binding molecule 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 a VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VH) sequence of one of SEQ ID NOs: 303-314) may be employed.

[0349] In various embodiments of the disclosed methods, the antigen-binding molecule of the anti-CD20 scFv-14 comprises a light chain (LC), and the LC may comprise a light chain variable region (LH) sequence comprising one of SEQ ID NOs: 11-20. In various embodiments of the disclosed methods, the light chain comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3. In addition, in embodiments of the disclosed methods, an antigen-binding molecule 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 a VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VL) comprising any of SEQ ID NOs: 11-20).

[0350] In various embodiments of the disclosed methods, the antigen-binding molecule for GALV gp70 comprises a light chain (LC), and the LC may comprise a light chain variable region (LH) sequence comprising one of SEQ ID NOs: 315-324. 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, in embodiments of the disclosed methods, an antigen-binding molecule 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 a VH of an antigen-binding molecule of the claims disclosed herein (e.g., an antigen-binding molecule comprising a variable region (VL) comprising any of SEQ ID NOs: 315-324).

[0351] In specific embodiments of the disclosed methods, the variable heavy chain comprises SEQ ID NO: 5.

[0352] In specific embodiments of the disclosed methods, the variable light chain comprises SEQ ID NO: 15.

[0353] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0354] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0355] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0356] In particular embodiments of the disclosed methods, with respect to a GALV gp70 binding molecule, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NO: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; a HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215; a HCDR1 according to any one of SEQ ID NOs: 132, 162, and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174, and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184, and 216; a HCDR1 according to any one of SEQ ID NOs: 133, 163, and 193; a HCDR2 according to any one of SEQ ID NOs: 145, 175, and 205; a HCDR3 according to any one of SEQ ID NOs: 155, 185, and 217; a HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; a HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; a HCDR3 according to SEQ ID NO: 218 or DYY; a HCDR1 according to any one of SEQ ID NOs: 135, 165, and 195; a HCDR2 according to any one of SEQ ID NOs: 147, 177, and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186, and 219; a HCDR1 according to any one of SEQ ID NOs: 136, 166, and 196;a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY.

[0357] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0358] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the light chain variable region and the heavy chain variable region of the binding molecule comprise: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR3 according to any one of SEQ ID NOs: 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: 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; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215;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: 133, 163 and 193; HCDR2 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; 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; 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0359] 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.

[0360] a) Methods for determining the number of cells or viral particles presenting a molecule of interest

[0361] There are situations where it may be desirable to determine the number of cells expressing a molecule of interest present in a sample. For example, it may be desirable to determine the number of immune cells expressing a molecule of interest present in a sample obtained from a subject. Alternatively, it may be desirable to determine the number of cells that are transfected and express a molecule of interest, which can be used as a measure of the level of transfection efficiency. The disclosed methods can be used in these and other applications where it is desirable to determine the number of cells expressing a molecule of interest present in a sample.

[0362] Thus, provided is a method of determining the number of cells presenting a molecule in a sample, wherein the molecule comprises an amino acid sequence selected from the group consisting of any one or more of the amino acid sequences described in Tables 3A to 3C, 4A to 4C, or 7A to 7C, 8A to 8C, 9 to 10.

[0363] In one embodiment, a sample is provided comprising cells known 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.

[0364] In specific 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 NO: 25, 46, 70, 32, 54, 39, 62, 80, 90, 98, 104, 116) or any combination.

[0365] 在具体实施方案中,所选择的氨基酸序列是MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLLSTIAGPLLLLLLLLILGPCIINKLVQFINDRISAVKILVLRQKYQALENEGNL(SEQ ID NO: 325)。

[0366] 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 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 specific embodiments, 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. T cells can be autologous, allogeneic, or heterologous. T cells can be CD4+ T cells or CD8+ T cells. When T cells are used in the disclosed methods, they can be in vivo T cells or in vitro T cells. Furthermore, cells may be placed in or isolated from any environment capable of maintaining the cells in a viable form, such as blood, tissue or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, a suitable buffer, and the like.

[0367] In a specific embodiment, the target molecule is a CAR. When the molecule is a CAR, it may comprise a molecule or a fragment thereof selected from the group consisting of: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, 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 (ITGA2), CD49f (ITGA3), CD49f (ITGA4), CD49f (ITGA5), CD49f (ITGA6), CD49f (ITGA7), CD49f (ITGA8), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA1), CD49f (ITGA9 ...18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA9), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA9), CD49f (ITGA9), CD49f (ITGA9), CD TGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β 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), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (B TLA), 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 co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα,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, Toll-like receptor, and combinations thereof.

[0368] The sample is then contacted with an antigen-binding molecule that specifically binds to the molecule of interest and comprises a detectable label under conditions that allow the formation of a binding complex comprising the cells present in the sample and the antigen-binding molecule. The antigen-binding molecule is preferably an antigen-binding molecule (or fragment thereof) disclosed herein, e.g., in the figures, sequence listing, or this section of the disclosure. Any antigen-binding molecule that specifically binds to all or a portion of the anti-CD20 scFv-14 molecule can be used in the disclosed methods. A number of examples of suitable antigen-binding molecules are provided herein, e.g., those having one or more of the CDRs shown in any of the tables presented herein, such as Tables 3A-3C, 4A-4C, or 7A-7C, 8A-8C.

[0369] Any detectable label can be used in the method, and a desired set of criteria can be used to select an appropriate label. Examples of types of detectable labels include fluorescent dyes selected from the group consisting of Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation) , GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midurish Blue, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGF P, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azomei Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mcitrine, YPet, TurboYFP, ZsYellow1, Kusabila 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, Peridin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Other types of detectable labels include optical dyes described in Johnson, Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition, Life Technologies, (2010), which is hereby expressly incorporated by reference, radiolabels (e.g., isotopic labels such as 3 H. 11 C. 14 C.15 N. 18 F. 35 S. 64 CU, 90 Y. 99 Tc, 111 In, 124 I. 125 I. 131 I), photochromic compounds, magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are known in the art and can be used in the disclosed methods.

[0370] The label can be associated with the antigen-binding molecule at any position in the molecule, but it is preferred that the label be associated with the molecule at one point (or multiple positions if multiple labels are used) so that the binding properties of the molecule are not modified (unless such modified 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 other aspects, 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.

[0371] In specific embodiments of the disclosed methods, with respect to the 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.

[0372] In specific embodiments of the disclosed methods, with respect to the 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.

[0373] In specific embodiments of the disclosed methods, with respect to the 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.

[0374] In specific embodiments of the disclosed methods, for the GALV 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: The sequence of any one of 243-252, 273-282 and 293-302.

[0375] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR2 according to any one of SEQ ID NOs: 153, 183, and 214; a HCDR3 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; HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; 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; HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196;a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY.

[0376] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0377] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the light chain variable region and the heavy chain variable region of the binding molecule comprise: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR3 according to any one of SEQ ID NOs: 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: 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; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215;a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 132, 162 and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174 and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184 and 216; a LCDR1 according to any one of SEQ ID NOs: 226, 256 and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276 and 296; a HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193 ... according to any one of SEQ ID NOs: 145, 175 and 205; according to any one of SEQ ID NOs: 155, 185 and 217; according to any one of SEQ ID NOs: 225, 255 and 285; according to any one of SEQ ID NOs: 235, 265 and RAS; according to any one of SEQ ID NOs: 245, 275 and 295; according to any one of SEQ ID NOs: 134, 164 and 194; according to any one of SEQ ID NOs: 146, 176 and 206; according to any one of SEQ ID NOs: 218 or DYY; according to any one of SEQ ID NOs: 224, 254 and 284; according to any one of SEQ ID NOs: 234, 264 and GTN; according to any one of SEQ ID NOs: 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0378] The antigen binding molecules may be disposed on any surface or not at all. For example, the antigen binding molecules may be present in a buffer, and the buffer-antigen binding molecules may be contacted with the sample. Alternatively, the antigen binding molecules may be associated with a surface. Suitable surfaces include agarose beads, magnetic beads (such as DYNABEADS ™ ) or plastic, glass or ceramic plates (such as well plates), bags (such as cell culture bags), etc. The surface itself can be arranged in another structure, such as a column.

[0379] Conditions that allow formation of the binding complex will depend on a variety of factors, however generally aqueous buffers at physiological pH and ionic strength, such as in phosphate buffered saline (PBS), will facilitate formation of the binding complex and are preferred in the disclosed methods.

[0380] Next, the number of cells present in the bound complex in the sample is determined. The specific method used to determine the number of cells present in the bound complex will depend on the nature of the label selected. For example, when a fluorescent label is selected, FACS can be used; when an isotopic label is selected, mass spectrometry, NMR or other techniques can be used; when 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 the cell number to be calculated based on the output.

[0381] b) Methods for determining the presence or absence of a molecule

[0382] It is valuable to have the ability to separate populations of different molecules, and particularly biologically related molecules, from one another. Using the antigen binding molecules provided herein, such separations can be achieved and used in a range of biotechnology, biopharmaceutical and therapeutic applications.

[0383] In one aspect of the present disclosure, a method of isolating a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule is provided. In another aspect of the present disclosure, a method of isolating a molecule comprising one or more of the amino acid sequences described in Tables 3A to 3C and Tables 4A to 4C is provided.

[0384] In another aspect of the present disclosure, a method of isolating a molecule comprising all or a portion of a GALV gp70 molecule is provided. In another aspect of the present disclosure, a method of isolating a molecule comprising one or more of the amino acid sequences described in Tables 7A to 7C and Tables 8A to 8C is provided.

[0385] 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. In one embodiment, the method comprises providing a sample known or suspected to contain all or a portion of a GALV gp70 molecule.

[0386] In one embodiment, the method comprises providing a sample known or suspected to contain one or more of the amino acid sequences of interest.

[0387] In specific 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 NO: 25, 46, 70, 32, 54, 39, 62, 80, 90, 98, 104, 116) or any combination.

[0388] 在具体实施方案中,所选择的氨基酸序列是MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLLSTIAGPLLLLLLLLILGPCIINKLVQFINDRISAVKILVLRQKYQALENEGNL(SEQ ID NO: 325)。

[0389] In a specific embodiment, the target molecule is a CAR. When the molecule is a CAR, it may comprise a molecule or a fragment thereof selected from the group consisting of: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, 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 (ITGA2), CD49f (ITGA3), CD49f (ITGA4), CD49f (ITGA5), CD49f (ITGA6), CD49f (ITGA7), CD49f (ITGA8), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA1), CD49f (ITGA9 ...18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA9), CD49f (ITGA9), CD49f (ITGA1), CD49f (ITGA9), CD49f (ITGA9), CD49f (ITGA9), CD TGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β 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), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (B TLA), 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 co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα,LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, Toll-like receptor, and combinations thereof.

[0390] Provided is an antigen binding molecule that specifically binds to all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein and optionally comprises a detectable label. Where a detectable label is determined, any detectable label may be employed in the method, as described herein, and a desired set of criteria may be used to select an appropriate label. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, cherry red, coumarin, methylcoumarin, 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, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Pharmacology, 2002). 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 hereby expressly incorporated by reference, radiolabeled (e.g., isotopically labeled such as 3 H. 11 C. 14 C. 15 N. 18 F. 35 S. 64 CU, 90 Y. 99 Tc, 111 In,124 I. 125 I. 131 I). Photochromic compounds, Halo tags, Atto dyes, Tracy dyes, protein fluorescent markers (e.g., protein fluorescent markers also include, but are not limited to, green fluorescent proteins, including GFP from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6: 178-182), enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150: 5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be used in the disclosed methods.

[0391] The label can be associated with the antigen-binding molecule at any position in the molecule, but it is preferred to associate the label with the molecule at one point at one position (or multiple positions if multiple labels are used) so that the binding properties of the molecule are not modified (unless such modified binding activity is desired). With respect 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, such as those disclosed herein, for example, those having one or more of the CDRs shown in Tables 3A to 3C and Tables 4A to 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, such as those disclosed herein, for example, those having one or more of the CDRs shown in Tables 7A to 7C and Tables 8A to 8C.

[0392] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0393] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0394] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0395] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR2 according to any one of SEQ ID NOs: 153, 183, and 214; a HCDR3 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; HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; 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; HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196;a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY.

[0396] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0397] In particular embodiments 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: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NO: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a LCDR1 according to any one of SEQ ID NOs: 226, 256, and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266, and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276, and 296; a HCDR1 according to any one of SEQ ID NOs: 130, 160, and 190; a HCDR2 according to any one of SEQ ID NOs: 142, 172, and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161, and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173, and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183, and 215;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: 133, 163 and 193; HCDR2 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; 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; 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0398] The antigen binding molecules may be disposed on any surface or not at all. For example, the antigen binding molecules may be present in a buffer, and the buffer-antigen binding molecules may be contacted with the sample. Alternatively, the antigen binding molecules may be associated with a surface. Suitable surfaces include agarose beads, magnetic beads (such as DYNABEADS ™ ) or plastic, glass or ceramic plates (such as well plates), bags (such as cell culture bags), etc. The surface itself can be arranged in another structure, such as a column.

[0399] The sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex comprising a molecule containing the selected amino acid sequence and the antigen-binding molecule. The conditions that allow the formation of the binding complex will depend on a variety of factors, but generally, an aqueous buffer at physiological pH and ionic strength, such as phosphate-buffered saline (PBS), will facilitate the formation of the binding complex and is preferred in the disclosed methods. Since the component parts of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on the surface.

[0400] At this stage, a binding complex may not yet have formed, or a plurality of binding complexes may have formed comprising one or more antigen-binding molecules bound to a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule or GALV gp70 protein. Unbound molecules comprising the selected amino acid sequence and / or unbound antigen-binding molecules may also be present in the local environment of any formed binding complex.

[0401] Then separate any molecule that is not a part of the binding complex from any formation.Removal method will depend on the structure and / or local environment of the binding complex.For example, if the antigen binding molecules are arranged on a pearl, plate or bag, the unbound components of the reaction mixture can be washed away using the complete solution of the binding complex formed.If the binding complex is arranged on a pearl, the pearl itself can be located in a post or other structure, and the same method can be used.

[0402] The solution used to induce the formation of the binding complex can be used, for example, as a washing solution to remove unbound components. Any suitable buffer or solution that does not destroy the formed binding complex can be used. Generally, when performing this step of the method, it is preferred to avoid buffers with high salt concentrations, non-physiological pH, containing chaotropic agents or denaturants.

[0403] The resulting binding complex is then separated into (a) a molecule comprising all or a portion of the anti-CD20 scFv-14 molecule or GALV gp70 protein, and (b) an antigen binding molecule (e.g., one or more of SEQ ID NOs. 21-120) or a sequence comprising one or more of SEQ ID NOs: 127-324. Separation can be achieved using standard methods known to those skilled in the art. For example, a solution having an appropriate pH and composition can be washed over the complex. A solution commonly used for this purpose is 0.1 M glycine HCl, pH 2.5-3.0, and this solution can be used to achieve separation. Other solutions that can be used include 100 mM citric acid (pH 3.0), 50 mM to 100 mM triethylamine or triethanolamine (pH 11.5); 150 mM ammonium hydroxide (pH 10.5); 0.1 M glycine NaOH (pH 10.0); 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 M to 3.0 M sodium thiocyanate, 0.1 M Tris-acetate with 2.0 M NaCl (pH 7.7); 2 M to 6 M guanidine HCl, 2 M to 8 M urea, 1.0 M ammonium thiocyanate, 1% sodium deoxycholate, 1% SDS; and 10% dioxane, 50% ethylene glycol (pH 8-11.5).

[0404] After separation, if a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule or GALV gp70 protein is of primary interest, that molecule can be collected; alternatively, if an antigen-binding molecule is of primary interest, that antigen-binding molecule can be collected.

[0405] c) Methods for determining the presence or absence of molecules

[0406] As disclosed herein, it may sometimes 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, knowing that such a molecule is being expressed, regardless of expression level, may be beneficial. In other cases, it may be desirable to know whether a purification process or step designed to remove such a molecule is effective. Thus, qualitatively determining the presence or absence of a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule can be used in a variety of applications. The amino acid sequences described in Tables 3A to 3C and Tables 4A to 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.

[0407] As disclosed herein, it may sometimes be desirable to isolate molecules comprising all or a portion of a GALV gp70 protein. In other cases, simply knowing the presence or absence of a molecule comprising all or a portion of a GALV gp70 protein molecule in a sample may be sufficient information. For example, knowing that such a molecule is being expressed, regardless of expression level, may be beneficial. In other cases, it may be desirable to know whether a purification process or step designed to remove such a molecule is effective. Thus, qualitatively determining the presence or absence of a molecule comprising all or a portion of a GALV gp70 protein can be used in a variety of applications. The amino acid sequences described in Tables 7A to 7C and Tables 8A to 8C 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 a GALV gp70 protein.

[0408] In view of this, a method is provided for determining the presence or absence of a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein in a sample.

[0409] 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 or a GALVgp70 protein.

[0410] In a specific embodiment, all or a portion of the anti-CD20 scFv-14 molecule is a CAR. When the molecule is a CAR, it may comprise a molecule or fragment thereof selected from the group consisting of CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, 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), CD 49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158 A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (K IR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), 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 co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1),IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, Toll-like receptor and combinations thereof.

[0411] Provided are antigen-binding molecules (e.g., SEQ ID NOs: 20-120) that specifically bind to an antigen-binding molecule (e.g., all or a portion of an anti-CD20 scFv-14 molecule). In other aspects, provided are antigen-binding molecules (e.g., SEQ ID NOs: 127-324) that specifically bind to an antigen-binding molecule (e.g., all or a portion of a GALV gp70 protein). A suitable 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, edible cherry red, coumarin, methylcoumarin, 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, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Pharmacol. 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 hereby expressly incorporated by reference, radiolabeled (e.g., isotopically labeled such as 3 H. 11 C. 14 C. 15 N. 18 F. 35 S. 64 CU,90 Y. 99 Tc, 111 In, 124 I. 125 I. 131 I). Photochromic compounds, Halo tags, Atto dyes, Tracy dyes, protein fluorescent markers (for example, protein fluorescent markers also include but are not limited to green fluorescent protein, including GFP from Renilla, Cirrhilabrus or Aequorea species (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150: 5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be used in the disclosed methods.

[0412] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0413] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0414] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0415] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR2 according to any one of SEQ ID NOs: 153, 183, and 214; a HCDR3 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; HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; 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; HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196;a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY.

[0416] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0417] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the light chain variable region and the heavy chain variable region of the binding molecule comprise: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR3 according to any one of SEQ ID NOs: 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: 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; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215;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: 133, 163 and 193; HCDR2 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; 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; 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0418] The label can be associated with the antigen-binding molecule at any position in the molecule, but it is preferred to associate the label with the molecule at one point at one position (or multiple positions if multiple labels are used) so that the binding properties of the molecule are not modified (unless such modified binding activity is desired). Any antigen-binding molecule that specifically binds to all or a portion of an anti-CD20 scFv-14 molecule or GALV gp70 protein, such as those disclosed herein, for example, those having one or more of the CDRs shown in Tables 3A to 3C, Tables 4A to 4C, or Tables 7A to 7C, Tables 8A to 8C, can be used.

[0419] Next, the sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex comprising the molecules present in the sample and the antigen-binding molecule. The antigen-binding molecule may be disposed on any surface or not at all. For example, the antigen-binding molecule may be present in a buffer, and the buffer-antigen-binding molecule may be contacted with the sample. Alternatively, the antigen-binding molecule may be associated with a surface. Suitable surfaces include agarose beads, magnetic beads (such as DYNABEADS™ ) or plastic, glass or ceramic plates (such as well plates), bags (such as cell culture bags), etc. The surface itself can be arranged in another structure, such as a column.

[0420] The sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex comprising a molecule comprising all or a portion of an anti-CD20 scFv-14 molecule or GALVgp70 protein and the antigen-binding molecule. The conditions that allow the formation of the binding complex will depend on a variety of factors, but generally, an aqueous buffer at physiological pH and ionic strength, such as phosphate-buffered saline (PBS), will facilitate the formation of the binding complex and is preferred in the disclosed methods. Since the components of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on the surface.

[0421] At this stage, a binding complex may not have formed, or a plurality of binding complexes may have formed 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]. Unbound molecules comprising all or a portion of an anti-CD20 scFv-14 molecule and / or unbound antigen-binding molecules may also be present in the local environment of any formed binding complexes.

[0422] In other aspects, at this stage, no binding complexes may have formed, or multiple binding complexes may have formed comprising one or more antigen-binding molecules bound to molecules comprising all or a portion of a GALV gp70 protein. Unbound molecules comprising all or a portion of a GALV gp70 protein and / or unbound antigen-binding molecules may also be present in the local environment of any formed binding complexes.

[0423] Then separate any molecule that is not a part of the binding complex from any formation.Removal method will depend on the structure and / or local environment of the binding complex.For example, if the antigen binding molecules are arranged on a pearl, plate or bag, the unbound components of the reaction mixture can be washed away using the complete solution of the binding complex formed.If the binding complex is arranged on a pearl, the pearl itself can be located in a post or other structure, and the same method can be used.

[0424] The solution used to induce the formation of the binding complex can be used, for example, as a washing solution to remove unbound components. Any suitable buffer or solution that does not destroy the formed binding complex can also be used. Generally, when performing this step of the method, buffers with high salt concentrations, non-physiological pH, or containing chaotropic agents or denaturants should be avoided.

[0425] Finally, the presence or absence of a binding complex is detected, which will comprise 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 those described in Tables 3A to 3C, 4A to 4C, or 7A to 7C, 8A to 8C, or 9 to 10). The specific method used to detect the presence or absence of the binding complex will depend on the nature of the label selected. For example, when a fluorescent label is selected, FACS can be used; when an isotopic label is selected, mass spectrometry, NMR, or other techniques can be used; when a magnetic label is selected, magnetic-based cell sorting can be used; or microscopy can be used. The end result of this method is a qualitative assessment of the presence or absence of a detectably labeled antigen-binding molecule, and therefore the presence or absence of its binding partner, which comprises all or a portion of an anti-CD20 scFv-14 molecule or a GALV gp70 protein.

[0426] As is the case with all of the disclosed methods, the molecules comprising the anti-CD20 scFv-14 molecule or all or a portion of the GALV gp70 protein can be disposed in any environment. In preferred embodiments, the molecules comprising the anti-CD20 scFv-14 molecule or all or a portion of the GALV gp70 protein are expressed on the surface of a cell. In this embodiment, the cell can be of any type and can be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In one preferred embodiment, the cell is an immune cell. The immune cell of the method can be any type of immune cell (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). T cells, including T cytotoxic, T helper, and Treg cells, are particularly preferred. In specific embodiments, the cell is a T cell, which can be obtained as described herein and by methods known in the art. Any type of immune cell can be employed in this embodiment of the disclosed methods, and the cell can be a human or non-human cell. Exemplary cells include, but are not limited to, immune cells such as T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells. The T cell can be autologous, allogeneic, or heterologous. In further embodiments, the cell is a CAR-presenting T cell. The T cell can be a CD4+ T cell or a CD8+ T cell. When a T cell is employed in the disclosed methods, the T cell can be an in vivo T cell or an in vitro T cell.

[0427] In additional embodiments, the cell can be disposed in or isolated from any environment capable of maintaining the cell in a viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture media, ex vivo grown tissue, a suitable buffer, and the like.

[0428] d) Methods for increasing molecular concentration

[0429] Typically, the molecule of interest is present in the sample at a lower level than desired. For example, when cells are transfected with a foreign gene, the expression level of the protein encoded by the foreign gene is lower. This is also true for molecules secreted from cells; if the molecule comprises an anti-CD20 scFv-14 molecule from all or a portion of an anti-CD20 scFv-14 molecule and / or one or more of the molecules described in Tables 3A to 3C and 4A to 4C, such molecules are typically present in lower amounts but can still be detected using the methods provided herein. This is also true for viral particles; if the viral particles comprise all or a portion of the GALV gp70 protein, such viral particles are typically present in lower amounts but can still be detected using the methods provided herein. One solution to the problem of lower expression levels is to increase the concentration of the molecule of interest, which can be free in solution or expressed on the cell surface. The concentration of the molecule of interest expressed intracellularly can also be increased, however, the cells must first be lysed to release the molecule.

[0430] To address this issue, a method is provided 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 to 3C and 4A to 4C. In another aspect, described herein is a method for increasing the concentration of viral particles presenting molecules comprising all or a portion of a GALV gp70 protein and / or one or more molecules.

[0431] In one embodiment, the method comprises providing a sample comprising cells known or suspected of comprising a molecule comprising an amino acid sequence selected from the group consisting of all or a portion of an anti-CD20 scFv-14 molecule and / or one or more molecules described in Tables 3A to 3C and 4A to 4C. In another embodiment, the method comprises providing a sample comprising cells known or suspected of comprising a molecule comprising an amino acid sequence selected from the group consisting of all or a portion of a GALV gp70 protein and / or one or more molecules described in Tables 7A to 7C and 8A to 8C.

[0432] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0433] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0434] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0435] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR2 according to any one of SEQ ID NOs: 153, 183, and 214; a HCDR3 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; HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; 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; HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a 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.

[0436] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0437] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the light chain variable region and the heavy chain variable region of the binding molecule comprise: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR3 according to any one of SEQ ID NOs: 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: 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; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215;a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 132, 162 and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174 and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184 and 216; a LCDR1 according to any one of SEQ ID NOs: 226, 256 and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276 and 296; a HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193 ... according to any one of SEQ ID NOs: 145, 175 and 205; according to any one of SEQ ID NOs: 155, 185 and 217; according to any one of SEQ ID NOs: 225, 255 and 285; according to any one of SEQ ID NOs: 235, 265 and RAS; according to any one of SEQ ID NOs: 245, 275 and 295; according to any one of SEQ ID NOs: 134, 164 and 194; according to any one of SEQ ID NOs: 146, 176 and 206; according to any one of SEQ ID NOs: 218 or DYY; according to any one of SEQ ID NOs: 224, 254 and 284; according to any one of SEQ ID NOs: 234, 264 and GTN; according to any one of SEQ ID NOs: 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0438] In a specific embodiment, the molecule comprising all or a portion of an anti-CD20 scFv-14 molecule and / or one or more of the molecules described in Tables 3A to 3C and 4A to 4C is a CAR. When the molecule is a CAR, it may comprise a molecule or fragment thereof selected from the group consisting of CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, 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), CD49 ... TGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD1 50 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F 2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LI GHT), CD268 (BAFFR), 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 co-stimulator (ICOS), LFA-1 (CD11a / CD18),NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll-like receptor, and combinations thereof.

[0439] Provided is an antigen-binding molecule that specifically binds to all or a portion of an anti-CD20 scFv-14 molecule (e.g., one or more of the molecules described in Tables 3A to 3C and 4A to 4C) or an antigen-binding molecule that specifically binds to a GALV gp70 protein and optionally comprises a detectable label. Where a detectable label is preferred, any detectable label can be used in the method, as described herein, and a desired set of criteria can be used to select an appropriate label. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, edible cherry red, coumarin, methylcoumarin, 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, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Pharmacol. 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 hereby expressly incorporated by reference, radiolabeled (e.g., isotopically labeled such as 3 H. 11 C. 14 C. 15 N. 18 F. 35 S.64 CU, 90 Y. 99 Tc, 111 In, 124 I. 125 I. 131 I). Photochromic compounds, Halo tags, Atto dyes, Tracy dyes, protein fluorescent markers (for example, protein fluorescent markers also include but are not limited to green fluorescent protein, including GFP from Renilla, Cirrhilabrus or Aequorea species (Chalfie et al., (1994) Science 263:802-805), EGFP (Clon-tech Labs., Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6: 178-182), enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150: 5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be used in the disclosed methods.

[0440] The label can be associated with the antigen-binding molecule at any position in the molecule, but it is preferred that the label be associated with the molecule at one point at one position (or multiple positions if multiple labels are used) so that the binding properties of the molecule are not modified (unless such modified 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 an anti-CD20 scFv-14 molecule (e.g., one or more molecules described in Tables 3A to 3C and 4A to 4C; or one or more molecules comprising all or a portion of an anti-CD20 scFv-14 molecule bound to an antigen-binding molecule or fragment thereof) can be used, such as those disclosed herein, for example, those having one or more of the CDRs described in Tables 3A to 3C and 4A to 4C. With respect to GALV gp70 protein, any antigen-binding molecule or fragment thereof that specifically binds to all or a portion of GALV gp70 protein, such as those disclosed herein, for example, those having one or more of the CDRs shown in Tables 7A to 7C and Tables 8A to 8C, or fragments thereof, can be used.

[0441] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0442] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0443] In specific embodiments of the disclosed methods, the antigen binding molecule of the 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.

[0444] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the heavy chain variable region of the binding molecule comprises: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a HCDR1 according to any one of SEQ ID NOs: 129, 159, and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171, and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182, and 213; a HCDR2 according to any one of SEQ ID NOs: 153, 183, and 214; a HCDR3 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; HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; 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; HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196;a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NO: 221 or DYY; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY.

[0445] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecule, the light chain variable region of the binding molecule comprises: a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a LCDR1 according to any one of SEQ ID NOs: 228, 258, and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268, and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278, and 298; a LCDR1 according to any one of SEQ ID NOs: 227, 257, and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267, and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277, and 297; a LCDR1 according to any one of SEQ ID NOs: 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: 235, 265 and RAS; LCDR3 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: 235, 265 and RAS; 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; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300. ;

[0446] In specific embodiments of the disclosed methods, with respect to the GALV gp70 binding molecules, the light chain variable region and the heavy chain variable region of the binding molecule comprise: a HCDR1 according to any one of SEQ ID NOs: 127, 157, and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169, and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262, and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272, and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282, and 302; a HCDR1 according to any one of SEQ ID NOs: 128, 158, and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170, and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181, and 212; a LCDR3 according to any one of SEQ ID NOs: 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: 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; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215;a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; a HCDR1 according to any one of SEQ ID NOs: 132, 162 and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174 and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184 and 216; a LCDR1 according to any one of SEQ ID NOs: 226, 256 and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276 and 296; a HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193 ... according to any one of SEQ ID NOs: 145, 175 and 205; according to any one of SEQ ID NOs: 155, 185 and 217; according to any one of SEQ ID NOs: 225, 255 and 285; according to any one of SEQ ID NOs: 235, 265 and RAS; according to any one of SEQ ID NOs: 245, 275 and 295; according to any one of SEQ ID NOs: 134, 164 and 194; according to any one of SEQ ID NOs: 146, 176 and 206; according to any one of SEQ ID NOs: 218 or DYY; according to any one of SEQ ID NOs: 224, 254 and 284; according to any one of SEQ ID NOs: 234, 264 and GTN; according to any one of SEQ ID NOs: 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; 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; LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; 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; 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; 251, 281 and 301; or a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

[0447] The antigen binding molecules (e.g., one or more molecules described in Tables 3A to 3C, 4A to 4C, 7A to 7C, 8A to 8C) can be disposed on any surface or not at all. For example, the antigen binding molecules can be present in a buffer, and the buffer-antigen binding molecules can be contacted with the sample. Alternatively, the antigen binding molecules 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 plates), bags (such as cell culture bags), etc. The surface itself can be arranged in another structure, such as a column.

[0448] 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 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 specific embodiments, 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. T cells can be autologous, allogeneic, or heterologous. In another embodiment, the cells are CAR-presenting T cells. 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. In addition, the cells can be placed in or isolated from any environment capable of maintaining the cells in a viable form, such as blood, tissue or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, suitable buffer, etc.

[0449] 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 a GALV gp70 protein and a molecule of the antigen-binding molecule (e.g., one or more molecules described in Tables 3A to 3C, 4A to 4C, 7A to 7C, or 8A to 8C). The conditions that allow the formation of the binding complex will depend on a variety of factors, but generally, an aqueous buffer at physiological pH and ionic strength, such as phosphate-buffered saline (PBS), will facilitate the formation of the binding complex and is preferred in the disclosed methods. Since the component parts of the binding complex can be disposed on a surface as described herein, the formed binding complex can also be disposed on the surface.

[0450] At this stage, a binding complex may not have formed, or a plurality of binding complexes may have formed comprising one or more antigen-binding molecules (e.g., one or more molecules described in Tables 3A to 3C, 4A to 4C, 7A to 7C, 8A to 8C) bound to all or a portion of an anti-CD20 scFv-14 molecule or GALV gp70 protein. Unbound molecules comprising an anti-CD20 scFv-14 molecule, all or a portion of a GALV gp70 protein and / or unbound antigen-binding molecules (e.g., one or more molecules described in Tables 3A to 3C, 4A to 4C, 7A to 7C, 8A to 8C) may also be present in the local environment of any formed binding complex.

[0451] In some embodiments, the present invention relates to a method for removing antigen binding molecules from a plurality of target genes. The method comprises the steps of separating the target genes from the target genes and / or the binding complex of the target gene ...

[0452] The solution used to induce the formation of the binding complex can be used, for example, as a washing solution to remove unbound components. Any suitable buffer or solution that does not destroy the formed binding complex can also be used. Generally, when performing this step of the method, buffers with high salt concentrations, non-physiological pH, or containing chaotropic agents or denaturants should be avoided.

[0453] At this stage of the method, there will be a population of cells presenting molecules comprising all or a portion of the anti-CD20 scFv-14 molecule or viral particles presenting the GALV gp70 protein. If a detectable label is employed, the concentration of cells or viral particles can be readily determined, consistent with the nature of the label. Cells or viral particles that do not express molecules comprising all or a portion of the anti-CD20 scFv-14 molecule or the GALV gp70 protein will not be present, and thus the population (or concentration) of cells presenting all or a portion of the anti-CD20 scFv-14 molecule or viral particles presenting the GALV gp70 protein will be increased compared to the level prior to performing the method.

[0454] If the concentration of the molecule comprising all or a portion of the anti-CD20 scFv-14 molecule and / or one or more molecules described in Tables 3A to 3C, 4A to 4C, or viral particles presenting GALV gp70 protein and / or one or more molecules described in Tables 7A to 7C and 8A to 8C is not at the desired level, the above steps can be repeated a desired number of times. In the context of this step of the method, the desired number of times can also be zero if the desired cell concentration is already present.

[0455] Example

[0456] Hyperimmune mice were immunized with scFv14 fused to the crystallizable fragment (Fc) domain of mouse immunoglobulin protein isotype G2a. These culture supernatants were screened for the presence of antibodies that specifically bind to scFv14. Antibodies were generated using antibody heavy and light chain gene sequences (e.g., SEQ ID NOs: 1-20) from hybridomas selected from this screen, and these antibodies were again tested for their specificity for scFv14. Selected antibodies from this screen were conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC) fluorophores and characterized by flow cytometry.

[0457] Of 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 (an anti-CD19 CAR carrying FMC63) or to untransduced T cells from the same healthy donor. Ten of the 14 clones were found suitable for antibody production. Following antibody production, all 10 clones were retested for selectivity and sensitivity against scFv14 and an irrelevant anti-CD20 CAR, as well as an anti-CD19 CAR carrying FMC63. Five of these clones were conjugated to fluorophores for characterization by flow cytometry. One of these clones, 24C12, demonstrated robust, sensitive, and specific binding to scFv14, but not to an irrelevant anti-CD20 CAR or an anti-CD19 CAR carrying FMC63, and was selected as a flow cytometry reagent to characterize scFv14 expression in anti-CD20 samples.

[0458] Antibody Production and Characterization Activities Antibody clone 24C12 was generated that strongly bound to the anti-CD20 scFv contained in the anti-CD20 CAR, but not to an irrelevant anti-CD20 CAR or anti-CD19 CAR scFv, whether conjugated to PE or FITC. The methods described in detail in the Examples section below.

[0459] 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 Water Stain, 24C12 PE, and 24C12 FITC. Abbreviations: AF, Alexa Fluor; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; PE, phycoerythrin. LC is a custom-made antibody that binds to the peptide linker between the light and heavy chains of the chimeric antigen receptor (CAR) single-chain variable fragment (scFv), enabling 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 a peptide linker between the light and heavy chains of the chimeric antigen receptor (CAR) single-chain variable fragment (scFv), enabling assessment of overall CAR transduction efficiency. Equipment used in this study included a Sorvall LegendXTR centrifuge, a Vi-Cell XR, and a FACs Fortessa X-20 II. Abbreviation: FACS, fluorescence-activated cell sorter.

[0460] These studies used an anti-CD19 CAR and three anti-CD20 CARs (Table 14). The anti-CD19 CAR (FMC63-28z) includes the FMC63 anti-CD19 targeting scFv. The three anti-CD20 CARs differ only in the scFv used to target CD20; the scFvs include scFv2, scFv14, and Leu16. scFv14 and scFv2 are fully human anti-CD20 scFvs that differ in their complementarity determining regions. Leu16 is a murine anti-human CD20 scFv. See, for example, 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 using IgBLAST (Ig Basic Local Alignment Search Tool) was performed for Leu16 and FMC63 germline gene identification.

[0461] Table 14. CAR scFv used in this study

[0462]

[0463] Abbreviations: CAR, chimeric antigen receptor; scFv, single-chain variable fragment Vh; variable heavy chain; Vl, variable light chain; Vk, variable region gene segment.

[0464] I. Example 1: Generation of Hybridomas

[0465] Abveris DiversimAb was immunized with scFv-Fc protein derived from scFv14 and mouse IgG2a Fc ™ Hyperimmunized mice (Canton, MA). Hybridoma supernatants were first tested for sensitivity to CARs carrying scFv14 in a dilution series and, as a negative control, to CARs carrying FMC63, and then tested in a selective screen against CARs containing scFv2, scFv14, and Leu16. Binders that were selected to bind to scFv14 but not FMC63, scFv2, or Leu16 underwent antibody sequencing and recombinant protein production. Purified recombinant antibodies were retested to confirm specificity for scFv14 and subsequently conjugated to a fluorophore. The conjugated antibodies were rescreened for specificity and sensitivity, and one clone was selected for detection of scFv14.

[0466] II. Example 2: Hybridoma Sensitivity Screening

[0467] To test the sensitivity of hybridoma supernatants, a dilution series screen was performed. Healthy donor T cells, either untransduced (NTD) or transduced with a scFv14-carrying CAR or a FMC63-carrying CAR, were incubated with hybridoma supernatants and serially diluted in staining buffer. Negative controls consisted of an immunoglobulin (Ig) isotype control, conditioned medium from an unrelated hybridoma, and pooled serum from pre-immunized mice. Polyclonal serum from immunized mice was used as a positive control.

[0468] The cells were incubated with the diluted supernatant at room temperature (RT) for 45 minutes, then harvested and washed twice with staining buffer. The samples were stained. The supernatant and control samples were stained with goat anti-mouse IgG conjugated to Alexa Fluor (AF)-488 (1:4,000). A control for determining overall CAR expression was 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). The cells were stained at room temperature for 45 minutes, harvested, and washed twice with staining buffer, then immediately detected by BD Fortessa. ™ Read on flow cytometer. ™Data were analyzed using BD Biosciences software (BD Biosciences, version 10.6), and events were systematically gated on cells (using forward scatter [FSC]-area versus side scatter [SSC]-area plots), single cells (using FSC-area versus FSC-height plots), live cells (viability dye), and phycoerythrin (PE) (for CAR control antibodies) or AF-488 (for supernatant samples), where gating thresholds were set based on NTD control cells.

[0469] III. Example 3: Hybridoma Specificity Test

[0470] For specificity testing, human T cells transduced with CARs carrying scFv2, scFv14, or Leu16 scFv were used. To ensure robust staining in specificity screening, supernatants showing binding to scFv14 were tested at the highest concentration used in the dilution screen. The cells were stained and analyzed.

[0471] IV. Example 4: Characterization of recombinant antibodies

[0472] After sequencing the variable heavy (VH) and variable light (VL) domains of antibodies from hybridomas selected from dilution series and specificity screening, antibodies were manufactured by Genscript (Piscataway, NJ) according to standard procedures. Briefly, proteins were expressed using Expi293F cells and affinity purified using MabSelect SuRe LX (GE Healthcare, catalog number 17-5474-02). Purity was assessed by SDS-PAGE and SEC-HPLC. Antibodies were sterile-filtered using a 0.22-µm filter, packaged aseptically, and stored at −80°C.

[0473] For recombinant antibody specificity and selection studies, healthy human donor T cells transduced with CARs carrying scFv2, scFv14, or FMC63 were harvested, washed twice with staining buffer, and then incubated with either anti-scFv14 antibody or mouse IgG isotype control (both at 300 ng / mL), or in the absence of primary antibody as a negative control. NTD healthy donor T cell controls were also included. The cells were stained and analyzed.

[0474] V. Example 5: Conjugation of fluorophores to recombinant antibodies

[0475] The anti-scFv14 antibody 24C12 was sent to BD Pharmingen ™Custom conjugation with the fluorophores polyethylene (PE) and fluorescein isothiocyanate (FITC) was performed. Briefly, the heterobifunctional cross-linking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was coupled to PE, and SMCC-PE was covalently conjugated to the reduced antibody. Subsequently, the 1:1 PE-conjugated antibody was purified and the buffer exchanged to PBS using size exclusion chromatography. A reactive FITC molecule was coupled to EV-aFMC63 according to standard protocols to couple the fluorophore to primary amines on the antibody. Unconjugated fluorophore was removed, and the buffer was exchanged to PBS (pH 7.4) using standard size exclusion chromatography.

[0476] VI. Example 6: Characterization of Fluorophore-Conjugated Antibodies

[0477] To characterize the fluorophore-conjugated 24C12 antibody, donor T cells transduced with CARs carrying scFv2, scFv14, or FMC63 were harvested, washed twice with staining buffer, and then incubated with serial dilutions of 24C12 labeled with PE or FITC. NTD T cells from the same healthy donor, isotype controls, and unstained controls 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 purchased from BD Fortessa. ™ Read on flow cytometer. ™ Data were analyzed using BD Biosciences software (BD Biosciences, version 10.6), and events were systematically gated for cells (using forward scatter [FSC]-area versus side scatter [SSC]-area plots), single cells (using FSC-area versus FSC-height plots), live cells (vitality dye), and PE, FITC, or AF-647, with gating thresholds set based on NTD control cells.

[0478] VII. Example 7: Hybridoma Supernatant Characteristics

[0479] A panel of 25 cryopreserved hybridomas and associated culture supernatants (Research 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, the hybridomas (Table 15) were sequenced and recombinant antibodies were produced and characterized.

[0480] Table 15. Hybridoma supernatant characteristics

[0481]

[0482] Abbreviations: NB, no binding.

[0483] Note: This table lists the corresponding total protein concentration in the hybridoma, supernatant and affinity coefficient (KD (M)) as estimated by Octet equilibrium binding assay (see ELN EXP-20-BD1634) for the supernatant.

[0484] VIII. Example 8: Hybridoma Sensitivity and Dilution Series Screening

[0485] To assess the relative binding sensitivity of the hybridoma culture supernatants to scFv-14, supernatants were serially diluted and screened against NTD T cells, CAR T cells containing scFv-14, or CAR T cells containing FMC63. Polyclonal serum from mice immunized (Abveris ™ KIT19025-1) was used as a positive control. Pooled normal serum from unimmunized mice, conditioned media (CM) from an unrelated hybridoma, and mouse IgG isotype control were used as negative controls.

[0486] Figure 1 Figure A in shows that both CARs carrying scFv14 and FMC63 were expressed by the transduced T cells (73.3% and 83.7% LC + , respectively), as determined by staining with control anti- linker antibody LC. LC is a custom antibody that binds the peptide linker between the light and heavy chains of the CAR scFv, enabling assessment of total CAR transduction efficiency. CM, isotype control, and normal mouse serum controls showed no binding to scFv-14 ( Figure 1 Figures B-D in ). No binding of these controls to FMC63-28z CAR cells was observed (data not shown). As expected, CAR T cells carrying scFv14 stained positively with polyclonal anti-serum ( Figure 1 Figure E in ). None of the supernatants showed binding to NTD T cells (data not shown) or to T cells expressing CAR carrying FMC63 (data not shown).

[0487] Because low antibody concentrations in the supernatant could result in relatively poor binding, a range-finding preliminary screen was performed to determine an appropriate range for testing relative sensitivity in the serial dilution assay. Based on the results of the preliminary screen (data not shown), 25 hybridoma culture supernatants were binned into two groups; 9 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 supernatants 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).

[0488] 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 2 supernatants that showed binding to the scFv14-carrying CAR but not to the NTD control (see Figure 2 ) or with CAR carrying FMC63 ( Figure 16 The hybridoma that showed binding to scFv14 but not to NTDT cells or FMC63-28z at the lowest dilution in each of its corresponding series was selected for specificity screening.

[0489] Results from the dilution series showed that a total of 14 hybridoma supernatants bound to scFv-14, but none bound to the NTD control or anti-CD19 CAR T cells. The following hybridomas were further selected for specificity screening: 13C2, 22H5, 23C11, 24C12, 25B2, 25F10, 29F8, 29F1, 23D4, 23E1, 24C7, 26E1, 18D7, and 24D9.

[0490] There was a large shift in the mean fluorescence intensity (MFI) between the selected hybridomas. Figure 3 The binding of each of the 14 hybridomas selected for specificity screening at the highest concentration tested is summarized compared to the MFI of the sample. + The cell fraction (see Figure 3 A) and AF-488 + The MFI of the population (see Figure 3B).

[0491] IX. Example 9: Hybridoma-specific screening

[0492] To assess specificity for scFv14, supernatants were screened for binding to T cells expressing CAR carrying scFv14, carrying an irrelevant anti-CD20 scFv2 or a CAR carrying Leu16, or NTD T cell controls. Pooled polyclonal post-immune antisera served as positive controls, while negative controls consisted of pooled pre-immune mouse sera, CM from an irrelevant hybridoma, and a mouse IgG isotype control (see Figure 4 As determined by LC-PE staining, the overall CAR expression of CAR T cells carrying 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 antiserum. Since the immunogen was an anti-CD20 CAR, the polyclonal antiserum was expected to react with various shared domains in the CARs tested in this assay. Subsequently, the 14 hybridomas that showed binding to scFv14 in the dilution series (see Figure 5 ). Including LC PE + The control was used as a reference to determine the expression percentage of each CAR. The results indicated that all 14 hybridoma supernatants specifically bound to scFv-14 but not to any of the negative controls.

[0493] X. Example 10: Selection of Hybridomas for Sequencing and Antibody Production

[0494] The target of the dilution series and selection screening described in this paper embodiment is to select the candidate hybridoma for generation.Based on these results, 10 of the 14 test specific hybridomas are selected for sequencing and antibody production.If the sequence is too similar to another clone or appears to be a weak binder in the hybridoma supernatant screening, some clones are not selected.10 hybridomas include 18D7, 22H5, 23C11, 23E1, 24C12, 24C7, 25B2, 25F10, 29F1, 29F8.

[0495] XI. Example 11: Antibody Binding Confirmation and Selection for Characterization

[0496] To confirm the selectivity of the antibody clones for scFv-14, the antibody clones were screened against NTD T cells or T cells expressing CARs carrying 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 showed specific binding to scFv-14. Based on these results, all 8 clones were sent to BD Biosciences. ™ (San Diego, CA) for conjugation with PE and FITC.

[0497] XII. Example 12: Post-conjugation characterization of 24C12

[0498] Send to BD ™ Only 5 of the 8 recombinant antibodies intended for conjugation with fluorescent dyes arrived at Kite Pharma in time. ™ Although all five anti-scFv-14 antibodies were found to be selective for the anti-CD20 scFv component of KITE-363 based on their overall excellent binding characteristics (data not shown), one antibody, 24C12, was selected for final characterization.

[0499] Antibody clone 24C12 conjugated to PE or FITC was screened against healthy donor T cells that either retained the NTD or were transduced to express a CAR carrying scFv2, scFv14, or FMC63. Antibody 24C12 showed specific binding to scFv14 regardless of the fluorophore conjugate (see Figure 6 At the highest concentration of antibody tested (256 ng / mL), PE + (See Figure 6 A) or FITC + (See Figure 6 B) The fraction of cells showing selective binding to the CAR carrying scFv14 and lacking binding to irrelevant CARs carrying scFv2 or FMC63. For reference, overall CAR expression in T cells transduced with CARs carrying scFv2, scFv14, and FMC63, as determined by staining with LC-AF647, was 80.6%, 78.7%, and 80.7%, respectively (see Figure 6 ).

[0500] 24C12 PE (see Figure 7 A) and 24C12 FITC (see Figure 7 B) Serial dilutions showed that both were selective for scFv14.

[0501] XIII. Example 13: Generation of recombinant protein immunogens and screening reagents

[0502] 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 a mouse Fc, human mono Fc, or His tag for use as potential immunogens and screening agents. These proteins included the receptor binding domain (RBD) of GALV gp70 (residues 42–474), the coiled-coil (CC) domain of GALV gp70 (residues 505–616), or the entire predicted viral surface-exposed portion of GALV gp70 encompassing the RBD + CC domains (residues 42–616) ( Figure 8 The CC domain consists of heptad repeats that associate to form a non-covalent trimer. Analytical size exclusion chromatography demonstrated that recombinant proteins containing the CC domain form trimers.

[0503] 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 fluorescently conjugated anti-human Fc secondary antibody. An anti-SLC20A1 antibody (Proteintech, catalog number 12423-1-AP) was included as a control. Recombinant GALV gp70 protein containing the RBD bound to K562 cells in a dose-dependent manner, whereas protein consisting of only the CC domain did not bind to K562 cells. No binding was observed to negative control cells ( Figures 9A to 9B Together, this data demonstrates correct structure and function, giving us confidence in our reagents for antibody generation and screening.

[0504] XIV. Example 14: Hybridoma Activity and Screening

[0505] Immunization with Abveris DiversimAb using replication-incompetent empty retroviral (RVV) particles (without payload) containing the envelope protein GALV gp70 ™ and DiverGimab ™Superimmunized mice (Canton, MA). Mice were boosted with recombinant murine IgG2a Fc tagged GALV gp70 protein (residues 42-616) or empty RVV particles. Mouse sera were titered by dilution series by indirect ELISA coated with recombinant soluble huIgG1 monoFc tagged long arm GALV gp70 protein (residues 42-616, residues 42-474, or residues 505-616). An irrelevant huIgG1 monoFc tagged protein was used as a negative control. Mouse sera were also tested for sensitivity to a PG13-based stable packaging cell line that constitutively produces virus particles containing GALV gp70 envelope protein by flow cytometry in dilution series. PG13 cells were stained with normal mouse sera (NMS), no stain (NS), or isotype control as negative controls. Two mice were selected for hybridoma fusion based on positive ELISA and flow cytometry data FIG. 10A to FIG. 10B ).

[0506] XV. Example 15: Hybridoma Supernatant Screening

[0507] Supernatants from thousands of hybridoma fusions were screened using indirect ELISA coated with recombinant soluble huIgG1 monoFc tagged long arm GALV gp70 protein (residues 42-616). An irrelevant huIgG1 monoFc tagged protein was used as a negative control. Only 60 positive binders were identified, which were then subjected to secondary ELISA screening coated with recombinant soluble huIgG1 monoFc tagged long arm GALV gp70 protein (residues 42-616, residues 505-616, or residues 42-474). An irrelevant 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, one of the hybridoma cell lines was found to be non-viable, reducing the number of positive hybrids to 16.

[0508] Supernatants from the 16 live positive hybridoma lines tested in the ELISA-based screen were screened for sensitivity to a PG13-based stable packaging cell line that constitutively produces virus particles containing GALV gp70 envelope protein by flow cytometry in dilution series (residues 42-474) Figure 12). NIH-3T3 parental cells were used as negative controls and incubated with the same hybridoma supernatants and serially diluted with staining buffer. Negative controls consisted of immunoglobulin (Ig) isotype controls and normal mouse serum from two different mice (unimmunized). Cells were incubated with diluted supernatants for 45 minutes at room temperature (RT) and then harvested and washed twice with staining buffer. Supernatant and control samples were stained with PE-conjugated secondary antibodies. Data were analyzed using FlowJo ™ software (BD, version 10.6) and cells (using forward scatter [FSC]-area versus side scatter [SSC]-area plots), single cells (using FSC-area versus FSC-height plots), and phycoerythrin (PE) on events were systematically gated, with gating thresholds set based on negative control cells. Eight of the 16 live 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.

[0509] XVI. Example 16: Recombinant Antibody Sensitivity and Specificity Testing

[0510] All hybridoma clones that were positive for binding to GALV gp70 by ELISA were sequenced using next-generation sequencing (NGS). After sequencing the antibody variable heavy chain (VH) and variable light chain (VL) domains from the hybridomas, the antibodies were cloned into standard mammalian expression vectors as murine IgG2a format and manufactured at small scale. All unique VH and VL sequences produced a set of 12 recombinant clone antibodies that were functionally tested at small scale if the purification yield was high enough. 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 6). Figure 13 The following clones had sufficient purification yield for testing and were positive for PG13 staining: 8G8, 9A1, 40A3, 4F1, 35C11, and 9G11.

[0511] XVII. Example 17: Purified Antibody Sensitivity and Dilution Series Screening

[0512] To confirm the small-scale manufacturing and screening results and to retest clones with poor small-scale purification yields, antibodies were manufactured on a larger scale. Briefly, antibodies were expressed in ExpiCHO cells using the ExpiFectamine CHO Transfection Kit (ThermoFisher, Catalog No. A29133). Antibodies were affinity purified on HiTrap MabSelect SuRe columns (Cytiva, Catalog No. 11003493), followed by size exclusion chromatography on HiLoad Superdex 200 16 / 600 (Cytiva, Catalog No. 28989335). A panel of nine antibodies was successfully produced, and the purity of each antibody was >95% as determined by SDS-PAGE gels and analytical SEC (SEC-UPLC). Antibodies were sterile filtered using 0.22 µm filters and stored at −80°C. The following clones were selected for further analysis: 35C11, 3C8, 40A3, 40A6, 8G8, 9A1, 9G11, 2D3, and 4F1.

[0513] To test the sensitivity and specificity of the purified antibodies, a titration / 3-fold dilution series (from the highest concentration of 10 μg / mL down to 0.005 μg / mL) was performed, and PG13 cells were incubated with each antibody or the highest concentration of mouse IgG isotype control ( 14A to 14I NIH-3T3 cells served as a negative control and were incubated with the same purified antibody dilution series in staining buffer. Both cell lines were also stained with the secondary antibody alone (in the absence of the primary antibody) as a negative control. The cells were stained and analyzed.

[0514] XVIII. Example 18: Affinity ranking and specificity testing of purified antibodies using bio-layer interferometry

[0515] The relative affinity ranking, epitope binning, and specificity of the purified antibodies were measured by biolayer interferometry using Octet Red96 (Sartorius). Briefly, purified antibodies were loaded onto AMC biosensors (Cat. No. 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, residues 42-474, or residues 505-616). Figures 15A to 15H A negative control analyte, soluble VSV-G (residues 17-467) huIgG1 monoFc-tagged protein, was used to confirm antibody specificity. Competition assays were performed to identify clones with unique or non-overlapping epitopes. These experiments identified clones 35C11, 8G8, and 40A3, which harbor non-overlapping or non-competing epitopes within residues 42-474 of GALV-gp70.

[0516] XIX. Example 19: Purified Antibody Characteristics

[0517] A panel of nine recombinant antibodies was successfully generated. Antibodies were characterized and screened according to the methods described in Example 18. The affinity values ​​in Table 16 are for the antibody clones to huIgG1 monoFc-tagged Gibbon GALV gp70 protein (residues 42-474).

[0518] Table 16. Characteristics of purified antibodies

[0519]

[0520] Abbreviation: NB, no binding.

[0521] Note: This table lists the antibody clones and purification yields (mg / L) when produced in ExpiCHO cells and purified in two steps. Affinity values ​​(KD(M)) were estimated by Octet binding assay.

[0522] XX. Example 20: Conjugation of fluorophores to recombinant antibodies

[0523] Three anti-GALV gp70 antibody clones, 8G8, 40A3, and 35C11, were selected as lead antibody candidates due to their binding properties. These antibodies were then immunoprecipitated with Dylight following standard protocols. ™ The fluorophore was conjugated to primary amines on the antibody using a 650 (Thermo Fisher). Unconjugated fluorophores were removed using size exclusion chromatography. The purity of each conjugated antibody was determined to be >95% by analytical SEC (SEC-UPLC), and the extent of labeling for each antibody was determined spectrophotometrically.

[0524] XXI. Example 21: Characterization of Fluorophore-Conjugated Antibodies

[0525] To characterize the fluorophore-conjugated 8G8, 40A3, and 35C11 antibodies, PG13 cells were incubated with 200 ng of each fluorophore-labeled antibody. NIH-3T3 cells were used as a negative control. Both cell lines were also stained with the secondary antibody alone (in the absence of the primary antibody) as a negative control. The cells were stained and analyzed ( Figures 15A to 15H ).

[0526] Incorporated by reference

[0527] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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, no admission is made that any reference is prior art to the present disclosure. To the extent that any definitions or terminology used in any reference incorporated herein by reference differs from the definitions or terminology used in this specification, the definitions and terminology in this specification prevail.

[0528] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present application. The foregoing specific embodiments and examples are illustrative of the preferred embodiments of the present application and are not meant to limit the scope of the application as set forth in the appended claims. However, it will be appreciated that the present application can be practiced in many ways, and that the application should not be construed as limited to the embodiments set forth in this specification unless any claim limits it so.

Claims

1. An isolated antigen-binding molecule that binds to an anti-CD20 binding region, the isolated antigen-binding molecule 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 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%, or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-10.

3. The isolated antigen binding molecule of claim 1, 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%, or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-20.

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

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

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

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

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

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

10. The isolated antigen binding molecule of any preceding claim, 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 that has 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 that has 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. The isolated antigen binding molecule of any one of the preceding claims, wherein the isolated antigen binding molecule comprises a heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 25, 32, and 39.

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

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

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

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

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

116.

22. An antigen binding system, antibody, or antigen binding fragment thereof, comprising a GALV gp70 binding motif, wherein the GALV gp70 binding motif comprises the sequence 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 sequence of 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 antigen binding system, antibody, or antigen binding fragment thereof according to claim 22, wherein the GALVgp70 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), wherein (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; and (vi) the LCDR3 has a sequence according to any one of SEQ ID NOs: 243-252, 273-282 and 293-302.

24. The antigen binding system, antibody, or antigen binding fragment thereof according to claim 22 or 23, wherein the HCDR comprises: (i) a HCDR1 according to any one of SEQ ID NOs: 127, 157 and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169 and 199; a HCDR3 according to SEQ ID NO: 211 or DYY; (ii) a HCDR1 according to any one of SEQ ID NOs: 128, 158 and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170 and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181 and 212; (iii) a HCDR1 according to any one of SEQ ID NOs: 129, 159 and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171 and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182 and 213; (iv) a HCDR1 according to any one of SEQ ID NOs: 130, 160 and 190; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NO: 214 or DYY; (v) a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; (vi) a HCDR1 according to any one of SEQ ID NOs: 132, 162 and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174 and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184 and 216; (vii) a HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; a HCDR2 according to any one of SEQ ID NOs: 145, 175 and 205; a HCDR3 according to any one of SEQ ID NOs: 155, 185 and 217; (viii) a HCDR1 according to any one of SEQ ID NOs: 134, 164 and 194; a HCDR2 according to any one of SEQ ID NOs: 146, 176 and 206; a HCDR3 according to SEQ ID NO: 218 or DYY; (ix) a HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195; a HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; (x) a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196; a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NO: 220 or DYY; (xi) a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NOs: 221 or DYY; or (xii) a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NO: 222 or DYY, and The LCDR comprises: (i) LCDR1 according to any one of SEQ ID NOs: 232, 262 and 292; LCDR2 according to any one of SEQ ID NOs: 242, 272 and GTN; LCDR3 according to any one of SEQ ID NOs: 252, 282 and 302; (ii) 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; (iii) LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; 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; (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) 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; (vi) 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; (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) 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; (ix) 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 (x) LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

25. The antigen binding system, antibody, or antigen binding fragment thereof according to any one of claims 22 to 24, wherein the antigen binding system, antibody, or 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), wherein: The HCDR and the LCDR comprise: (i) a HCDR1 according to any one of SEQ ID NOs: 127, 157 and 187; a HCDR2 according to any one of SEQ ID NOs: 139, 169 and 199; a HCDR3 according to SEQ ID NOs: 211 or DYY; a LCDR1 according to any one of SEQ ID NOs: 232, 262 and 292; a LCDR2 according to any one of SEQ ID NOs: 242, 272 and GTN; a LCDR3 according to any one of SEQ ID NOs: 252, 282 and 302; (ii) a HCDR1 according to any one of SEQ ID NOs: 128, 158 and 188; a HCDR2 according to any one of SEQ ID NOs: 140, 170 and 200; a HCDR3 according to any one of SEQ ID NOs: 151, 181 and 212; a LCDR1 according to any one of SEQ ID NOs: 228, 258 and 288; a LCDR2 according to any one of SEQ ID NOs: 238, 268 and KVS; a LCDR3 according to any one of SEQ ID NOs: 248, 278 and 298; (iii) a HCDR1 according to any one of SEQ ID NOs: 129, 159 and 189; a HCDR2 according to any one of SEQ ID NOs: 141, 171 and 201; a HCDR3 according to any one of SEQ ID NOs: 152, 182 and 213; a LCDR1 according to any one of SEQ ID NOs: 226, 256 and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276 and 296 (iv) a HCDR1 according to any one of SEQ ID NOs: 130, 160 and 190; a HCDR2 according to any one of SEQ ID NOs: 142, 172 and 202; a HCDR3 according to SEQ ID NOs: 214 or DYY; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; (v) a HCDR1 according to any one of SEQ ID NOs: 131, 161 and 191; a HCDR2 according to any one of SEQ ID NOs: 143, 173 and 203; a HCDR3 according to any one of SEQ ID NOs: 153, 183 and 215; a LCDR1 according to any one of SEQ ID NOs: 227, 257 and 287; a LCDR2 according to any one of SEQ ID NOs: 237, 267 and GTN; a LCDR3 according to any one of SEQ ID NOs: 247, 277 and 297; (vi) a HCDR1 according to any one of SEQ ID NOs: 132, 162 and 192; a HCDR2 according to any one of SEQ ID NOs: 144, 174 and 204; a HCDR3 according to any one of SEQ ID NOs: 154, 184 and 216; a LCDR1 according to any one of SEQ ID NOs: 226, 256 and 286; a LCDR2 according to any one of SEQ ID NOs: 236, 266 and DTS; a LCDR3 according to any one of SEQ ID NOs: 246, 276 and 296; (vii) a HCDR1 according to any one of SEQ ID NOs: 133, 163 and 193; a HCDR2 according to any one of SEQ ID NOs: 145, 175 and 205; a HCDR3 according to any one of SEQ ID NOs: 155, 185 and 217; a LCDR1 according to any one of SEQ ID NOs: 225, 255 and 285; a LCDR2 according to any one of SEQ ID NOs: 235, 265 and RAS; a LCDR3 according to any one of SEQ ID NOs: 245, 275 and 295; (viii) a HCDR1 according to any one of SEQ ID NOs: 134, 164, and 194; a HCDR2 according to any one of SEQ ID NOs: 146, 176, and 206; a HCDR3 according to SEQ ID NOs: 218 or DYY; a LCDR1 according to any one of SEQ ID NOs: 224, 254, and 284; a LCDR2 according to any one of SEQ ID NOs: 234, 264, and GTN; a LCDR3 according to any one of SEQ ID NOs: 244, 274, and 294 (ix) a HCDR1 according to any one of SEQ ID NOs: 135, 165 and 195; a HCDR2 according to any one of SEQ ID NOs: 147, 177 and 207; a HCDR3 according to any one of SEQ ID NOs: 156, 186 and 219; a LCDR1 according to any one of SEQ ID NOs: 223, 253 and 283; a LCDR2 according to any one of SEQ ID NOs: 233, 263 and SGS; a LCDR3 according to any one of SEQ ID NOs: 243, 273 and 293; (x) a HCDR1 according to any one of SEQ ID NOs: 136, 166 and 196; a HCDR2 according to any one of SEQ ID NOs: 148, 178 and 208; a HCDR3 according to SEQ ID NOs: 220 or DYY; a LCDR1 according to any one of SEQ ID NOs: 229, 259 and 289; a LCDR2 according to any one of SEQ ID NOs: 239, 269 and GTN; a LCDR3 according to any one of SEQ ID NOs: 249, 279 and 299; (xi) a HCDR1 according to any one of SEQ ID NOs: 137, 167 and 197; a HCDR2 according to any one of SEQ ID NOs: 149, 179 and 209; a HCDR3 according to SEQ ID NOs: 221 or DYY; a LCDR1 according to any one of SEQ ID NOs: 231, 261 and 291; a LCDR2 according to any one of SEQ ID NOs: 241, 271 and GTN; a LCDR3 according to any one of SEQ ID NOs: 251, 281 and 301; or (xii) a HCDR1 according to any one of SEQ ID NOs: 138, 168 and 198; a HCDR2 according to any one of SEQ ID NOs: 150, 180 and 210; a HCDR3 according to SEQ ID NOs: 222 or DYY; a LCDR1 according to any one of SEQ ID NOs: 230, 260, 290; a LCDR2 according to any one of SEQ ID NOs: 240, 270 and GTN; a LCDR3 according to any one of SEQ ID NOs: 250, 280 and 300.

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

27. The antigen binding system, antibody, or antigen binding fragment thereof of claim 26, wherein the antigen binding system, antibody, or 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, wherein: (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.

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

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

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 according to claim 31.

33. A method of producing an engineered cell, the method 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 according to any one of claims 22 to 30, optionally wherein the cell is an immune cell.

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

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 antigen binding system, antibody, or antigen binding fragment thereof of claim 36, wherein the fluorescent label is selected from the group consisting of: Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation) , GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midurish Blue, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGF P, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azomeiner Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabila 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, Peridinyl Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

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

39. 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, the method 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 formation of a binding complex between GALV gp70 and the antigen-binding protein; (d) separating from the binding complex any molecules that are not part of the binding complex; and (e) Detecting the presence or absence of the bound complex.

40. The method of claim 38 or 39, wherein the antigen binding molecules are 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. 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 method of claim 41, wherein the fluorescent label is selected from the group consisting of: Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation) , GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midurish Blue, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGF P, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azomeiner Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabila 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, Peridinyl Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

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

44. The method of claim 43, wherein the flow-based detection method is a flow cytometry viral assay.

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

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