CA3085351A1 - Bispecific hiv-1-neutralizing antibodies - Google Patents
Bispecific hiv-1-neutralizing antibodies Download PDFInfo
- Publication number
- CA3085351A1 CA3085351A1 CA3085351A CA3085351A CA3085351A1 CA 3085351 A1 CA3085351 A1 CA 3085351A1 CA 3085351 A CA3085351 A CA 3085351A CA 3085351 A CA3085351 A CA 3085351A CA 3085351 A1 CA3085351 A1 CA 3085351A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- imab
- seq
- amino acid
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 171
- 108090000623 proteins and genes Proteins 0.000 claims description 44
- 102000004169 proteins and genes Human genes 0.000 claims description 41
- 150000001413 amino acids Chemical class 0.000 claims description 24
- 230000035772 mutation Effects 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 21
- 229950010245 ibalizumab Drugs 0.000 claims description 20
- 230000004075 alteration Effects 0.000 claims description 18
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 12
- 230000003472 neutralizing effect Effects 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 101710091045 Envelope protein Proteins 0.000 claims description 6
- 101710188315 Protein X Proteins 0.000 claims description 6
- 210000000170 cell membrane Anatomy 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- 238000001990 intravenous administration Methods 0.000 claims description 4
- 238000007920 subcutaneous administration Methods 0.000 claims description 4
- 238000007918 intramuscular administration Methods 0.000 claims description 3
- 102220581211 Oxidized purine nucleoside triphosphate hydrolase_I45K_mutation Human genes 0.000 claims description 2
- 238000007912 intraperitoneal administration Methods 0.000 claims description 2
- 230000002685 pulmonary effect Effects 0.000 claims description 2
- 102100021696 Syncytin-1 Human genes 0.000 claims 1
- 102000006240 membrane receptors Human genes 0.000 claims 1
- 108020004084 membrane receptors Proteins 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 5
- 230000002265 prevention Effects 0.000 abstract description 2
- 101001108927 Rattus norvegicus Nuclear pore complex protein Nup155 Proteins 0.000 description 117
- 102100031876 SRC kinase signaling inhibitor 1 Human genes 0.000 description 117
- 241000725303 Human immunodeficiency virus Species 0.000 description 116
- 238000001542 size-exclusion chromatography Methods 0.000 description 37
- 230000000694 effects Effects 0.000 description 35
- 239000000872 buffer Substances 0.000 description 32
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 24
- 230000027455 binding Effects 0.000 description 23
- 230000005764 inhibitory process Effects 0.000 description 21
- 238000000034 method Methods 0.000 description 21
- 241000700605 Viruses Species 0.000 description 18
- 238000006386 neutralization reaction Methods 0.000 description 17
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 16
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 16
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 14
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 14
- 230000003389 potentiating effect Effects 0.000 description 14
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 13
- 108010091769 Shiga Toxin 1 Proteins 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 230000001976 improved effect Effects 0.000 description 12
- 230000000840 anti-viral effect Effects 0.000 description 11
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 10
- 241000282414 Homo sapiens Species 0.000 description 10
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 10
- 238000004873 anchoring Methods 0.000 description 10
- 230000036436 anti-hiv Effects 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 230000003612 virological effect Effects 0.000 description 9
- 102000018697 Membrane Proteins Human genes 0.000 description 8
- 108010052285 Membrane Proteins Proteins 0.000 description 8
- 101100288142 Mus musculus Klkb1 gene Proteins 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 238000012494 forced degradation Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 230000002776 aggregation Effects 0.000 description 7
- 238000004220 aggregation Methods 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 230000005540 biological transmission Effects 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 230000008646 thermal stress Effects 0.000 description 7
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 6
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 6
- 101001018259 Homo sapiens Microtubule-associated serine/threonine-protein kinase 1 Proteins 0.000 description 6
- 101000693728 Homo sapiens S-acyl fatty acid synthase thioesterase, medium chain Proteins 0.000 description 6
- 102100025541 S-acyl fatty acid synthase thioesterase, medium chain Human genes 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 102100034349 Integrase Human genes 0.000 description 5
- -1 coatings Substances 0.000 description 5
- 238000000113 differential scanning calorimetry Methods 0.000 description 5
- 230000005714 functional activity Effects 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 4
- 241001112090 Pseudovirus Species 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000002869 basic local alignment search tool Methods 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 238000005199 ultracentrifugation Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 208000020322 Gaucher disease type I Diseases 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 208000031886 HIV Infections Diseases 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 125000001165 hydrophobic group Chemical group 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000002864 sequence alignment Methods 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 229920002785 Croscarmellose sodium Polymers 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 208000037357 HIV infectious disease Diseases 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 2
- 108070000030 Viral receptors Proteins 0.000 description 2
- 230000010530 Virus Neutralization Effects 0.000 description 2
- 230000023445 activated T cell autonomous cell death Effects 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000011260 co-administration Methods 0.000 description 2
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 2
- 238000011577 humanized mouse model Methods 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 2
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001757 thermogravimetry curve Methods 0.000 description 2
- OKMWKBLSFKFYGZ-UHFFFAOYSA-N 1-behenoylglycerol Chemical compound CCCCCCCCCCCCCCCCCCCCCC(=O)OCC(O)CO OKMWKBLSFKFYGZ-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 108010017088 CCR5 Receptors Proteins 0.000 description 1
- 102000004274 CCR5 Receptors Human genes 0.000 description 1
- 108010041397 CD4 Antigens Proteins 0.000 description 1
- 108010061299 CXCR4 Receptors Proteins 0.000 description 1
- 102000012000 CXCR4 Receptors Human genes 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 102100034353 Integrase Human genes 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 230000010757 Reduction Activity Effects 0.000 description 1
- 241001274197 Scatophagus argus Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- SSZBUIDZHHWXNJ-UHFFFAOYSA-N Stearinsaeure-hexadecylester Natural products CCCCCCCCCCCCCCCCCC(=O)OCCCCCCCCCCCCCCCC SSZBUIDZHHWXNJ-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000003655 absorption accelerator Substances 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000003429 anti-cardiolipin effect Effects 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 229960001681 croscarmellose sodium Drugs 0.000 description 1
- 229960000913 crospovidone Drugs 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 239000001767 crosslinked sodium carboxy methyl cellulose Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 108010078428 env Gene Products Proteins 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000012537 formulation buffer Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229960002737 fructose Drugs 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- 229940049654 glyceryl behenate Drugs 0.000 description 1
- 238000011194 good manufacturing practice Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000001253 polyvinylpolypyrrolidone Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000003340 retarding agent Substances 0.000 description 1
- 102220224782 rs74315446 Human genes 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000009637 wintergreen oil Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- AIDS & HIV (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Communicable Diseases (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Hematology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
In various embodiments, the present invention relates generally to using bispecific antibodies in the prevention and treatment of HIV.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Patent Application No.
15/414,822, filed January 25, 2017, which is a divisional of U.S. Patent Application No.
14/558,341, filed December 2, 2014, which is patented as U.S. Patent No. 9,587,012, which claims priority to and the benefit of U.S. Provisional Patent Application No. 61/910,685, filed December 2, 2013, each of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
In various embodiments, the present invention relates generally to using bispecific antibodies in the prevention and treatment of HIV.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created December 21, 2017, is named ADR-001CP 5T25.txt and is 145,851 bytes in size.
BACKGROUND
Passive immunization with antibodies (Abs) is a recognized method of prophylaxis and treatment of infectious diseases. This approach may involve preparing human immunoglobulins from donors who recovered from an infectious disease and utilizing such preparations, containing Abs specific for the infectious organism, to protect a recipient against the same disease.
Alternatively, therapeutic antibodies can be made by immunizing mice with an antigen, and then engineering/humanizing the mouse Ab into a human version. Monoclonal antibodies (mAbs) are homogeneous in terms of physical characteristics and immunochemical reactivity, and so offer the possibility of absolute specific activity.
That specificity can ultimately be a limitation for some targets, so practitioners have developed "bispecific" mAbs composed of fragments of two different mAbs and which bind to two different types of antigen. This facilitates binding to antigens expressed only weakly, for example. Some bispecific mAbs can stimulate strong immune responses, limiting their clinical application. One recent approach to ameliorating this effect is "CrossMab" methodology, a bispecific antibody format that adopts a more native antibody-like structure.
The prospects for generating a highly potent bispecific or bivalent antibody against a pathogen, such as HIV, for clinical use involves many uncertainties. The low spike density and spike structure on HIV may impede bivalent binding of antibodies to HIV, for example, and the .. geometry and spatial relationship of cell surface anchoring are not well-characterized. Nor is it known whether sufficient epitope accessibility on the HIV envelope exists.
CrossMab bispecific antibodies that are anchored to a host cell membrane offer the possibility of improved local antibody concentration, targeting of sequential and/or interdependent entry steps, and compensating for monovalent binding.
Further still, large-scale, commercial production of antibodies remains challenging. For example, the production of therapeutic antibodies often requires the use of very large cell cultures followed by extensive purification steps, under Good Manufacturing Practice conditions, thereby resulting in extremely high production costs. Other limitations such as poor insolubility, protein aggregation, and protein instability can also make manufacturing of antibodies less than optimal.
Accordingly, there remains a need for therapeutically effective HIV antibodies that can be easily produced at a commercial scale.
SUMMARY
In one aspect, the present invention pertains to a bispecific antibody for neutralizing HIV. The bispecific antibody includes portions of a first and a second antibody, in which the first antibody binds to a HIV envelope protein. In certain embodiments, the first antibody is selected from
This application is a continuation-in-part of U.S. Patent Application No.
15/414,822, filed January 25, 2017, which is a divisional of U.S. Patent Application No.
14/558,341, filed December 2, 2014, which is patented as U.S. Patent No. 9,587,012, which claims priority to and the benefit of U.S. Provisional Patent Application No. 61/910,685, filed December 2, 2013, each of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
In various embodiments, the present invention relates generally to using bispecific antibodies in the prevention and treatment of HIV.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created December 21, 2017, is named ADR-001CP 5T25.txt and is 145,851 bytes in size.
BACKGROUND
Passive immunization with antibodies (Abs) is a recognized method of prophylaxis and treatment of infectious diseases. This approach may involve preparing human immunoglobulins from donors who recovered from an infectious disease and utilizing such preparations, containing Abs specific for the infectious organism, to protect a recipient against the same disease.
Alternatively, therapeutic antibodies can be made by immunizing mice with an antigen, and then engineering/humanizing the mouse Ab into a human version. Monoclonal antibodies (mAbs) are homogeneous in terms of physical characteristics and immunochemical reactivity, and so offer the possibility of absolute specific activity.
That specificity can ultimately be a limitation for some targets, so practitioners have developed "bispecific" mAbs composed of fragments of two different mAbs and which bind to two different types of antigen. This facilitates binding to antigens expressed only weakly, for example. Some bispecific mAbs can stimulate strong immune responses, limiting their clinical application. One recent approach to ameliorating this effect is "CrossMab" methodology, a bispecific antibody format that adopts a more native antibody-like structure.
The prospects for generating a highly potent bispecific or bivalent antibody against a pathogen, such as HIV, for clinical use involves many uncertainties. The low spike density and spike structure on HIV may impede bivalent binding of antibodies to HIV, for example, and the .. geometry and spatial relationship of cell surface anchoring are not well-characterized. Nor is it known whether sufficient epitope accessibility on the HIV envelope exists.
CrossMab bispecific antibodies that are anchored to a host cell membrane offer the possibility of improved local antibody concentration, targeting of sequential and/or interdependent entry steps, and compensating for monovalent binding.
Further still, large-scale, commercial production of antibodies remains challenging. For example, the production of therapeutic antibodies often requires the use of very large cell cultures followed by extensive purification steps, under Good Manufacturing Practice conditions, thereby resulting in extremely high production costs. Other limitations such as poor insolubility, protein aggregation, and protein instability can also make manufacturing of antibodies less than optimal.
Accordingly, there remains a need for therapeutically effective HIV antibodies that can be easily produced at a commercial scale.
SUMMARY
In one aspect, the present invention pertains to a bispecific antibody for neutralizing HIV. The bispecific antibody includes portions of a first and a second antibody, in which the first antibody binds to a HIV envelope protein. In certain embodiments, the first antibody is selected from
2 PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8 and a variant thereof. In certain embodiments, the bispecific antibody includes portions of a second antibody, in which the second antibody binds to a cell membrane protein. For example, the second antibody binds to a cell receptor protein or a cell membrane co-receptor protein. In an embodiment, the second antibody is selected from a CD4 antibody, a CCR5 antibody and a CXCR4 antibody, such as Pro 140, ibalizumab, 515H7, or a variant thereof. In various embodiments, the bispecific antibody has a CrossMab format.
In another aspect, the present invention provides a bispecific antibody including portions of a first antibody and a second antibody, wherein the first antibody binds to a HIV envelope protein and the second antibody binds to a cell membrane protein. In various embodiments, the bispecific antibody has a CrossMab format.
In various embodiments, pharmaceutical compositions including the bispecific antibodies disclosed herein are also provided. The pharmaceutical composition may be formulated for oral, intranasal, pulmonary, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous delivery.
In a further aspect, methods for neutralizing HIV are provided. The methods include the steps of contacting an antigen binding site with a bispecific antibody that binds a HIV
envelope protein and contacting another antigen binding site with a bispecific antibody that binds a cell membrane protein.
In another aspect, methods for treating a patient infected with HIV are also provided. The methods include administering to the patient any of the bispecific antibodies or pharmaceutical compositions as disclosed herein. In an embodiment, the patient is human.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings, like reference characters generally refer to the same parts throughout the different views. Also, the drawings are not necessarily to scale, with an emphasis instead
In another aspect, the present invention provides a bispecific antibody including portions of a first antibody and a second antibody, wherein the first antibody binds to a HIV envelope protein and the second antibody binds to a cell membrane protein. In various embodiments, the bispecific antibody has a CrossMab format.
In various embodiments, pharmaceutical compositions including the bispecific antibodies disclosed herein are also provided. The pharmaceutical composition may be formulated for oral, intranasal, pulmonary, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous delivery.
In a further aspect, methods for neutralizing HIV are provided. The methods include the steps of contacting an antigen binding site with a bispecific antibody that binds a HIV
envelope protein and contacting another antigen binding site with a bispecific antibody that binds a cell membrane protein.
In another aspect, methods for treating a patient infected with HIV are also provided. The methods include administering to the patient any of the bispecific antibodies or pharmaceutical compositions as disclosed herein. In an embodiment, the patient is human.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings, like reference characters generally refer to the same parts throughout the different views. Also, the drawings are not necessarily to scale, with an emphasis instead
3 generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present invention are described with reference to the following drawings, in which:
Figure 1 is a diagram illustrating a CrossMab antibody derived from two IgG
monoclonal antibodies.
Figure 2A is a diagram illustrating an iMab antibody (shorthand for the monoclonal antibody ibalizumab) that targets CD4 and a Pro 140 antibody that targets CCR5.
Figure 2B is a diagram illustrating mAbs that target the HIV envelope gp120.
Figure 3 is a graph comparing the maximum percentage inhibition (MPI) against cell-to-cell HIV
transmission using a combination of iMab and 10E8 antibodies with CrossMab bispecific 10E8/iMab antibodies. Except otherwise stated, all iMab-based bispecific antibodies were constructed using the MV1 variant.
Figures 4A-J are a series of graphs comparing the inhibition of various strains of X4 and dual-tropic HIV using varying concentrations of 10E8, Pro 140 or 10E8/P140 antibodies. P140 is shorthand for Pro 140.
Figures 5A-G are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, Pro 140, 10E8/P140 or a combination of the individual 10E8 and Pro 140 monoclonal antibodies.
Figures 6A-D are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, X19, 10E8/X19 or 10E8/P140 antibodies.
Figures 7A-H are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, Pro 140, 10E8/P140 and 10E8/aHer2 antibodies.
Figure 8A is a graph comparing the binding of CrossMab bispecific antibodies 10E8/iMab and A 10E8/iMab to the HIV-1 glycoprotein MPER.
Figure 1 is a diagram illustrating a CrossMab antibody derived from two IgG
monoclonal antibodies.
Figure 2A is a diagram illustrating an iMab antibody (shorthand for the monoclonal antibody ibalizumab) that targets CD4 and a Pro 140 antibody that targets CCR5.
Figure 2B is a diagram illustrating mAbs that target the HIV envelope gp120.
Figure 3 is a graph comparing the maximum percentage inhibition (MPI) against cell-to-cell HIV
transmission using a combination of iMab and 10E8 antibodies with CrossMab bispecific 10E8/iMab antibodies. Except otherwise stated, all iMab-based bispecific antibodies were constructed using the MV1 variant.
Figures 4A-J are a series of graphs comparing the inhibition of various strains of X4 and dual-tropic HIV using varying concentrations of 10E8, Pro 140 or 10E8/P140 antibodies. P140 is shorthand for Pro 140.
Figures 5A-G are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, Pro 140, 10E8/P140 or a combination of the individual 10E8 and Pro 140 monoclonal antibodies.
Figures 6A-D are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, X19, 10E8/X19 or 10E8/P140 antibodies.
Figures 7A-H are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, Pro 140, 10E8/P140 and 10E8/aHer2 antibodies.
Figure 8A is a graph comparing the binding of CrossMab bispecific antibodies 10E8/iMab and A 10E8/iMab to the HIV-1 glycoprotein MPER.
4 Figures 8B-E are a series of graphs comparing the inhibition percentages of 10E8 (light gray lines) and A10E8 (dark gray lines) against iMab resistant R5 viruses (Figure 8B) and X4 viruses (Figure 8C), as well as the inhibition percentages of 10E8/iMab (light gray lines) and A10E8/iMab (dark gray lines) against iMab resistant R5 viruses (Figure 8D) and X4 viruses (Figure 8E).
Figures 9A-G are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, A10E8, 4E10, 10E8/P140, A10E8/P140 and antibodies.
Figure 10 is a graph comparing the antiviral coverage of the CrossMab antibodies 10E8/Pro140 and 10E8/iMab, their parental monoclonal antibodies 10E8, Pro140 and iMab, and various other HIV envelope-targeting monoclonal antibodies against a large panel of HIV
envelope pseudotyped viruses.
Figures 11A-E are a series of graphs comparing the maximum percentage inhibition (MPI) of a large panel of HIV envelope pseudotyped viruses with the monoclonal antibody iMab (grey bars in all panels) and the CrossMab antibodies PGT145/ibalizumab (145/iMab; Figure 11A), PGT128/ibalizumab (128/iMab; Figure 11B), PGT151/ibalizumab (151/iMab; Figure 11C), 3BNC117/ibalizumab (117/iMab; Figure 11D) and 10E8/ibalizumab (10E8/iMab;
Figure 11E).
Figures 12A-E are a series of graphs comparing the maximum percentage inhibition (MPI) and IC80 antibody concentrations of the CrossMab antibodies PGT145/ibalizumab (145/iMab;
Figure 12A), PGT128/ibalizumab (128/iMab; Figure 12B), PGT151/ibalizumab (151/iMab;
Figure 12C), 3BNC117/ibalizumab (117/iMab; Figure 12D) and 10E8/ibalizumab (10E8/iMab;
Figure 12E) against a large panel of HIV envelope pseudotyped viruses.
Figures 13A-E are a series of graphs comparing the IC80 antibody concentrations for iMab- and Pro140-based CrossMab bispecific antibodies and their parent antibodies for PGT145/iMab and PGT145/Pro140 (Figure 13A), 3BNC117/iMab and 3BNC117/Pro140 (Figure 13B),
Figures 9A-G are a series of graphs comparing the inhibition of various strains of HIV using varying concentrations of 10E8, A10E8, 4E10, 10E8/P140, A10E8/P140 and antibodies.
Figure 10 is a graph comparing the antiviral coverage of the CrossMab antibodies 10E8/Pro140 and 10E8/iMab, their parental monoclonal antibodies 10E8, Pro140 and iMab, and various other HIV envelope-targeting monoclonal antibodies against a large panel of HIV
envelope pseudotyped viruses.
Figures 11A-E are a series of graphs comparing the maximum percentage inhibition (MPI) of a large panel of HIV envelope pseudotyped viruses with the monoclonal antibody iMab (grey bars in all panels) and the CrossMab antibodies PGT145/ibalizumab (145/iMab; Figure 11A), PGT128/ibalizumab (128/iMab; Figure 11B), PGT151/ibalizumab (151/iMab; Figure 11C), 3BNC117/ibalizumab (117/iMab; Figure 11D) and 10E8/ibalizumab (10E8/iMab;
Figure 11E).
Figures 12A-E are a series of graphs comparing the maximum percentage inhibition (MPI) and IC80 antibody concentrations of the CrossMab antibodies PGT145/ibalizumab (145/iMab;
Figure 12A), PGT128/ibalizumab (128/iMab; Figure 12B), PGT151/ibalizumab (151/iMab;
Figure 12C), 3BNC117/ibalizumab (117/iMab; Figure 12D) and 10E8/ibalizumab (10E8/iMab;
Figure 12E) against a large panel of HIV envelope pseudotyped viruses.
Figures 13A-E are a series of graphs comparing the IC80 antibody concentrations for iMab- and Pro140-based CrossMab bispecific antibodies and their parent antibodies for PGT145/iMab and PGT145/Pro140 (Figure 13A), 3BNC117/iMab and 3BNC117/Pro140 (Figure 13B),
5 PGT128/iMab and PGT128/Pro140 (Figure 13C), PGT151/iMab and PGT151/Pro140 (Figure 13D) and 10E8/iMab and 10E8/Pro140 (Figure 13E).
Figures 14A-E are a series of graphs comparing the IC50 antibody concentrations for iMab- and Pro140-based CrossMab bispecific antibodies and their parent antibodies for PGT145/iMab and PGT145/Pro140 (Figure 14A), 3BNC117/iMab and 3BNC117/Pro140 (Figure 14B), PGT128/iMab and PGT128/Pro140 (Figure 14C), PGT151/iMab and PGT151/Pro140 (Figure 14D) and 10E8/iMab and 10E8/Pro140 (Figure 14E).
Figures 15A-E are graphs displaying the IC80 antibody concentrations for iMab-based CrossMab bispecific antibodies and their parent antibodies against cell-to-cell transmission of HIV for 10E8/iMab (Figure 15A), 3BNC117/iMab (Figure 15B), PGT145/iMab (Figure 15C), PGT128/iMab (Figure 15D) and PGT151/iMab (Figure 15E).
Figure 16 is a graph displaying the maximum percent inhibition (MPI) of CrossMab bispecific antibodies and parental antibodies against cell-to-cell transmission of HIV.
Figure 17A is a graph comparing the inhibition of an HIV strain against varying concentrations of 10E8, Pro 140, 10E8/P140 CrossMab bispecific antibody, and a combination of individual 10E8 and Pro 140 monoclonal antibodies.
Figure 17B is a graph comparing the inhibition of an HIV strain against varying concentrations of iMab, 10E8, 10E8/iMab CrossMab bispecific antibody, and a combination of individual 10E8 and iMab monoclonal antibodies.
Figures 18A-D are a series of graphs comparing the inhibition of various HIV
R5 strains against varying concentrations of 10E8, Pro140, 10E8/P140 and 10E8/515H7 antibodies.
Figures 18E-H are a series of graphs comparing the inhibition of various HIV
X4 strains against various concentrations of 10E8, 515H7 and 10E8/515H7 antibodies.
Figures 14A-E are a series of graphs comparing the IC50 antibody concentrations for iMab- and Pro140-based CrossMab bispecific antibodies and their parent antibodies for PGT145/iMab and PGT145/Pro140 (Figure 14A), 3BNC117/iMab and 3BNC117/Pro140 (Figure 14B), PGT128/iMab and PGT128/Pro140 (Figure 14C), PGT151/iMab and PGT151/Pro140 (Figure 14D) and 10E8/iMab and 10E8/Pro140 (Figure 14E).
Figures 15A-E are graphs displaying the IC80 antibody concentrations for iMab-based CrossMab bispecific antibodies and their parent antibodies against cell-to-cell transmission of HIV for 10E8/iMab (Figure 15A), 3BNC117/iMab (Figure 15B), PGT145/iMab (Figure 15C), PGT128/iMab (Figure 15D) and PGT151/iMab (Figure 15E).
Figure 16 is a graph displaying the maximum percent inhibition (MPI) of CrossMab bispecific antibodies and parental antibodies against cell-to-cell transmission of HIV.
Figure 17A is a graph comparing the inhibition of an HIV strain against varying concentrations of 10E8, Pro 140, 10E8/P140 CrossMab bispecific antibody, and a combination of individual 10E8 and Pro 140 monoclonal antibodies.
Figure 17B is a graph comparing the inhibition of an HIV strain against varying concentrations of iMab, 10E8, 10E8/iMab CrossMab bispecific antibody, and a combination of individual 10E8 and iMab monoclonal antibodies.
Figures 18A-D are a series of graphs comparing the inhibition of various HIV
R5 strains against varying concentrations of 10E8, Pro140, 10E8/P140 and 10E8/515H7 antibodies.
Figures 18E-H are a series of graphs comparing the inhibition of various HIV
X4 strains against various concentrations of 10E8, 515H7 and 10E8/515H7 antibodies.
6 Figures 19A-B are a series of graphs comparing inhibition of various HIV
strains against varying concentrations of 10E8/Pro140, 10E8/iMab, 10E8/515H7 and 10E8/X19 antibodies.
Figure 19C indicates the density of CD4, CCR5 and CXCR4 receptors present on TZM-bl cells.
Figure 20 compares the binding of CrossMab bispecific antibodies 10E8/Pro140, A10E8/Pro140 and 4E10/Pro140 to the HIV-1 glycoprotein MPER.
Figures 21A-G are a series of graphs comparing the inhibition of various strains of HIV against varying concentrations of 4E10, Pro140 and 4E10/P140 and 10E8/P140 antibodies.
Figure 22A is size exclusion chromatography analysis of the CrossMab antibodies 10E8/iMab, 10E8/P140 and 3BNC117/iMab.
Figure 22B is size exclusion chromatography analysis of the monoclonal antibodies iMab, 10E8 and Pro140.
Figure 23 is size exclusion chromatography analysis of monoclonal antibody 10E8 and a chimeric antibody comprised of the 10E8 heavy chain paired with the 4E10 light chain.
Figures 24A-C are a series of size exclusion chromatography graphs of: the monoclonal antibodies 10E8 and 4E10 and a chimeric antibody comprised of the 10E8 heavy chain paired with the 4E10 light chain (Figure 24A), the monoclonal antibody 10E8 and 10E8 mutants with potentially stabilizing mutations genetically engineered in the 10E8 light chain (Figure 24B), and the monoclonal antibody 10E8 and 10E8 mutants genetically grafted with the kappa light chain of non-10E8 antibodies (Figure 24C).
Figure 25 is a size exclusion chromatography graph of the monoclonal antibody 4E10 and 4E10 mutants genetically grafted with the light regions of 10E8 that included the CDR1 region, CDR2 region, CDR3 region, or combined CDR1, CDR2 and CDR3 regions.
Figure 26A is a size exclusion chromatography graph of 10E8 chimeric antibodies. CDR123 is a chimeric antibody of the 10E8 heavy chain paired with a 10E8 light chain genetically grafted
strains against varying concentrations of 10E8/Pro140, 10E8/iMab, 10E8/515H7 and 10E8/X19 antibodies.
Figure 19C indicates the density of CD4, CCR5 and CXCR4 receptors present on TZM-bl cells.
Figure 20 compares the binding of CrossMab bispecific antibodies 10E8/Pro140, A10E8/Pro140 and 4E10/Pro140 to the HIV-1 glycoprotein MPER.
Figures 21A-G are a series of graphs comparing the inhibition of various strains of HIV against varying concentrations of 4E10, Pro140 and 4E10/P140 and 10E8/P140 antibodies.
Figure 22A is size exclusion chromatography analysis of the CrossMab antibodies 10E8/iMab, 10E8/P140 and 3BNC117/iMab.
Figure 22B is size exclusion chromatography analysis of the monoclonal antibodies iMab, 10E8 and Pro140.
Figure 23 is size exclusion chromatography analysis of monoclonal antibody 10E8 and a chimeric antibody comprised of the 10E8 heavy chain paired with the 4E10 light chain.
Figures 24A-C are a series of size exclusion chromatography graphs of: the monoclonal antibodies 10E8 and 4E10 and a chimeric antibody comprised of the 10E8 heavy chain paired with the 4E10 light chain (Figure 24A), the monoclonal antibody 10E8 and 10E8 mutants with potentially stabilizing mutations genetically engineered in the 10E8 light chain (Figure 24B), and the monoclonal antibody 10E8 and 10E8 mutants genetically grafted with the kappa light chain of non-10E8 antibodies (Figure 24C).
Figure 25 is a size exclusion chromatography graph of the monoclonal antibody 4E10 and 4E10 mutants genetically grafted with the light regions of 10E8 that included the CDR1 region, CDR2 region, CDR3 region, or combined CDR1, CDR2 and CDR3 regions.
Figure 26A is a size exclusion chromatography graph of 10E8 chimeric antibodies. CDR123 is a chimeric antibody of the 10E8 heavy chain paired with a 10E8 light chain genetically grafted
7 with the 10E8 antibody germline CDR region sequences. FW123 is a chimeric antibody of the 10E8 heavy chain paired with a 10E8 light chain genetically grafted with the 10E8 antibody germline framework region sequences.
Figure 26B is a table indicating the expression, HIV MPER binding ability, size exclusion chromatography profile, and HIV neutralization profile of the CDR123 and FW123 antibodies.
Figure 27 is a size exclusion chromatography graph of monoclonal antibody 10E8, its somatic variant H6L10, and a CrossMab bispecific antibody consisting of H6L10 paired with Pro140.
Figure 28 is a graph depicting the pharmacokinetics profiles of 10E8, H6L10/Pro 140 and its parental antibodies in a mouse model.
Figure 29 is a graph comparing the potency of 10E8v i.o/iMab or P140 CrossMab antibodies with 10E8/iMab or P140 antibodies.
Figure 30 is a graph depicting the pharmacokinetics of 10E8 and CrossMab antibodies derived from several 10E8 variants and iMab or P140 in a mouse model.
Figures 31A-B are a series of graphs depicting the HIV viral coverage of 10E8vid/P140 and 10E8v2.0/iMab antibodies.
Figures 31C-D are a series of graphs depicting size exclusion chromatography stability graphs of 10E8vid/P140 and 10E8v2.o/iMab antibodies.
Figures 32A-B are a series of graphs depicting the size exclusion stability graphs of 10E8vid/P140 and 10E8v2.o/iMab antibodies stored in PBS at 4 C.
Figure 33 depicts a native mass spectroscopy analysis of the 10E8v2.o/iMab (N297A) antibody.
Figures 34A-C are a series of graphs comparing the activity of 10E8vid/P140 and 10E8v2.0/iMab on a HIV Clade C panel, and the IC50 and IC80 activities of the antibodies.
Figure 26B is a table indicating the expression, HIV MPER binding ability, size exclusion chromatography profile, and HIV neutralization profile of the CDR123 and FW123 antibodies.
Figure 27 is a size exclusion chromatography graph of monoclonal antibody 10E8, its somatic variant H6L10, and a CrossMab bispecific antibody consisting of H6L10 paired with Pro140.
Figure 28 is a graph depicting the pharmacokinetics profiles of 10E8, H6L10/Pro 140 and its parental antibodies in a mouse model.
Figure 29 is a graph comparing the potency of 10E8v i.o/iMab or P140 CrossMab antibodies with 10E8/iMab or P140 antibodies.
Figure 30 is a graph depicting the pharmacokinetics of 10E8 and CrossMab antibodies derived from several 10E8 variants and iMab or P140 in a mouse model.
Figures 31A-B are a series of graphs depicting the HIV viral coverage of 10E8vid/P140 and 10E8v2.0/iMab antibodies.
Figures 31C-D are a series of graphs depicting size exclusion chromatography stability graphs of 10E8vid/P140 and 10E8v2.o/iMab antibodies.
Figures 32A-B are a series of graphs depicting the size exclusion stability graphs of 10E8vid/P140 and 10E8v2.o/iMab antibodies stored in PBS at 4 C.
Figure 33 depicts a native mass spectroscopy analysis of the 10E8v2.o/iMab (N297A) antibody.
Figures 34A-C are a series of graphs comparing the activity of 10E8vid/P140 and 10E8v2.0/iMab on a HIV Clade C panel, and the IC50 and IC80 activities of the antibodies.
8 Figures 35 and 36 are graphs comparing the potency of 10E8vi.i/P140, 10E8v2.o/iMab, and various monoclonal antibodies against HIV.
Figures 37A-C demonstrate that a select number of 10E8V2.0/iMab (also referred to as 10E8.2/iMab) variants retained functional antiviral activity and increased solubility. Figure 37A
demonstrates that some of the 10E8.2/iMab variants retained functional activity in an in vitro HIV-1 neutralization assay. Figure 37B shows that 10E8.2/iMab and some of the 10E8.2/iMab variants have similar in vivo pharmacokinetic profiles. Figure 37C shows the precipitation profiles of 10E8.2/iMab and some of the 10E8.2/iMab variants under thermal stress-inducing conditions.
Figures 38A-B show the results of size exclusion chromatography, which was used to identify 10E8.2/iMab variants with the least aggregation after thermal stress-inducing conditions.
Figures 39A-B illustrate the solubility of 10E8.2/iMab and the 10E8.4/iMab variant at 4 C after ultracentrifugation.
Figure 40 demonstrates the turbidity of 10E8.2/iMab and the 10E8.4/iMab variant at different concentrations over time.
Figure 41 shows the thermostability of 10E8.2/iMab and the 10E8.4/iMab variant as assessed by differential scanning calorimetry.
Figure 42 demonstrates the turbidity of 10E8.2/iMab and the 10E8.4/iMab variant after forced degradation at 50 C for six days. For each set of histograms, the bars from left to right represent 10E8.2/iMab (pre-centrifugation), 10E8.4/iMab (pre-centrifugation), 10E8.2/iMab (post-centrifugation), and 10E8.4/iMab (post-centrifugation).
Figure 43 shows the anti-HIV coverage of 10E8.2/iMab and the 10E8.4/iMab variant.
Figure 44 is a graph showing the in vivo antiviral activity of 10E8.2/iMab and the 10E8.4/iMab variant in a humanized mouse model of HIV-1 infection.
Figures 37A-C demonstrate that a select number of 10E8V2.0/iMab (also referred to as 10E8.2/iMab) variants retained functional antiviral activity and increased solubility. Figure 37A
demonstrates that some of the 10E8.2/iMab variants retained functional activity in an in vitro HIV-1 neutralization assay. Figure 37B shows that 10E8.2/iMab and some of the 10E8.2/iMab variants have similar in vivo pharmacokinetic profiles. Figure 37C shows the precipitation profiles of 10E8.2/iMab and some of the 10E8.2/iMab variants under thermal stress-inducing conditions.
Figures 38A-B show the results of size exclusion chromatography, which was used to identify 10E8.2/iMab variants with the least aggregation after thermal stress-inducing conditions.
Figures 39A-B illustrate the solubility of 10E8.2/iMab and the 10E8.4/iMab variant at 4 C after ultracentrifugation.
Figure 40 demonstrates the turbidity of 10E8.2/iMab and the 10E8.4/iMab variant at different concentrations over time.
Figure 41 shows the thermostability of 10E8.2/iMab and the 10E8.4/iMab variant as assessed by differential scanning calorimetry.
Figure 42 demonstrates the turbidity of 10E8.2/iMab and the 10E8.4/iMab variant after forced degradation at 50 C for six days. For each set of histograms, the bars from left to right represent 10E8.2/iMab (pre-centrifugation), 10E8.4/iMab (pre-centrifugation), 10E8.2/iMab (post-centrifugation), and 10E8.4/iMab (post-centrifugation).
Figure 43 shows the anti-HIV coverage of 10E8.2/iMab and the 10E8.4/iMab variant.
Figure 44 is a graph showing the in vivo antiviral activity of 10E8.2/iMab and the 10E8.4/iMab variant in a humanized mouse model of HIV-1 infection.
9 Figure 45A, shows a sequence alignment of the light chains of 10E8.2/iMab (SEQ
ID NO:33) and the 10E8.4/iMab variant (SEQ ID NO:44).
Figure 45B shows a sequence alignment of the heavy chains of 10E8.2/iMab (SEQ
ID NO:34) and the 10E8.4/iMab variant (SEQ ID NO:42). Underlined sequences denote CDR1, CDR2, and CDR3. Italicized sequences denote constant light chain or constant heavy chain sequences.
Figure 46 is a graph showing exemplary variants of 10E8 antibodies that are stable while retaining anti-HIV activity.
DETAILED DESCRIPTION
Embodiments of the present invention provide for inhibition of HIV. In various implementations, bispecific antibodies are formed, each including heavy chain and light chain components from two different parent antibodies. In various embodiments, one parent antibody specifically binds HIV, for example, the HIV envelope protein Env. In various embodiments, the other parent antibody specifically binds a cell membrane protein, for example CD4 and CCR5.
In various embodiments, the bispecific antibody (e.g., a HIV CrossMab antibody) of the present invention has the natural architecture of an IgG molecule, but with bispecificity. In a bispecific antibody, a heavy chain and light chain from each of two parental antibodies are combined, providing an antibody in which the antigen binding sites of fragment antigen-binding 1 (Fabl) and Fab2 have different binding specificities. In certain embodiments, the bispecific antibody is a CrossMab format antibody, as shown in Figure 1. In a CrossMab format, one heavy chain includes a "knob" structure and the other heavy chain includes a corresponding "hole" structure, and the positions of the constant domains (i.e., CL and CH1) from one parental antibody are switched, which together ensure correct pairing of heavy chains and light chains during assembly.
Various mAbs have been shown to block HIV infection by targeting and binding to the HIV
envelope protein Env (Figures 2B and 10). These mAbs include, for example, PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, and 10E8. Figure 2B
(adapted from www.scripps.edu/news/press/2014/20140424hiv.html) illustrates how the mAb targets the V1/V2 epitope on the HIV viral envelope gp120; how mAb PGT128 targets the glycan on the V3 stem region of HIV gp120; how mAb 3BNC117 targets the CD4 binding site of HIV gp120; how mAb 10E8 targets the membrane proximal external region (MPER) of HIV
gp41; and how mAb PGT151 targets an epitope on both HIV gp120 and HIV gp41.
In addition, monoclonal antibodies Pro 140 ("P140"), Ibalizumab ("iMab") and 515H7 have been shown to block HIV infection by targeting and binding to CCR5, CD4 and CXCR4 human cell membrane proteins, respectively (Figure 2A). Specifically, Figure 2A
shows how iMab targets CD4, the primary receptor for HIV-1 entry that is expressed on human T-cells; and how Pro 140 targets CCR5, a co-receptor for HIV-1 entry by CCR5 tropic HIV-1.
Although the ensuing discussion focuses on the use of bispecific antibodies directed to Env and the cell membrane proteins CD4 and CCR5, it is to be understood that this is solely for ease of presentation, and that any suitable antibody directed to any HIV epitope and any suitable antibody directed to any suitable cell membrane protein may be used and are within the scope of the invention.
Accordingly, in various embodiments, the present invention provides bispecific antibodies that target and bind to the HIV Env protein as well as the cell membrane proteins CCR5, CD4 and/or CXCR4. In certain embodiments, the bispecific antibodies include sequences (for example, heavy and light chain sequences) derived from, but not limited to, the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, and/or 10E8 antibodies and variants thereof.
The amino acid sequences defining the heavy and light chains of the PGT145 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/3U15 H and http://www.ncbi.nlm.nih.gov/protein/3U1S L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PG9 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/3U4E H and www dot ncbi dot nlm dot nih dot gov/protein/3MUH L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT128 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov /protein/3TYG H
and www dot ncbi dot nlm dot nih dot gov protein/3TYG L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT121 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov /protein/4FQC H
and www dot ncbi dot nlm dot nih dot gov/protein/4FQC L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 10-1074 antibody can be found, for example, in Mouquet H., et at., (2012) PNAS, 109(47): E3268-77 (including supplementary information), the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 3BNC117 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LSV H and www dot ncbi dot nlm dot nih dot gov/protein/4LSV L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the VRC01 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LST H and www dot ncbi dot nlm dot nih dot gov/protein/4LST L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT151 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4NUG H and www dot ncbi dot nlm dot nih dot gov/protein/4NUG L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 4E10 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LLV H and www dot ncbi dot nlm dot nih dot gov/protein/4LLV_L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 10E8 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4G6F_B and www dot ncbi dot nlm dot nih dot gov/protein/4G6F_D, respectively, the entire contents of which are incorporated herein by reference.
In certain embodiments, the bispecific antibodies include sequences (for example, heavy and light chain sequences) derived from, but not limited to, the P140, iMab (or the MV1 variant) .. and/or 515H7 antibodies and variants thereof. The heavy and light chain sequences of the Pro 140, iMab (or its MV1 variant), and 515H7 antibodies are further described, for example, in Olson, W. C. et at., (1999) J Virol., 73(5):4145-55, Trkola, A. et at., (2001) J Virol., 75(2):579-88, U.S. Patent No. 7,122,185, Burkly L. C. et al., (1992) J Immunol., 149(5):1779-87, Moore J.
P. et at., (1992) J Virol., 66(8):4784-93, Reimann K. A., et at., (1997) AIDS
Res Hum Retroviruses, 13(11):933-43, International Patent Publication No.
W02014100139, and European Patent Publication No. EP2246364, the entire contents of all of which are incorporated herein by reference.
As used herein, an antibody "variant" refers to an antibody which has an amino acid sequence which differs from the amino acid sequence of a parent antibody from which it is derived. In various embodiments, the variant has one or more amino acid alterations with respect to the parent antibody.
In various embodiments, the bispecific antibody of the present invention includes a heavy and light chain sequence from the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody or a variant thereof and a heavy and light chain sequence from the P140, iMab (or the MV1 variant), or 515H7 antibody or a variant thereof.
In exemplary embodiments, a series of HIV CrossMab antibodies have been constructed including but not limited to, for example, 145/MV1, 117/MV1, 128/MV1, 10E8/MV1, 145/P140, 128/P140, 117/P140, 10E8/P140, 10E8/alpha-Her2, 10E8/X19, and 4E10/P140.
("145"), 3BNC117 ("117"), PGT128 ("128"), and 10E8 are four different HIV
envelope antibodies. Pro 140 ("P140") is a mAb that binds to the cell surface receptor CCR5. MV1 is a CD4 antibody that is a modified variant of the mAb Ibalizumab ("iMab"; see, for example, International Patent Publication No. W02014100139, incorporated herein by reference in its entirety). X19 is one of the antibody variants of the anti-cell surface receptor CXCR4 (see, for example, U.S. Patent No. 8,329,178, incorporated herein by reference in its entirety) that does not bind to cells expressing CXCR4 (and is therefore used as a non-surface binding control).
Alpha-Her2 is a mAb that binds to the Her2 receptor expressed on cells. Many of these CrossMab antibodies increase the breadth of HIV neutralization as compared to their parental antibodies (i.e., monoclonal antibodies MV1, 145, 117 or 10E8). In various embodiments, the bispecific antibodies of the invention significantly increase the potency of neutralization against HIV as compared to their parental antibodies.
The amino acid sequences defining the heavy and light chains of various exemplary HIV
CrossMab antibodies are shown below.
145/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 145/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQSPDSLAVSLGERVIMNCKSSQSLLYSTNQKNYLATNYQQKPGQSPKLLIYTNASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 145/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT145 derived light chain of the 145/MV1 antibody -PGT145-LC (SEQ ID NO:3):
EVVITQSPLFLPVT PGEAASLSCKCSHSLQHSTGANYLAWYLQRPGQT PRLL IHLATHRASGVPDRFSGS
GSGTDFTLKI SRVESDDVGTYYCMQGLHSPWT FGQGTKVE IKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT145 derived heavy chain of the 145/MV1 antibody -PGT145-HC-Knob (SEQ ID NO:4):
QVQLVQSGAEVKKPGSSVKVSCKASGNS FSNHDVHWVRQATGQGLEWMGWMSHEGDKTGLAQKFQGRVT I
TRDSGASTVYMELRGLTADDTAIYYCLIGSKHRLRDY FLYNEYGPNYEEWGDYLATLDVWGHGTAVTVSS
ASTKGPSVFPLAPSSKST SGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT
VP S S SLGTQT Y ICNVNHKPSNT KVDKKVE PKSCDKT HTCP PC PAPE
FEGGPSVFLEPPKPKDILMISRT P
EVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKT KPRE EQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKA
LPAS IEKT I SKAKGQPRE PQVYTL PPCRDELT KNQVSLWCLVKGFY PS DIAVEWE SNGQPENNY KIT
PPV
LDSDGS FFLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
117/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 117/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 117/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 3BNC117 derived light chain of the 117/MV1 antibody -3BNC117-LC (SEQ ID NO:5):
DI QMTQ SP S SLSASVGDTVT ITCQANGYLNWYQQRRGKAPKLL I YDGSKLERGVPSRFSGRRWGQEYNLT
INNLQPEDIATY FCQVYE FVVFGQGT KVQVDI KRTVAAPSVF I FPP SDEQLKSGTASVVCLLNN FY
PREA
KVQWKVDNALQSGNSQE SVT EQDSKDST Y SLS STLTLSKADY EKHKVYACEVTHQGLS SPVT KS
FNRGEC
Amino acid sequence defining the 3BNC117 derived heavy chain of the 117/MV1 antibody -3BNC117-HC-Knob (SEQ ID NO:6):
QVQLLQ SGAAVT KPGASVRVSCEASGYN I RDY FIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSL
TRHASWDFDT FS FYMDLKALRSDDTAVY FCARQRSDYWDEDVWGQGTLVTVS SAST KGPSVFPLAPS SKS
TSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRI PEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQPR
EPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKLTVDK
S RWQQGNV FS CS VL HEAL HS HY TQ KSLSLS PGK
128/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 128/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 128/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT128 derived light chain of the 128/MV1 antibody -PGT128-LC (SEQ ID NO:7):
QSALTQ PP SASGSPGQ SIT I SCIGTSNNEVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSKSGNTASL
TVSGLQTDDEAVYYCGSLVGNWDVI FGGGTKLTVLGQPKAAPSVTL FP PS SE ELQANKATLVCL I SD FY
P
GAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPTEC
Amino acid sequence defining the PGT128 derived heavy chain of the 128/MV1 antibody -PGT128-HC-Knob (SEQ ID NO:8):
QPQLQE SGPTLVEASETL SLTCAVSGDSTAACNS FWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPS
LKSRLTLALDTPKNLVELKLNSVTAADTATYYCAREGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SL SSVVTVP SS S
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLS PGK
10E8/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 10E8/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/MV1 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/MV1 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
A10E8/MV1 antibody Amino acid sequence defining the MV1 derived light chain of the A10E8/MV1 antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the A10E8/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the A 10E8 derived light chain of the A 1 0E8/MV1 antibody A 1 0E8-LC (SEQ ID NO:21):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGASGNRASL
T I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I SDF
YPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPT
EC S
Amino acid sequence defining the A 10E8 derived heavy chain of the A10E8/MV1 antibody 10E8-HC-Knob (SEQ ID NO:22):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
151/MV1 antibody Amino acid sequence defining the MV1 derived light chain of the 151/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 151/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT151 derived light chain of the 151/MV1 antibody PGT151-LC (SEQ ID NO:23):
DIVMTQTPLSLSVT PGQPAS I SCKS S E SLRQSNGKT SLYWYRQKPGQS PQLLVFEVSNRFSGVS
DRFVGS
GSGTDFTLRI SRVEAE DVGFYYCMQS KD FPLT FGGGTKVDLKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT151 derived heavy chain of the 151/MV1 antibody PGT151-HC-Knob (SEQ ID NO:24):
RVQLVE SGGGVVQPGKSVRL SCVVSD FP FS KY PMYWVRQAPGKGLEWVAAISGDAWHVVY SNSVQGRFLV
SRDNVKNTLYLEMNSLKIEDTAVYRCARMFQESGPPRLDRWSGRNYYYYSGMDVWGQGTIVIVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSS
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHS HYTQKSLSLS PGK
145/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 145/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGE PAS I SCRS SQRLL S SYGHT YLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 145/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
.. QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT145 derived light chain of the 145/P140 antibody -PGT145-LC (SEQ ID NO:3):
EVVITQSPLFLPVT PGEAASLSCKCSHSLQHSTGANYLAWYLQRPGQT PRLL IHLATHRASGVPDRFSGS
GSGTDFTLKI SRVESDDVGTYYCMQGLHSPWT FGQGTKVE IKRTVAAPSVFI FP PSDEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT145 derived heavy chain of the 145/P140 antibody -PGT145-HC-Knob (SEQ ID NO:4):
QVQLVQSGAEVKKPGSSVKVSCKASGNS FSNHDVHWVRQATGQGLEWMGWMSHEGDKTGLAQKFQGRVT I
TRDSGASTVYMELRGLTADDTAIYYCLIGSKHRLRDY FLYNEYGPNYEEWGDYLATLDVWGHGTAVTVSS
ASTKGPSVFPLAPSSKST SGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT
VP S S SLGTQT Y ICNVNHKPSNT KVDKKVE PKSCDKT HTCP PC PAPE
FEGGPSVFLEPPKPKDILMISRT P
EVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKT KPRE EQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKA
LPAS IEKT I SKAKGQPRE PQVYTL PPCRDELT KNQVSLWCLVKGFY PSDIAVEWE SNGQPENNY KIT
PPV
LDSDGS FFLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
117/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 117/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGE PAS I SCRS SQRLL S SYGHT YLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 117/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 3BNC117 derived light chain of the 117/P140 antibody -3BNC117-LC (SEQ ID NO:5):
DI QMTQ SP S SLSASVGDTVT ITCQANGYLNWYQQRRGKAPKLL I YDGSKLERGVPSRFSGRRWGQEYNLT
INNLQPEDIATY FCQVYE FVVFGQGT KVQVDI KRTVAAPSVF I FPP SDEQLKSGTASVVCLLNN FY
PREA
KVQWKVDNALQSGNSQE SVT EQDSKDST Y SLS STLTLSKADY EKHKVYACEVTHQGLS SPVT KS
FNRGEC
Amino acid sequence defining the 3BNC117 derived heavy chain of the 117/P140 antibody -3BNC117-HC-Knob (SEQ ID NO:6):
QVQLLQ SGAAVT KPGASVRVSCEASGYN I RDY FIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSL
TRHASWDFDT FS FYMDLKALRSDDTAVY FCARQRSDYWDEDVWGQGTLVTVS SAST KGPSVFPLAPS SKS
TSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRI PEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQPR
EPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKLTVDK
S RWQQGNV FS CS VL HEAL HS HY TQ KSLSLS PGK
128/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 128/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRS SQRLL S SYGHTYLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 128/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT128 derived light chain of the 128/P140 antibody -PGT128-LC (SEQ ID NO:7):
QSALTQ PP SASGSPGQ SIT I SCIGTSNNEVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSKSGNTASL
TVSGLQTDDEAVYYCGSLVGNWDVI FGGGTKLTVLGQPKAAPSVTL FP PS SE ELQANKATLVCL I SD FY
P
GAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPTEC
Amino acid sequence defining the PGT128 derived heavy chain of the 128/P140 antibody -PGT128-HC-Knob (SEQ ID NO:8):
QPQLQE SGPTLVEASETL SLTCAVSGDSTAACNS FWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPS
LKSRLTLALDTPKNLVELKLNSVTAADTATYYCAREGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SL SSVVTVP SS S
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLS PGK
10E8/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 10E8/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 10E8/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/P140 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/P140 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
A 1 0E8/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the A10E8/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the A10E8/P140 antibody -PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the A10E8 derived light chain of the A10E8/P140 antibody -Al 0E8-LC (SEQ ID NO:21):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGASGNRASL
T I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I SDF
YPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPT
EC S
Amino acid sequence defining the A10E8 derived heavy chain of the A10E8/P140 antibody -10E8-HC-Knob (SEQ ID NO:22):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
151/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 151/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the 151/P140 antibody -PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKG FY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT151 derived light chain of the 151/P140 antibody -PGT151-LC (SEQ ID NO:23):
DIVMTQTPLSLSVT PGQPAS I SCKS S E SLRQSNGKT SLYWYRQKPGQS PQLLVFEVSNRFSGVS
DRFVGS
GSGTDFTLRI SRVEAE DVGFYYCMQS KD FPLT FGGGTKVDLKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT151 derived heavy chain of the 151/P140 antibody -PGT151 -HC -Knob (SEQ ID NO:24):
RVQLVE SGGGVVQPGKSVRL SCVVSD FP FS KY PMYWVRQAPGKGLEWVAAISGDAWHVVY SNSVQGRFLV
SRDNVKNTLYLEMNSLKIEDTAVYRCARMFQESGPPRLDRWSGRNYYYYSGMDVWGQGTIVIVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSS
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHS HYTQKSLSLS PGK
10E8/Alpha-Her2 antibody:
Amino acid sequence defining the alpha-Her2 derived light chain of the 10E8/Alpha-Her2 antibody - antiHer2-VLCH1 (SEQ ID NO:13):
DIVMTQSHKFMSTSVGDRVS ITCKASQDVNTAVAWYQQKPGHS PKLL I Y SAS FRYTGVPDRFTGNRSGTD
FT FT I S SVQAEDLAVYYCQQHYTT PPT FGGGT KVE I KS SAST KGPSVFPLAP S S KST
SGGTAALGCLVKD
Y FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPK
Sc Amino acid sequence defining the alpha-Her2 derived heavy chain of the 10E8/Alpha-Her2 antibody - antiHer2-HC-Hole-Cross (SEQ ID NO:14):
QVQLQQSGPELVKPGASLKLSCTASGENIKDTY I HWVKQRPEQGLEWIGRIY PINGYTRYDPKFQDKAT I
TADT SSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYWGQGASVIVSSASTAAPSVFI FP PS DEQLKS
GTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVT
HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDPEV
KFNWYVDGVEVHNAKT KPRE EQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAS I E KT I S KAKGQ
P
RE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FELVSKLTVD
KS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/Alpha-Her2 antibody -10E8-LC (SEQ ID NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/Alpha-Her2 antibody -10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
4E10/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 4E10/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 4E10/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 4E10 derived light chain of the 4E10/P140 antibody - 4E10-LC
(SEQ ID NO:17):
E IVLTQ SPGTQSLS PGERATLSCRASQSVGNNKLAWYQQRPGQAPRLL IYGASSRPSGVADRFSGSGSGT
DFTLT I SRLE PEDFAVYYCQQYGQ SL ST FGQGTKVEVKRTVAAPSVFI FP PSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SL SSTLTL SKADYEKHKVYACEVT HQGL SS PVT KS F
NRGEC
Amino acid sequence defining the 4E10 derived heavy chain of the 4E10/P140 antibody -PGT145-HC-Knob (SEQ ID NO:18):
QVQLVQSGAEVKRPGSSVTVSCKASGGS FSTYALSWVRQAPGRGLEWMGGVI PLLT ITNYAPRFQGRIT I
TADRST STAYLELNSLRPEDTAVYYCAREGTTGAGWLGKP IGAFAHWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY IC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I S KAK
GQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKL
TVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
10E8/X19 antibody:
Amino acid sequence defining the X19 derived light chain of the 10E8/X19 antibody - X19-VLCH1 (SEQ ID NO:19):
E IVLTQ SPATLSVS PGRRATLSCRASQSVNTNLAWYQQKPGQAPRLL I YGAS SRATGI PDRFSGSGSGTD
=I I SRLEPEDFAVYYCQHYGSS PLT FGGGT KLE I KS SAST KGPSVFPLAP SSKST
SGGTAALGCLVKD
Y FPE PVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPK
Sc Amino acid sequence defining the X19 derived heavy chain of the 10E8/X19 antibody - X19-HC-Hole-Cross (SEQ ID NO:20):
QVQLVQ SGGGVVQPGRSLRL SCAASG FT FS SY PMHWVRQAPGKGLEWMTVI S SDGRNKYY PDSVKGRFT
I
SRDNSKNTLYLQMNSLRPEDTAVYYCARGGYHDEWSGPDYWGQGTLVTVS SASTAAPSVF I FPPSDEQLK
SGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVT EQDSKDSTY SLS STLTLSKADY EKHKVYACEV
THQGLSSPVTKS FNRGECDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQ
PREPQVCTLP PSRDELTKNQVSLSCAVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLVSKLTV
DKSRWQQGNV FS CSVL HEAL HS HY TQ KSLSLS PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/X19 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/X19 antibody - PGT145-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8/515H7 antibody Amino acid sequence defining the 515H7 derived light chain of the 10E8/515H7 antibody ¨
515H7-VLCH1 (SEQ ID NO:25):
DIVMSQ SP SSLAVSAGEKVTMSCKSSQSL FNSRT RKNYLAWYQQKPGQ SPKLL I YWASARDSGVPARFTG
SGSETY FTLT I SRVQAEDLAVYYCMQ S FNLRT FGGGTKLE IKASTKGPSVFPLAPSSKST SGGTAALGCL
VKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDKKV
EPKSC
Amino acid sequence defining the 515H7 derived heavy chain of the 10E8/515H7 antibody ¨
515H7-Hole-Cross (SEQ ID NO:26):
EVNLVE SGGGLVQPGGSLRL SCAT SG FT FT DNYMSWVRQP PGKALEWLGF I RNKANGYTT DY
SASVRGRF
T I SRDNSQSILYLQMNALRAEDSATYYCARDVGSNY FDYWGQGTTLTVSSASTAAPSVFI FP PSDEQLKS
GTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVT
HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDPEV
KFNWYVDGVEVHNAKT KPRE EQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAS I E KT I S KAKGQ
P
RE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FELVSKLTVD
KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/515H7 antibody - 10E8-LC (SEQ ID NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/515H7 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
T I SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
Chimeric CDR123 antibody (SEQ ID NO:27):
SELTQDPAVSVALGQTVRITCRGDSLRSHYASWYQQKPGQAPVLVI YGKNNRPSGI PDRFSGSSSGNTAS
LT ITGAQAEDEADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVTLEPPSSEELQANKATLVCL I SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Chimeric FW123 (SEQ ID NO:28):
YELTQETGVSVALGRTVT ITCQGDSLRSYYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYCNSRDSSGNHLVVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
10E8V1.0/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v1.0/MV1 antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v1.0/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v1.0 derived light chain of the 10E8v1.0/iMab antibody -10E8v1.0-LC (SEQ ID NO:29):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS S SGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.0 derived heavy chain of the 10E8v1.0/iMab antibody - 10E8v1.0-HC-Knob (SEQ ID NO:30):
.. EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE
SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.1/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v1.1/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v1.1/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v1.1 derived light chain of the 10E8v1.1/iMab antibody ¨
10E8v1.1-LC (SEQ ID NO:31):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS SSGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.1 derived heavy chain of the 10E8v1.1/iMab antibody - 10E8v1.1 HC-Knob (SEQ ID NO:32):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKYYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V2.0/iMab antibody (also referred to as 10E8.2/iMab antibody) Amino acid sequence defining the MV1 derived light chain of the 10E8v2.0/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v2.0/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v2.0 derived light chain of the 10E8v2.0/iMab antibody ¨
10E8v2.0-LC (SEQ ID NO:33):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v2.0 derived heavy chain of the 10E8v2.0/iMab antibody - 10E8v2.0-HC-Knob (SEQ ID NO:34):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V3.0/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v3.0/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v3.0/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v3.0 derived light chain of the 10E8v3.0/iMab antibody ¨
10E8v3.0-LC (SEQ ID NO:15):
SELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGI HDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8v3.0 derived heavy chain of the 10E8v3.0/iMab antibody - 10E8v3.0-HC-Knob (SEQ ID NO:16):
EVQLVE SGGDLVKPGGSLRL SC SASG FS FKNTWMTWVRQAPGKGLEWVGRITGPGEGWTSDYAATVQGRF
T I SRNNMIDMLYLEMNRLRTDDTGLYYCVHTEKYYNFWGGYPPGEEY FQHWGRGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.0/P140 (H6L10/PRO140) antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8V1.0/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the 10E8V1.0/P140 antibody - PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the L10 derived light chain of the 10E8V1.0/P140 antibody ¨ L10-LC (SEQ ID NO:29):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS S SGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the H6 derived heavy chain of the 10E8V1.0/P140 antibody ¨ H6-HC-Knob (SEQ ID NO:30):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.1/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v1.1/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
-- CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY
ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v1.1/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v1.1 derived light chain of the 10E8v1.1/P140 antibody ¨
10E8v1.1-LC (SEQ ID NO:31):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS SSGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.1 derived heavy chain of the 10E8v1.1/P140 antibody - 10E8v1.1 HC-Knob (SEQ ID NO:32):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKYYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V2.0/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v2.0/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRS SQRLL S SYGHTYLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v2.0/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v2.0 derived light chain of the 10E8v2.0/P140 antibody ¨
10E8v2.0-LC (SEQ ID NO:33):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v2.0 derived heavy chain of the 10E8v2.0/P140 antibody - 10E8v2.0 HC-Knob (SEQ ID NO:34):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V3.0/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v3.0/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v3.0/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v3.0 derived light chain of the 10E8v3.0/P140 antibody ¨
10E8v3.0-LC (SEQ ID NO:15):
SELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGI HDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8v3.0 derived heavy chain of the 10E8v3.0/P140 antibody - 10E8v3.0 HC-Knob (SEQ ID NO:16):
EVQLVE SGGDLVKPGGSLRL SC SASG FS FKNTWMTWVRQAPGKGLEWVGRITGPGEGWTSDYAATVQGRF
.. T I SRNNMIDMLYLEMNRLRTDDTGLYYCVHTEKYYNFWGGYPPGEEY FQHWGRGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.1/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.1 derived heavy chain of the 10E8.2.1/iMab antibody -10E8.2.1-HC-Knob (SEQ ID NO:35) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.2/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.2 derived heavy chain of the 10E8.2.2/iMab antibody -10E8.2.2-HC-Knob (SEQ ID NO:36) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTh<VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.3/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKG FY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) AS ELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.3 derived heavy chain of the 10E8.2.3/iMab antibody -10E8.2.3-HC-Knob (SEQ ID NO:37) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
.. T I SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT
KVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT PPVLDS DGS
FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.4/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2.4/iMab antibody MV1-VLCH1-LM52 (SEQ ID NO:38) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWANST E SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.3 derived heavy chain of the 10E8.2.3/iMab antibody -10E8.2.3-HC-Knob (SEQ ID NO:39) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT I :VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.5/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.5 derived heavy chain of the 10E8.2.5 /iMab antibody - 10E8.2.5 -HC-Knob (SEQ ID NO:40) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRKNSINTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTKVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
.. 10E8.2.6/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.6 derived heavy chain of the 10E8.2.6/iMab antibody -10E8.2.6-HC-Knob (SEQ ID NO:41) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNSINTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT I :VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.7/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.4 derived heavy chain of the 10E8.4/iMab antibody -10E8.4-HC-Knob (SEQ ID NO:42) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRKNSKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT }<VIVS SAST
KGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.8/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.8 derived heavy chain of the 10E8.2.8/iMab antibody -10E8.2.8-HC-Knob (SEQ ID NO:43) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRDNSKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT [VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.10/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8.4 derived light chain of the 10E8.4/iMab antibody ¨
10E8.4-LC (SEQ ID NO:44) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKTGQAPKLL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.8 derived heavy chain of the 10E8.2.8/iMab antibody -10E8.2.8-HC-Knob (SEQ ID NO:45) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNSKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTKVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8 .4/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.4 derived light chain of the 10E8.4/iMab antibody ¨
10E8.4-LC (SEQ ID NO:46) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKTGQAPKLL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
.. DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY
SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.4 derived heavy chain of the 10E8.4/iMab antibody -10E8.4-HC-Knob (SEQ ID NO:47) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRKNSKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT }<VIVS SAST
KGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS
FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
In various embodiments, at least one of the heavy chain and/or light chain sequences derived from the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or the MV1 variant), 515H7 antibodies and variants thereof are paired together to form a bispecific antibody (e.g., a HIV CrossMab antibody). In an exemplary embodiment, at least one of the disclosed heavy and light chains selected from SEQ ID NOs: 1-36 are paired together to form a bispecific antibody (e.g., a HIV CrossMab antibody).
In various embodiments, the amino acid sequence of the bispecific antibody (e.g., HIV
CrossMab antibody) further includes an amino acid analog, an amino acid derivative, or other non-classical amino acids.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody. In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to a wild-type heavy chain or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody. In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to any of the sequences disclosed herein.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 wild-type heavy chain or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 wild-type heavy chain or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody.
In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%
identical to any of the sequences disclosed herein.
Homology or identity may be determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et at., (1990) PROC. NATL. ACAD. Sa. USA 87, 2264-2268; Altschul, (1993) J. MoL. EvoL.
36, 290-300; Altschul et at., (1997) NUCLEIC ACIDS RES. 25, 3389-3402, incorporated by reference) are tailored for sequence similarity searching. The approach used by the BLAST
program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases see Altschul et at., (1994) NATURE
GENETICS 6, 119-129 which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. Sa. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty);
R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=1;
and gapw=32.
Searches may also be conducted using the NCBI (National Center for Biotechnology .. Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default =
5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits:
.. default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default =
15 for all programs, not applicable to blastn; and ¨Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG
.. package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, .. PGT151, 4E10, or 10E8 antibody. In various embodiments, the bispecific antibody (e.g., HIV
CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to a wild-type heavy or light chain sequence of the P140, iMab (or the MV1 variant), 515H7 antibody. In exemplary embodiments, the bispecific antibody (e.g., HIV
CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to any of the sequences disclosed herein.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to a wild-type heavy or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody.
In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to any of the sequences disclosed herein.
The amino acid alteration can be an amino acid deletion, insertion, substitution, or modification.
In one embodiment, the amino acid alteration is an amino acid deletion. In another embodiment, the amino acid alteration is an amino acid substitution.
In various embodiments, the amino acid alteration may be in the Complementarity Determining Regions (CDRs) of the bispecific antibody (e.g., the CDR1, CDR2 or CDR3 regions). In another embodiment, the amino acid alteration may be in the framework regions (FWs) of the bispecific antibody (e.g., the FW1, FW2, FW3, or FW4 regions). In a further embodiment, the amino acid alteration may be in the joining regions (J regions) of the bispecific antibody (e.g., the J1, J2, J3, J4, J5, J6, or J7 regions).
Also provided herein are chimeric antibody derivatives of the bispecific antibodies, i.e., antibody molecules in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. For example, the bispecific antibody may include a heavy and/or light chain in which one or more CDRs or FWs derived from an antibody selected from a PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or the MV1 variant), or 515H7 antibody are replaced with one or more CDRs or FWs derived from a different antibody selected from a PGT145, PG9, PGT128, PGT121,
ID NO:33) and the 10E8.4/iMab variant (SEQ ID NO:44).
Figure 45B shows a sequence alignment of the heavy chains of 10E8.2/iMab (SEQ
ID NO:34) and the 10E8.4/iMab variant (SEQ ID NO:42). Underlined sequences denote CDR1, CDR2, and CDR3. Italicized sequences denote constant light chain or constant heavy chain sequences.
Figure 46 is a graph showing exemplary variants of 10E8 antibodies that are stable while retaining anti-HIV activity.
DETAILED DESCRIPTION
Embodiments of the present invention provide for inhibition of HIV. In various implementations, bispecific antibodies are formed, each including heavy chain and light chain components from two different parent antibodies. In various embodiments, one parent antibody specifically binds HIV, for example, the HIV envelope protein Env. In various embodiments, the other parent antibody specifically binds a cell membrane protein, for example CD4 and CCR5.
In various embodiments, the bispecific antibody (e.g., a HIV CrossMab antibody) of the present invention has the natural architecture of an IgG molecule, but with bispecificity. In a bispecific antibody, a heavy chain and light chain from each of two parental antibodies are combined, providing an antibody in which the antigen binding sites of fragment antigen-binding 1 (Fabl) and Fab2 have different binding specificities. In certain embodiments, the bispecific antibody is a CrossMab format antibody, as shown in Figure 1. In a CrossMab format, one heavy chain includes a "knob" structure and the other heavy chain includes a corresponding "hole" structure, and the positions of the constant domains (i.e., CL and CH1) from one parental antibody are switched, which together ensure correct pairing of heavy chains and light chains during assembly.
Various mAbs have been shown to block HIV infection by targeting and binding to the HIV
envelope protein Env (Figures 2B and 10). These mAbs include, for example, PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, and 10E8. Figure 2B
(adapted from www.scripps.edu/news/press/2014/20140424hiv.html) illustrates how the mAb targets the V1/V2 epitope on the HIV viral envelope gp120; how mAb PGT128 targets the glycan on the V3 stem region of HIV gp120; how mAb 3BNC117 targets the CD4 binding site of HIV gp120; how mAb 10E8 targets the membrane proximal external region (MPER) of HIV
gp41; and how mAb PGT151 targets an epitope on both HIV gp120 and HIV gp41.
In addition, monoclonal antibodies Pro 140 ("P140"), Ibalizumab ("iMab") and 515H7 have been shown to block HIV infection by targeting and binding to CCR5, CD4 and CXCR4 human cell membrane proteins, respectively (Figure 2A). Specifically, Figure 2A
shows how iMab targets CD4, the primary receptor for HIV-1 entry that is expressed on human T-cells; and how Pro 140 targets CCR5, a co-receptor for HIV-1 entry by CCR5 tropic HIV-1.
Although the ensuing discussion focuses on the use of bispecific antibodies directed to Env and the cell membrane proteins CD4 and CCR5, it is to be understood that this is solely for ease of presentation, and that any suitable antibody directed to any HIV epitope and any suitable antibody directed to any suitable cell membrane protein may be used and are within the scope of the invention.
Accordingly, in various embodiments, the present invention provides bispecific antibodies that target and bind to the HIV Env protein as well as the cell membrane proteins CCR5, CD4 and/or CXCR4. In certain embodiments, the bispecific antibodies include sequences (for example, heavy and light chain sequences) derived from, but not limited to, the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, and/or 10E8 antibodies and variants thereof.
The amino acid sequences defining the heavy and light chains of the PGT145 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/3U15 H and http://www.ncbi.nlm.nih.gov/protein/3U1S L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PG9 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/3U4E H and www dot ncbi dot nlm dot nih dot gov/protein/3MUH L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT128 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov /protein/3TYG H
and www dot ncbi dot nlm dot nih dot gov protein/3TYG L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT121 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov /protein/4FQC H
and www dot ncbi dot nlm dot nih dot gov/protein/4FQC L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 10-1074 antibody can be found, for example, in Mouquet H., et at., (2012) PNAS, 109(47): E3268-77 (including supplementary information), the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 3BNC117 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LSV H and www dot ncbi dot nlm dot nih dot gov/protein/4LSV L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the VRC01 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LST H and www dot ncbi dot nlm dot nih dot gov/protein/4LST L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the PGT151 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4NUG H and www dot ncbi dot nlm dot nih dot gov/protein/4NUG L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 4E10 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4LLV H and www dot ncbi dot nlm dot nih dot gov/protein/4LLV_L, respectively, the entire contents of which are incorporated herein by reference.
The amino acid sequences defining the heavy and light chains of the 10E8 antibody can be found, for example, at www dot ncbi dot nlm dot nih dot gov/protein/4G6F_B and www dot ncbi dot nlm dot nih dot gov/protein/4G6F_D, respectively, the entire contents of which are incorporated herein by reference.
In certain embodiments, the bispecific antibodies include sequences (for example, heavy and light chain sequences) derived from, but not limited to, the P140, iMab (or the MV1 variant) .. and/or 515H7 antibodies and variants thereof. The heavy and light chain sequences of the Pro 140, iMab (or its MV1 variant), and 515H7 antibodies are further described, for example, in Olson, W. C. et at., (1999) J Virol., 73(5):4145-55, Trkola, A. et at., (2001) J Virol., 75(2):579-88, U.S. Patent No. 7,122,185, Burkly L. C. et al., (1992) J Immunol., 149(5):1779-87, Moore J.
P. et at., (1992) J Virol., 66(8):4784-93, Reimann K. A., et at., (1997) AIDS
Res Hum Retroviruses, 13(11):933-43, International Patent Publication No.
W02014100139, and European Patent Publication No. EP2246364, the entire contents of all of which are incorporated herein by reference.
As used herein, an antibody "variant" refers to an antibody which has an amino acid sequence which differs from the amino acid sequence of a parent antibody from which it is derived. In various embodiments, the variant has one or more amino acid alterations with respect to the parent antibody.
In various embodiments, the bispecific antibody of the present invention includes a heavy and light chain sequence from the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody or a variant thereof and a heavy and light chain sequence from the P140, iMab (or the MV1 variant), or 515H7 antibody or a variant thereof.
In exemplary embodiments, a series of HIV CrossMab antibodies have been constructed including but not limited to, for example, 145/MV1, 117/MV1, 128/MV1, 10E8/MV1, 145/P140, 128/P140, 117/P140, 10E8/P140, 10E8/alpha-Her2, 10E8/X19, and 4E10/P140.
("145"), 3BNC117 ("117"), PGT128 ("128"), and 10E8 are four different HIV
envelope antibodies. Pro 140 ("P140") is a mAb that binds to the cell surface receptor CCR5. MV1 is a CD4 antibody that is a modified variant of the mAb Ibalizumab ("iMab"; see, for example, International Patent Publication No. W02014100139, incorporated herein by reference in its entirety). X19 is one of the antibody variants of the anti-cell surface receptor CXCR4 (see, for example, U.S. Patent No. 8,329,178, incorporated herein by reference in its entirety) that does not bind to cells expressing CXCR4 (and is therefore used as a non-surface binding control).
Alpha-Her2 is a mAb that binds to the Her2 receptor expressed on cells. Many of these CrossMab antibodies increase the breadth of HIV neutralization as compared to their parental antibodies (i.e., monoclonal antibodies MV1, 145, 117 or 10E8). In various embodiments, the bispecific antibodies of the invention significantly increase the potency of neutralization against HIV as compared to their parental antibodies.
The amino acid sequences defining the heavy and light chains of various exemplary HIV
CrossMab antibodies are shown below.
145/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 145/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQSPDSLAVSLGERVIMNCKSSQSLLYSTNQKNYLATNYQQKPGQSPKLLIYTNASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 145/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT145 derived light chain of the 145/MV1 antibody -PGT145-LC (SEQ ID NO:3):
EVVITQSPLFLPVT PGEAASLSCKCSHSLQHSTGANYLAWYLQRPGQT PRLL IHLATHRASGVPDRFSGS
GSGTDFTLKI SRVESDDVGTYYCMQGLHSPWT FGQGTKVE IKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT145 derived heavy chain of the 145/MV1 antibody -PGT145-HC-Knob (SEQ ID NO:4):
QVQLVQSGAEVKKPGSSVKVSCKASGNS FSNHDVHWVRQATGQGLEWMGWMSHEGDKTGLAQKFQGRVT I
TRDSGASTVYMELRGLTADDTAIYYCLIGSKHRLRDY FLYNEYGPNYEEWGDYLATLDVWGHGTAVTVSS
ASTKGPSVFPLAPSSKST SGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT
VP S S SLGTQT Y ICNVNHKPSNT KVDKKVE PKSCDKT HTCP PC PAPE
FEGGPSVFLEPPKPKDILMISRT P
EVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKT KPRE EQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKA
LPAS IEKT I SKAKGQPRE PQVYTL PPCRDELT KNQVSLWCLVKGFY PS DIAVEWE SNGQPENNY KIT
PPV
LDSDGS FFLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
117/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 117/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 117/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 3BNC117 derived light chain of the 117/MV1 antibody -3BNC117-LC (SEQ ID NO:5):
DI QMTQ SP S SLSASVGDTVT ITCQANGYLNWYQQRRGKAPKLL I YDGSKLERGVPSRFSGRRWGQEYNLT
INNLQPEDIATY FCQVYE FVVFGQGT KVQVDI KRTVAAPSVF I FPP SDEQLKSGTASVVCLLNN FY
PREA
KVQWKVDNALQSGNSQE SVT EQDSKDST Y SLS STLTLSKADY EKHKVYACEVTHQGLS SPVT KS
FNRGEC
Amino acid sequence defining the 3BNC117 derived heavy chain of the 117/MV1 antibody -3BNC117-HC-Knob (SEQ ID NO:6):
QVQLLQ SGAAVT KPGASVRVSCEASGYN I RDY FIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSL
TRHASWDFDT FS FYMDLKALRSDDTAVY FCARQRSDYWDEDVWGQGTLVTVS SAST KGPSVFPLAPS SKS
TSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRI PEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQPR
EPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKLTVDK
S RWQQGNV FS CS VL HEAL HS HY TQ KSLSLS PGK
128/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 128/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 128/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT128 derived light chain of the 128/MV1 antibody -PGT128-LC (SEQ ID NO:7):
QSALTQ PP SASGSPGQ SIT I SCIGTSNNEVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSKSGNTASL
TVSGLQTDDEAVYYCGSLVGNWDVI FGGGTKLTVLGQPKAAPSVTL FP PS SE ELQANKATLVCL I SD FY
P
GAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPTEC
Amino acid sequence defining the PGT128 derived heavy chain of the 128/MV1 antibody -PGT128-HC-Knob (SEQ ID NO:8):
QPQLQE SGPTLVEASETL SLTCAVSGDSTAACNS FWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPS
LKSRLTLALDTPKNLVELKLNSVTAADTATYYCAREGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SL SSVVTVP SS S
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLS PGK
10E8/MV1 antibody:
Amino acid sequence defining the MV1 derived light chain of the 10E8/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8/MV1 antibody - MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/MV1 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/MV1 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
A10E8/MV1 antibody Amino acid sequence defining the MV1 derived light chain of the A10E8/MV1 antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the A10E8/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the A 10E8 derived light chain of the A 1 0E8/MV1 antibody A 1 0E8-LC (SEQ ID NO:21):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGASGNRASL
T I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I SDF
YPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPT
EC S
Amino acid sequence defining the A 10E8 derived heavy chain of the A10E8/MV1 antibody 10E8-HC-Knob (SEQ ID NO:22):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
151/MV1 antibody Amino acid sequence defining the MV1 derived light chain of the 151/MV1 antibody - MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 151/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT151 derived light chain of the 151/MV1 antibody PGT151-LC (SEQ ID NO:23):
DIVMTQTPLSLSVT PGQPAS I SCKS S E SLRQSNGKT SLYWYRQKPGQS PQLLVFEVSNRFSGVS
DRFVGS
GSGTDFTLRI SRVEAE DVGFYYCMQS KD FPLT FGGGTKVDLKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT151 derived heavy chain of the 151/MV1 antibody PGT151-HC-Knob (SEQ ID NO:24):
RVQLVE SGGGVVQPGKSVRL SCVVSD FP FS KY PMYWVRQAPGKGLEWVAAISGDAWHVVY SNSVQGRFLV
SRDNVKNTLYLEMNSLKIEDTAVYRCARMFQESGPPRLDRWSGRNYYYYSGMDVWGQGTIVIVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSS
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHS HYTQKSLSLS PGK
145/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 145/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGE PAS I SCRS SQRLL S SYGHT YLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 145/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
.. QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT145 derived light chain of the 145/P140 antibody -PGT145-LC (SEQ ID NO:3):
EVVITQSPLFLPVT PGEAASLSCKCSHSLQHSTGANYLAWYLQRPGQT PRLL IHLATHRASGVPDRFSGS
GSGTDFTLKI SRVESDDVGTYYCMQGLHSPWT FGQGTKVE IKRTVAAPSVFI FP PSDEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT145 derived heavy chain of the 145/P140 antibody -PGT145-HC-Knob (SEQ ID NO:4):
QVQLVQSGAEVKKPGSSVKVSCKASGNS FSNHDVHWVRQATGQGLEWMGWMSHEGDKTGLAQKFQGRVT I
TRDSGASTVYMELRGLTADDTAIYYCLIGSKHRLRDY FLYNEYGPNYEEWGDYLATLDVWGHGTAVTVSS
ASTKGPSVFPLAPSSKST SGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT
VP S S SLGTQT Y ICNVNHKPSNT KVDKKVE PKSCDKT HTCP PC PAPE
FEGGPSVFLEPPKPKDILMISRT P
EVTCVVVDVS HE DPEVKFNWYVDGVEVHNAKT KPRE EQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKA
LPAS IEKT I SKAKGQPRE PQVYTL PPCRDELT KNQVSLWCLVKGFY PSDIAVEWE SNGQPENNY KIT
PPV
LDSDGS FFLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
117/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 117/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGE PAS I SCRS SQRLL S SYGHT YLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 117/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 3BNC117 derived light chain of the 117/P140 antibody -3BNC117-LC (SEQ ID NO:5):
DI QMTQ SP S SLSASVGDTVT ITCQANGYLNWYQQRRGKAPKLL I YDGSKLERGVPSRFSGRRWGQEYNLT
INNLQPEDIATY FCQVYE FVVFGQGT KVQVDI KRTVAAPSVF I FPP SDEQLKSGTASVVCLLNN FY
PREA
KVQWKVDNALQSGNSQE SVT EQDSKDST Y SLS STLTLSKADY EKHKVYACEVTHQGLS SPVT KS
FNRGEC
Amino acid sequence defining the 3BNC117 derived heavy chain of the 117/P140 antibody -3BNC117-HC-Knob (SEQ ID NO:6):
QVQLLQ SGAAVT KPGASVRVSCEASGYN I RDY FIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSL
TRHASWDFDT FS FYMDLKALRSDDTAVY FCARQRSDYWDEDVWGQGTLVTVS SAST KGPSVFPLAPS SKS
TSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRI PEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQPR
EPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKLTVDK
S RWQQGNV FS CS VL HEAL HS HY TQ KSLSLS PGK
128/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 128/P140 antibody - PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRS SQRLL S SYGHTYLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 128/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT128 derived light chain of the 128/P140 antibody -PGT128-LC (SEQ ID NO:7):
QSALTQ PP SASGSPGQ SIT I SCIGTSNNEVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSKSGNTASL
TVSGLQTDDEAVYYCGSLVGNWDVI FGGGTKLTVLGQPKAAPSVTL FP PS SE ELQANKATLVCL I SD FY
P
GAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPTEC
Amino acid sequence defining the PGT128 derived heavy chain of the 128/P140 antibody -PGT128-HC-Knob (SEQ ID NO:8):
QPQLQE SGPTLVEASETL SLTCAVSGDSTAACNS FWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPS
LKSRLTLALDTPKNLVELKLNSVTAADTATYYCAREGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SL SSVVTVP SS S
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLS PGK
10E8/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 10E8/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 10E8/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/P140 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/P140 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
A 1 0E8/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the A10E8/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the A10E8/P140 antibody -PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the A10E8 derived light chain of the A10E8/P140 antibody -Al 0E8-LC (SEQ ID NO:21):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGASGNRASL
T I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I SDF
YPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVAPT
EC S
Amino acid sequence defining the A10E8 derived heavy chain of the A10E8/P140 antibody -10E8-HC-Knob (SEQ ID NO:22):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
151/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 151/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the 151/P140 antibody -PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD I Y PGGNY I RNNE
KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FT YWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKG FY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the PGT151 derived light chain of the 151/P140 antibody -PGT151-LC (SEQ ID NO:23):
DIVMTQTPLSLSVT PGQPAS I SCKS S E SLRQSNGKT SLYWYRQKPGQS PQLLVFEVSNRFSGVS
DRFVGS
GSGTDFTLRI SRVEAE DVGFYYCMQS KD FPLT FGGGTKVDLKRTVAAPSVFI FP PS DEQLKSGTASVVCL
LNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKS FNRGEC
Amino acid sequence defining the PGT151 derived heavy chain of the 151/P140 antibody -PGT151 -HC -Knob (SEQ ID NO:24):
RVQLVE SGGGVVQPGKSVRL SCVVSD FP FS KY PMYWVRQAPGKGLEWVAAISGDAWHVVY SNSVQGRFLV
SRDNVKNTLYLEMNSLKIEDTAVYRCARMFQESGPPRLDRWSGRNYYYYSGMDVWGQGTIVIVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSS
LGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I
EKT I SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT
PPVLDSDG
S F FLY SKLTVDKSRWQQGNVESCSVLHEALHS HYTQKSLSLS PGK
10E8/Alpha-Her2 antibody:
Amino acid sequence defining the alpha-Her2 derived light chain of the 10E8/Alpha-Her2 antibody - antiHer2-VLCH1 (SEQ ID NO:13):
DIVMTQSHKFMSTSVGDRVS ITCKASQDVNTAVAWYQQKPGHS PKLL I Y SAS FRYTGVPDRFTGNRSGTD
FT FT I S SVQAEDLAVYYCQQHYTT PPT FGGGT KVE I KS SAST KGPSVFPLAP S S KST
SGGTAALGCLVKD
Y FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPK
Sc Amino acid sequence defining the alpha-Her2 derived heavy chain of the 10E8/Alpha-Her2 antibody - antiHer2-HC-Hole-Cross (SEQ ID NO:14):
QVQLQQSGPELVKPGASLKLSCTASGENIKDTY I HWVKQRPEQGLEWIGRIY PINGYTRYDPKFQDKAT I
TADT SSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYWGQGASVIVSSASTAAPSVFI FP PS DEQLKS
GTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVT
HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDPEV
KFNWYVDGVEVHNAKT KPRE EQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAS I E KT I S KAKGQ
P
RE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FELVSKLTVD
KS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/Alpha-Her2 antibody -10E8-LC (SEQ ID NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/Alpha-Her2 antibody -10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
4E10/P140 antibody:
Amino acid sequence defining the Pro 140 derived light chain of the 4E10/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the Pro 140 derived heavy chain of the 4E10/P140 antibody -PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 4E10 derived light chain of the 4E10/P140 antibody - 4E10-LC
(SEQ ID NO:17):
E IVLTQ SPGTQSLS PGERATLSCRASQSVGNNKLAWYQQRPGQAPRLL IYGASSRPSGVADRFSGSGSGT
DFTLT I SRLE PEDFAVYYCQQYGQ SL ST FGQGTKVEVKRTVAAPSVFI FP PSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SL SSTLTL SKADYEKHKVYACEVT HQGL SS PVT KS F
NRGEC
Amino acid sequence defining the 4E10 derived heavy chain of the 4E10/P140 antibody -PGT145-HC-Knob (SEQ ID NO:18):
QVQLVQSGAEVKRPGSSVTVSCKASGGS FSTYALSWVRQAPGRGLEWMGGVI PLLT ITNYAPRFQGRIT I
TADRST STAYLELNSLRPEDTAVYYCAREGTTGAGWLGKP IGAFAHWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY IC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I S KAK
GQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLY
SKL
TVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
10E8/X19 antibody:
Amino acid sequence defining the X19 derived light chain of the 10E8/X19 antibody - X19-VLCH1 (SEQ ID NO:19):
E IVLTQ SPATLSVS PGRRATLSCRASQSVNTNLAWYQQKPGQAPRLL I YGAS SRATGI PDRFSGSGSGTD
=I I SRLEPEDFAVYYCQHYGSS PLT FGGGT KLE I KS SAST KGPSVFPLAP SSKST
SGGTAALGCLVKD
Y FPE PVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPK
Sc Amino acid sequence defining the X19 derived heavy chain of the 10E8/X19 antibody - X19-HC-Hole-Cross (SEQ ID NO:20):
QVQLVQ SGGGVVQPGRSLRL SCAASG FT FS SY PMHWVRQAPGKGLEWMTVI S SDGRNKYY PDSVKGRFT
I
SRDNSKNTLYLQMNSLRPEDTAVYYCARGGYHDEWSGPDYWGQGTLVTVS SASTAAPSVF I FPPSDEQLK
SGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVT EQDSKDSTY SLS STLTLSKADY EKHKVYACEV
THQGLSSPVTKS FNRGECDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I EKT I SKAKGQ
PREPQVCTLP PSRDELTKNQVSLSCAVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS F FLVSKLTV
DKSRWQQGNV FS CSVL HEAL HS HY TQ KSLSLS PGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/X19 antibody - 10E8-LC
(SEQ NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/X19 antibody - PGT145-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
TI SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEY FQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8/515H7 antibody Amino acid sequence defining the 515H7 derived light chain of the 10E8/515H7 antibody ¨
515H7-VLCH1 (SEQ ID NO:25):
DIVMSQ SP SSLAVSAGEKVTMSCKSSQSL FNSRT RKNYLAWYQQKPGQ SPKLL I YWASARDSGVPARFTG
SGSETY FTLT I SRVQAEDLAVYYCMQ S FNLRT FGGGTKLE IKASTKGPSVFPLAPSSKST SGGTAALGCL
VKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDKKV
EPKSC
Amino acid sequence defining the 515H7 derived heavy chain of the 10E8/515H7 antibody ¨
515H7-Hole-Cross (SEQ ID NO:26):
EVNLVE SGGGLVQPGGSLRL SCAT SG FT FT DNYMSWVRQP PGKALEWLGF I RNKANGYTT DY
SASVRGRF
T I SRDNSQSILYLQMNALRAEDSATYYCARDVGSNY FDYWGQGTTLTVSSASTAAPSVFI FP PSDEQLKS
GTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVT
HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDPEV
KFNWYVDGVEVHNAKT KPRE EQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAS I E KT I S KAKGQ
P
RE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FELVSKLTVD
KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8 derived light chain of the 10E8/515H7 antibody - 10E8-LC (SEQ ID NO:9):
YELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8 derived heavy chain of the 10E8/515H7 antibody - 10E8-HC-Knob (SEQ ID NO:10):
EVQLVE SGGGLVKPGGSLRL SC SASG FD FDNAWMTWVRQP PGKGLEWVGRITGPGEGWSVDYAAPVEGRF
T I SRLNSINFLYLEMNNLRMEDSGLY FCARTGKYYDEWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
Chimeric CDR123 antibody (SEQ ID NO:27):
SELTQDPAVSVALGQTVRITCRGDSLRSHYASWYQQKPGQAPVLVI YGKNNRPSGI PDRFSGSSSGNTAS
LT ITGAQAEDEADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVTLEPPSSEELQANKATLVCL I SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
Chimeric FW123 (SEQ ID NO:28):
YELTQETGVSVALGRTVT ITCQGDSLRSYYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRAS
LT I SGAQAEDDAEYYCNSRDSSGNHLVVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I SD
FY PGAVTVAWKADS SPVKAGVETTT P SKQSNNKYAASSYL SLT PEQWKSHRSY SCQVT HEGSTVEKTVAP
TECS
10E8V1.0/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v1.0/MV1 antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v1.0/MV1 antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v1.0 derived light chain of the 10E8v1.0/iMab antibody -10E8v1.0-LC (SEQ ID NO:29):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS S SGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.0 derived heavy chain of the 10E8v1.0/iMab antibody - 10E8v1.0-HC-Knob (SEQ ID NO:30):
.. EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE
SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.1/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v1.1/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v1.1/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v1.1 derived light chain of the 10E8v1.1/iMab antibody ¨
10E8v1.1-LC (SEQ ID NO:31):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS SSGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.1 derived heavy chain of the 10E8v1.1/iMab antibody - 10E8v1.1 HC-Knob (SEQ ID NO:32):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKYYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V2.0/iMab antibody (also referred to as 10E8.2/iMab antibody) Amino acid sequence defining the MV1 derived light chain of the 10E8v2.0/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v2.0/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v2.0 derived light chain of the 10E8v2.0/iMab antibody ¨
10E8v2.0-LC (SEQ ID NO:33):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v2.0 derived heavy chain of the 10E8v2.0/iMab antibody - 10E8v2.0-HC-Knob (SEQ ID NO:34):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V3.0/iMab antibody Amino acid sequence defining the MV1 derived light chain of the 10E8v3.0/iMab antibody MV1-VLCH1 (SEQ ID NO:1):
DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8v3.0/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO:2):
QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v3.0 derived light chain of the 10E8v3.0/iMab antibody ¨
10E8v3.0-LC (SEQ ID NO:15):
SELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGI HDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8v3.0 derived heavy chain of the 10E8v3.0/iMab antibody - 10E8v3.0-HC-Knob (SEQ ID NO:16):
EVQLVE SGGDLVKPGGSLRL SC SASG FS FKNTWMTWVRQAPGKGLEWVGRITGPGEGWTSDYAATVQGRF
T I SRNNMIDMLYLEMNRLRTDDTGLYYCVHTEKYYNFWGGYPPGEEY FQHWGRGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.0/P140 (H6L10/PRO140) antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8V1.0/P140 antibody -PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the PRO140 derived heavy chain of the 10E8V1.0/P140 antibody - PRO140-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the L10 derived light chain of the 10E8V1.0/P140 antibody ¨ L10-LC (SEQ ID NO:29):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS S SGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the H6 derived heavy chain of the 10E8V1.0/P140 antibody ¨ H6-HC-Knob (SEQ ID NO:30):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V1.1/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v1.1/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
-- CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY
ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v1.1/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v1.1 derived light chain of the 10E8v1.1/P140 antibody ¨
10E8v1.1-LC (SEQ ID NO:31):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRP SGI PDRFSGS SSGNTA
SLT IAGAQAEDDADYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v1.1 derived heavy chain of the 10E8v1.1/P140 antibody - 10E8v1.1 HC-Knob (SEQ ID NO:32):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKYYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V2.0/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v2.0/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRS SQRLL S SYGHTYLHWYLQKPGQS PQLL TY
EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v2.0/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVS SASTAAP SVFI FP
PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8v2.0 derived light chain of the 10E8v2.0/P140 antibody ¨
10E8v2.0-LC (SEQ ID NO:33):
ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8v2.0 derived heavy chain of the 10E8v2.0/P140 antibody - 10E8v2.0 HC-Knob (SEQ ID NO:34):
EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8V3.0/P140 antibody Amino acid sequence defining the PRO140 derived light chain of the 10E8v3.0/P140 antibody PRO140-VLCH1 (SEQ ID NO:11):
DIVMTQSPLSLPVT PGEPAS I SCRSSQRLL SSYGHTYLHWYLQKPGQS PQLL TY EVSNRFSGVPDRFSGS
GSGTDFTLKI SRVEAEDVGVYYCSQSTHVPLT FGQGTKVE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the P140 derived heavy chain of the 10E8v3.0/P140 antibody PRO140-HC-Hole-Cross (SEQ ID NO:12):
EVQLVESGGGLVKPGGSLRLSCAASGYT FSNYWI GWVRQAPGKGLEWI GD TY PGGNY I RNNE KFKDKTTL
SADT SKNTAYLQMNSLKT EDTAVYYCGS S FGSNYVFAW FTYWGQGTLVTVSSASTAAP SVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8v3.0 derived light chain of the 10E8v3.0/P140 antibody ¨
10E8v3.0-LC (SEQ ID NO:15):
SELTQETGVSVALGRTVT ITCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGI HDRFSGSASGNRAS
LT I SGAQAEDDAEYYC SSRDKSGSRL SVEGGGTKLTVL SQ PKAAPSVTL FPP SSEELQANKATLVCL I
SD
FY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
TECS
Amino acid sequence defining the 10E8v3.0 derived heavy chain of the 10E8v3.0/P140 antibody - 10E8v3.0 HC-Knob (SEQ ID NO:16):
EVQLVE SGGDLVKPGGSLRL SC SASG FS FKNTWMTWVRQAPGKGLEWVGRITGPGEGWTSDYAATVQGRF
.. T I SRNNMIDMLYLEMNRLRTDDTGLYYCVHTEKYYNFWGGYPPGEEY FQHWGRGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.1/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.1 derived heavy chain of the 10E8.2.1/iMab antibody -10E8.2.1-HC-Knob (SEQ ID NO:35) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTLVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.2/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.2 derived heavy chain of the 10E8.2.2/iMab antibody -10E8.2.2-HC-Knob (SEQ ID NO:36) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRLNSINFLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTh<VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.3/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKG FY PS DIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) AS ELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.3 derived heavy chain of the 10E8.2.3/iMab antibody -10E8.2.3-HC-Knob (SEQ ID NO:37) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
.. T I SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT
KVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SD IAVEWE SNGQ PENNYKTT PPVLDS DGS
FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.4/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2.4/iMab antibody MV1-VLCH1-LM52 (SEQ ID NO:38) DIVMTQ SPDSLAVSLGERVININCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWANST E SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.3 derived heavy chain of the 10E8.2.3/iMab antibody -10E8.2.3-HC-Knob (SEQ ID NO:39) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNTKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT I :VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.5/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.5 derived heavy chain of the 10E8.2.5 /iMab antibody - 10E8.2.5 -HC-Knob (SEQ ID NO:40) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRKNSINTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTKVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
.. 10E8.2.6/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.6 derived heavy chain of the 10E8.2.6/iMab antibody -10E8.2.6-HC-Knob (SEQ ID NO:41) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNSINTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT I :VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.7/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFY PGAVTVAWKADSS PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.4 derived heavy chain of the 10E8.4/iMab antibody -10E8.4-HC-Knob (SEQ ID NO:42) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRKNSKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT }<VIVS SAST
KGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.8/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKS SQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRE SGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL S S PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.2 derived light chain of the 10E8.2/iMab antibody ¨
10E8.2-LC (SEQ ID NO: 33) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKPGQAP ILL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.8 derived heavy chain of the 10E8.2.8/iMab antibody -10E8.2.8-HC-Knob (SEQ ID NO:43) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
T I SRDNSKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGT [VIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8.2.10/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVTMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWESNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
Amino acid sequence defining the 10E8.4 derived light chain of the 10E8.4/iMab antibody ¨
10E8.4-LC (SEQ ID NO:44) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKTGQAPKLL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SEELQANKATLVCL I S
DFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLT PEQWKSHRSY SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.2.8 derived heavy chain of the 10E8.2.8/iMab antibody -10E8.2.8-HC-Knob (SEQ ID NO:45) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRDNSKNTLYLEMNNVRTEDTGYY FCARTGKHYDFWSGYPPGEEY FQDWGQGTKVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PREPQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
10E8 .4/iMab Amino acid sequence defining the MV1 derived light chain of the 10E8.2/iMab antibody MV1-VLCH1 (SEQ ID NO: 1) DIVMTQ SPDSLAVSLGERVIMNCKSSQSLLY STNQKNYLAWYQQKPGQ SPKLL I YWASTRESGVPDRFSG
SGSGTDFTLT I S SVQAEDVAVYYCQQYY SY RT FGGGTKLE IKSSASTKGPSVFPLAPSSKST SGGTAALG
CLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQ SSGLY SLS SVVTVP SS SLGTQTY ICNVNHKPSNTKVDK
KVEPKSC
Amino acid sequence defining the MV1 derived heavy chain of the 10E8.2/iMab antibody MV1-HC-Hole-Cross (SEQ ID NO: 2) QVQLQQSGPEVVKPGASVKNISCKASGYT FT SYVIHWVRQKPGQGLDWIGY INPYNDGT DY DE KFKGKATL
TSUI' ST STAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTAAPSVFI FP PSDEQL
KSGTASVVCLLNNFY PREAKVQWKVDNALQ SGNSQE SVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE
VT HQGL SS PVTKS FNRGECDKT HTCP PC PAPE FEGGPSVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS I E KT I SKAKG
QPRE PQVCTL PP SRDELT KNQVSL SCAVKGFY PSDIAVEWE SNGQPENNY KTTP PVLDSDGS
FFLVSKLT
VDKS RWQQGNVF SC SVLH EALH S HYT QKSL SL S PGK
Amino acid sequence defining the 10E8.4 derived light chain of the 10E8.4/iMab antibody ¨
10E8.4-LC (SEQ ID NO:46) ASELTQDPAVSVALKQTVT I TCRGDSLRSHYASWYQKKTGQAPKLL FYGKNNRPSGVPDRFSGSASGNRA
SLT I SGAQAEDDAEYYCSSRDKSGSRLSVEGGGTKLTVLSQPKAAPSVIL FP PS SE ELQANKATLVCL I S
.. DFY PGAVTVAWKADS S PVKAGVET TT PSKQSNNKYAASSYLSLT PEQWKSHRSY
SCQVTHEGSTVEKTVA
PT EC S
Amino acid sequence defining the 10E8.4 derived heavy chain of the 10E8.4/iMab antibody -10E8.4-HC-Knob (SEQ ID NO:47) EVRLVE SGGGLVKPGGSLRL SC SASG FN FDDAWMTWVRQP PGKGLEWVGRI SGPGEGWSVDYAE SVKGRF
TI SRKNSKNTLYLEMNNVRTEDTGYY FCARTGKHYD FWSGY P PGEEY FQDWGQGT }<VIVS SAST
KGPSVF
PLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALT SGVHT FPAVLQSSGLY SLSSVVTVPSSSLGTQ
TY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFL FP PKPKDTLMI SRT PEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKAL PAS I E KT I
SKAKGQ PRE PQVYTLP PCRDELTKNQVSLWCLVKGFY P SDIAVEWE SNGQ PENNYKTT PPVLDSDGS
FEL
YSKLTVDKSRWQQGNVESCSVLHEALHSHYTQKSLSLSPGK
In various embodiments, at least one of the heavy chain and/or light chain sequences derived from the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or the MV1 variant), 515H7 antibodies and variants thereof are paired together to form a bispecific antibody (e.g., a HIV CrossMab antibody). In an exemplary embodiment, at least one of the disclosed heavy and light chains selected from SEQ ID NOs: 1-36 are paired together to form a bispecific antibody (e.g., a HIV CrossMab antibody).
In various embodiments, the amino acid sequence of the bispecific antibody (e.g., HIV
CrossMab antibody) further includes an amino acid analog, an amino acid derivative, or other non-classical amino acids.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody. In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to a wild-type heavy chain or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody. In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that is at least 60% identical to any of the sequences disclosed herein.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 wild-type heavy chain or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 wild-type heavy chain or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody.
In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%
identical to any of the sequences disclosed herein.
Homology or identity may be determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et at., (1990) PROC. NATL. ACAD. Sa. USA 87, 2264-2268; Altschul, (1993) J. MoL. EvoL.
36, 290-300; Altschul et at., (1997) NUCLEIC ACIDS RES. 25, 3389-3402, incorporated by reference) are tailored for sequence similarity searching. The approach used by the BLAST
program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases see Altschul et at., (1994) NATURE
GENETICS 6, 119-129 which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. Sa. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty);
R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=1;
and gapw=32.
Searches may also be conducted using the NCBI (National Center for Biotechnology .. Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default =
5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits:
.. default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default =
15 for all programs, not applicable to blastn; and ¨Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG
.. package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, .. PGT151, 4E10, or 10E8 antibody. In various embodiments, the bispecific antibody (e.g., HIV
CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to a wild-type heavy or light chain sequence of the P140, iMab (or the MV1 variant), 515H7 antibody. In exemplary embodiments, the bispecific antibody (e.g., HIV
CrossMab antibody) comprises a sequence that includes at least one amino acid alteration with respect to any of the sequences disclosed herein.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to a wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody.
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to a wild-type heavy or light chain sequence of the P140, iMab (or the MV1 variant), or 515H7 antibody.
In exemplary embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to any of the sequences disclosed herein.
The amino acid alteration can be an amino acid deletion, insertion, substitution, or modification.
In one embodiment, the amino acid alteration is an amino acid deletion. In another embodiment, the amino acid alteration is an amino acid substitution.
In various embodiments, the amino acid alteration may be in the Complementarity Determining Regions (CDRs) of the bispecific antibody (e.g., the CDR1, CDR2 or CDR3 regions). In another embodiment, the amino acid alteration may be in the framework regions (FWs) of the bispecific antibody (e.g., the FW1, FW2, FW3, or FW4 regions). In a further embodiment, the amino acid alteration may be in the joining regions (J regions) of the bispecific antibody (e.g., the J1, J2, J3, J4, J5, J6, or J7 regions).
Also provided herein are chimeric antibody derivatives of the bispecific antibodies, i.e., antibody molecules in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. For example, the bispecific antibody may include a heavy and/or light chain in which one or more CDRs or FWs derived from an antibody selected from a PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or the MV1 variant), or 515H7 antibody are replaced with one or more CDRs or FWs derived from a different antibody selected from a PGT145, PG9, PGT128, PGT121,
10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or the MV1 variant), or 515H7 antibody.
In various embodiments, the present invention provides improved bispecific antibodies that demonstrate advantageous properties related to solubility, stability, and therapeutic activity. It is contemplated that such antibodies may be particularly suited for large-scale commercial production. For example, such antibodies may exhibit increased solubility, reduced aggregation, reduced precipitation, and/or enhanced stability or resistance to degradation during manufacturing.
In an exemplary embodiment, the improved bispecific antibody is a variant of the 10E8V2.0/iMab antibody (also referred to as 10E8.2/iMab antibody). In such embodiments, the variant may exhibit enhanced solubility, stability, and/or therapeutic activity (e.g., antiviral activity) compared to the parent 10E8V2.0/iMab antibody.
In some embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the heavy chain or light chain sequences of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO: 33, or SEQ ID NO:34).
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to the heavy chain or light chain sequences of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO: 33, or SEQ ID NO:34).
In various embodiments, the bispecific antibody may comprise one or more amino acid alterations in the Complementarity Determining Regions (CDRs) of the 10E8V2.0/iMab antibody (e.g., the CDR1, CDR2 or CDR3 regions). In another embodiment, the bispecific antibody may comprise one or more amino acid alterations in the framework regions (FWs) of the bispecific antibody (e.g., the FW1, FW2, FW3, or FW4 regions). In a further embodiment, the amino acid alterations may be in the joining regions (J regions) of the 10E8V2.0/iMab antibody (e.g., the J1, J2, J3, J4, J5, J6, or J7 regions).
In some embodiments, the bispecific antibody comprises a variant heavy chain derived from 10E8V2.0 (i.e., SEQ ID NO:34). In such embodiments, the bispecific antibody may include one or more mutations at positions selected from L72, 175, F77, L89, Y98, F100a, W100b, Y100e, P100f, P100g, L108, and/or L170 of the heavy chain (the mutation positions on SEQ ID NO: 34 are determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include one or more mutations at positions selected from L72, 175, F77, and/or L108. In some embodiment, the bispecific antibody may include the one or more mutations selected from L72K, I75K, F77T, and L108K. In an embodiment, the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47.
In some embodiments, the bispecific antibody comprises a variant light chain derived from 10E8V2.0 (i.e., SEQ ID NO:33). In such embodiments, the bispecific antibody may include one or more mutations at positions selected from L15, P40, 145, and P112 of the light chain (the mutation positions on SEQ ID NO: 33 are determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include one or more mutations at positions selected from P40 and 145. In some embodiments, the bispecific antibody may include one or more mutations selected from P4OT and I45K. In an embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46.
In some embodiments, the bispecific antibody comprises a variant light chain derived from MV1 (i.e., SEQ ID NO: 1). In such embodiments, the bispecific antibody may include mutations at positions 52-54 (the mutation position on SEQ ID NO: 1 is determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include amino acid mutations at positions 52-54. In some embodiments, the amino acid at position 52 is mutated to Asn (N), the amino acid at position 53 is mutated to Ser (S), and the amino acid at position 54 is mutated to Thr (T). In some embodiments, the Asn mutation at position 52 is N-linked glycosylated. In an embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.
In illustrative embodiment, the bispecific antibody comprises a heavy and light chain derived from 10E8 comprising the amino acid sequence of SEQ ID NO: 47 and SEQ ID
NO:46, respectively. The bispecific antibody further comprises a heavy and light chain derived from MV1 comprising the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2, respectively.
Modification of the amino acid sequence of recombinant binding protein is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described, for example, in Sambrook et at., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.
Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein.
Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.
Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains.
Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions. Specific expression and purification conditions will vary depending upon the expression system employed.
In various embodiments, the bispecific antibodies of the present invention (e.g., HIV CrossMab antibodies) are used in therapy. For example, the bispecific antibody (e.g., HIV CrossMab antibody) can be used to neutralize HIV in a mammal (e.g., a human patient).
For example, antibodies of the invention can bind to HIV so as to partially or completely inhibit one or more biological activities of the virus. In an embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a R5-tropic HIV. In another embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a X4-tropic HIV. In a further embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a R5X4 dual-tropic HIV. In some embodiments, use of the antibody to neutralize HIV in a mammal comprises administering to the mammal a therapeutically effective amount of the antibody.
Generally, a therapeutically effective amount of active component is in the range of, for example, about 0.1 mg/kg to about 100 mg/kg, e.g., about 1 mg/kg to about 100 mg/kg, e.g., about 1 mg/kg to about 10 mg/kg of the body weight of the patient. In various embodiments, a therapeutically effective amount of active component is in a range of about 0.01 mg/kg to about 30 mg/kg of the body weight of the patient, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg body weight, inclusive of all values and ranges there between.
In some embodiments, the therapeutically effective amount of active component is any value between about 1 to 10 mg/kg, between about 10 to 20 mg/kg, between about 20 to 30 mg/kg, between about 30 to 40 mg/kg, between about 40 to 50 mg/kg, between about 50 to 60 mg/kg, between about 60 to 70 mg/kg, between about 70 to 80 mg/kg, between about 80 to 90 mg/kg, or between about 90 to 100 mg/kg.
In some embodiments, the therapeutically effective amount of active component is about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 6 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 60 mg/kg delivered intravenously (IV).
In some embodiments, the therapeutically effective amount of active component is about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, or about 20 mg/kg delivered subcutaneously (s.c.) or intramuscularly (i.m).
The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level.
Alternatively, the initial dosage can be smaller than the optimum, and the dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from, for example, 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are more than once daily, about once per day, about twice a day, about three times a day, about four times a day, about five times a day, about every other day, about every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year. Formulation of antibody-based drugs is within ordinary skill in the art.
In various embodiments, the antibodies of the invention may be administered for a prolonged period. For example, the antibodies may be administered for at least about 1 week, at least about 4 weeks, about 8 weeks, or at least about 12 weeks. In some embodiments, the regimen is for at least about 1 month, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about years, at least about 15 years, at least about 20 years, at least about 30 years, at least about 40 years, or at least about 50 years.
For therapeutic use, an antibody may be combined with a pharmaceutically acceptable carrier.
10 As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
Pharmaceutical compositions containing antibodies, such as those disclosed herein, can be presented in a dosage unit form and can be prepared by any suitable method. A
pharmaceutical composition should be formulated to be compatible with its intended route of administration.
Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. In an embodiment, the route of administration for antibodies of the invention is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
In some embodiments, the pharmaceutical compositions are formulated as a composition adapted for oral administration. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, sprinkles, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
In some embodiments, the pharmaceutical compositions are formulated as a composition adapted for parenteral administration. Dosage forms suitable for parenteral administration (e.g.
-- intravenous, subcutaneous, intramuscular, or intraperitoneal injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use.
They may contain, for example, suspending or dispersing agents.
In some embodiments, the compositions may additionally include pharmaceutically acceptable excipients or carriers. Exemplary excipients include sodium citrate, dicalcium phosphate, etc., and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar, etc., b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpolypyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethyl cellulose etc., c) humectants such as glycerol, etc., d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers such as crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium (cross-linked sodium carboxymethylcellulose), sodium starch glycolate, -- etc., e) solution retarding agents such as paraffin, etc., f) absorption accelerators such as quaternary ammonium compounds, etc., g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, etc., h) absorbents such as kaolin and bentonite clay, etc., and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, glyceryl behenate, etc., and mixtures of such excipients. One of skill in the art will recognize that particular excipients may have two or more functions.
Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
EXAMPLES
Example 1. Construction and Characterization of HIV CrossMab Antibodies Figures [[13]] 13A-E and Figures 14A-E [[14]] demonstrate that some iMab-based CrossMabs have greater potency and breadth than parental Abs. Except otherwise stated, all iMab-based bispecific antibodies were constructed using the MV1 variant. IC80, the antibody concentration that confers 80% neutralization of viral infectivity, is one method to evaluate antibody potency against HIV. The lower the IC80 number (indicated in the y-axis of the graphs in term of antibody concentration (m/m1)), the more potent the antibody is at neutralizing a particular HIV
strain or isolate. IC50, the antibody concentration that confers 50%
neutralization of viral infectivity, is another method to evaluate antibody potency against HIV. The lower the IC50 number (indicated in the y-axis of the graphs in term of antibody concentration (m/m1)), the more potent the antibody is at neutralizing a particular HIV strain or isolate.
Various sets of antibodies were tested against a large panel of HIV-1 pseudoviruses (118 different HIV viral isolates) representative of HIV envelope diversity by geography, clade, tropism, and stage of infection. IC80 and IC50 were used to evaluate the strength of antiviral potency and breadth. Figures 13E and 14E clearly demonstrate that, as compared to the parental antibodies iMab and 10E8, the bispecific CrossMab of the two together (10E8/iMab) neutralizes almost all HIV viruses (each virus is indicated as a dot) more potently. The other antibody sets (used to make 145/iMab, 117/iMab, 128/iMab and 151/iMab) sometimes enhance HIV
potency compared to their parental components and sometimes do not.
As shown in Figures 15A-E, the antibody iMab is also relatively potent in cell-cell neutralizing assays. PGT145, 3BNC117, 10E8, PGT128 and PGT151 are relatively potent at neutralizing cell-free viral infection, but are poor in neutralizing viruses in cell-cell transmission assays.
Creating bispecific antibodies including PGT145, 3BNC117, 10E8, PGT128 and PGT151 with iMab makes these chimeric antibodies active at neutralizing viruses in a cell-cell transmission assay. It can be seen that 10E8/iMab is the most potent antibody in these comparative studies. It is also found that 10E8/iMab is most active in preventing cell-cell transmission in vitro.
As illustrated in Figure 16, the improved potency of 10E8/iMab is statistically significant.
Figure 3 shows that improved potency requires covalent linkage of the antibody, i.e., the CrossMab format (since co-administration of two parental antibodies, iMab and 10E8, provides a lower MPI than the fused and physically linked bispecific 10E8/iMab antibody).
Figures 10, 11A-E, 12A-E, 13A-E, and 14A-E provide further evidence of the improved potency of iMab-derived CrossMab antibodies over its parental antibodies.
In summary, it is found that, for the iMab-based CrossMabs (fused with PGT145, 3BNC117, PGT151, PGT128 and 10E8), 117/iMab improves breadth but not potency; 145/iMab, 151/iMab and 128/iMab improve breadth and potency; and 10E8/iMab markedly improves breadth and potency. In terms of epitope location/accessibility and potential models of neutralization, 10E8/iMab appears to exhibit pre- and post-attachment neutralization;
145/iMab, 151/iMab and 117/iMab appear to exhibit pre-attachment neutralization; and 117/iMab may show signs of steric restriction and potentially reduced potency for some viruses. 10E8/iMab also exhibits potent activity against HIV cell-to-cell transmission.
As also shown in Figures 13A-D and 14A-D, Pro 140-based CrossMab activities are sometimes weaker than their parental antibodies and corresponding iMab-based CrossMabs, as shown by the high concentrations required to reach IC80 and IC50. Anchoring of these four mAbs to the host cell receptor CCR5 via another host cell receptor-binding antibody called Pro 140 does not improve the antiviral potency or breadth (as measured by IC80 against a large panel of HIV
isolates) compared to their respective parental antibodies. These panels indicate that Pro140-based CrossMabs for these four antibodies are weaker than their corresponding iMab-based CrossMabs (IC50 and IC80 comparisons of Pro140-based vs. iMab-based CrossMabs).
As shown in Figures 13E and 14E, 10E8/P140, a fifth Pro 140-based CrossMab, is more potent than its parental antibodies and 10E8/iMab CrossMab. These panels illustrate a comparison of the potency (IC80 or IC50) of parental mAb Pro140 (right-most column of data points in Figures 13E and 14E), bispecific CrossMab 10E8/P140 (second from right column of data points in Figures 13E and 14E), and parental mAb 10E8 (center column of data points in Figures 13E and 14E) against a large panel of HIV isolates. These panels also illustrate a comparison of the potency (IC80 or IC50) of parental mAb iMab (left-most column of data points in Figures 13E
and 14E), bispecific CrossMab 10E8/iMab (second from left column of data points in Figures 13E and 14E), and parental mAb 10E8 (center column of data points in Figures 13E and 14E) against a large panel of HIV isolates. The second from left and second from right columns of data points in Figures 13E and 14E illustrate a comparison of the potency (IC80 or IC50) of the bispecific CrossMabs 10E8/iMab and 10E8/P140 against a large panel of HIV
isolates.
Pro 140 is known to not have activity against X4 HIV viruses, as X4 viruses use CXCR4 as a co-receptor for HIV-1 entry, and Pro 140 binds to CCR5. 10E8 alone has very weak activity against X4 viruses. However, the bispecific CrossMab 10E8/Pro 140 can neutralize all X4 viruses tested to date better than either of the parent antibodies. Figures 4A-J illustrate the effectiveness of 10E8, Pro 140, and 10E8/P140 bispecific CrossMab antibody in inhibition of various strains of HIV.
As shown in Figures 5A-G, 10E8/Pro140 CrossMab is a more potent inhibitor of various strains of HIV than the co-administration of the two parental antibodies, demonstrating a synergistic, not merely additive, enhancement of potency with this particular bispecific antibody.
As shown in Figures 6A-D, a CrossMab of 10E8 fused to a non-membrane bound antibody (X19) does not provide enhanced potency, as can be seen when compared to membrane bound 10E8/P140. Thus, the potency of the 10E8/P140 CrossMab appears to require anchoring of 10E8 to the cell membrane. However, membrane binding alone does not afford the enhanced potency of these CrossMabs. Figures 7A-H show that anchoring 10E8 on HER2 does not provide substantial potency enhancement as compared to anchoring 10E8 on CCR5.
Anchoring of 10E8 to a viral receptor specifically (in this case CCR5 via Pro 140 or CD4 via iMab) provides enhanced antiviral activity.
A10E8 is a mutant version of the 10E8 mAb that has a one amino acid deletion in the light chain FR3. Compared to 10E8, A10E8 has a much weaker epitope binding activity, as illustrated in Figures 8A-C. However, once the A10E8 was anchored on a cell receptor (by combining A10E8 and iMab in a CrossMab antibody - iMab specifically binds cell receptor CD4), Figures 8D-E, show that its inhibition activity is improved. These data suggest the contribution of specific cell receptor anchoring, i.e., anchoring on a viral receptor or a viral co-receptor, in enhancing the activity of this HIV antibody. Still, while A 10E8/P140 CrossMab has improved antiviral activity over A10E8, it is still not as potent as 10E8/P140 CrossMab. A10E8/P140 CrossMab is comparatively more effective in neutralizing R5 viruses than it is in neutralizing X4 viruses.
4E10 is an anti-gp41 MPER mAb known to be less potent than the anti-gp41 MPER
mAb 10E8.
Similar to the results for A10E8, Figures 20 and 21A-G show that anchoring 4E10 on co-receptor CCR5 (via Pro 140 in a CrossMab antibody) enhanced antiviral activity of 4E10 significantly.
Taken together, this suggests that the anchoring of a number of anti-gp41 MPER
Abs to either CCR5 or CD4 (via combining the MPER Abs with P140 or iMab in a CrossMab antibody bispecific) can greatly improve the potency and breadth of the respective anti-gp41 MPER Ab.
Multiple parameters contribute to enhanced activity of certain bispecific CrossMabs against HIV, including parental Ab potency, affinity, and pre- and post-attachment neutralization abilities. In particular, the 10E8/Pro140 CrossMab represents an effective combination in terms of overcoming energetic, spatial and temporal constraints, targeting sequential/interdependent steps in the entry process, epitope location/accessibility, binding affinity, pre-and post-attachment neutralization, and binding geometry. As shown in Figure 20, 4E10/Pro140 has a greater binding affinity for MPER than A10E8/Pro140 and 10E8/Pro140. Figures 9A-G show the inhibition potency of 10E8/Pro140, A10E8/Pro140 and 4E10/Pro140, and their parental antibodies 10E8, A10E8 and 4E10 against various strains of HIV. Figures 10, 13A-E, 14A-E, 15A-E, 16, and 17A-B provide additional evidence of the greater potency of CrossMab antibodies as compared to their parental antibodies individually and the parental antibodies in combination.
The enhanced antiviral coverage of 10E8/iMab and 10E8/Pro140 CrossMabs is illustrated in Figure 10, which depict the potency and breadth of several antibodies against HIV. The x-axis indicates the concentration of a particular antibody, the y-axis indicates the percent of a large panel of HIV viral isolates neutralized by a particular antibody at a specific concentration, and each line indicates a different antibody evaluated. The left-most lines along the x-axis and those that can closely approach or reach 100% on the y-axis indicate a highly potent and broad antibody against HIV. 10E8/P140 CrossMab and 10E8/iMab CrossMab are among the most effective antibodies with respect to both viral coverage and potency, and are significantly more effective than their parental antibodies.
Figures 18A-H and 19A-C show the potency of the CrossMab 10E8/515H7 antibody as compared to its parental antibodies and previously discussed antibodies. The potency of a CrossMab antibody does not appear to correlate directly with the density of cell membrane protein targets, as the density of CCR5 (the target of Pro140) is less than that of CD4 (the target of ibalizumab), yet the potency of 10E8/Pro140-derived CrossMab antibody is greater than that of 10E8/iMab-derived CrossMab antibody.
As shown in Figures 22A-B, the lack of single, sharp peaks in size exclusion chromatography indicates a type of instability indicative of multiple molecular species for 10E8 and 10E8-derived CrossMab antibodies. Table 1 recites various process and formulation modifications used to resolve the 10E8 instability. However, as indicated by the "X" in the SEC or Size Exclusion Chromatography column, the modifications were unsuccessful in providing a single, sharp peak.
Table 1: Process and formulation screen to resolve 10E8 instability Conditions SEC Purpose E DTA* X sequester metal ions, enzymatic activity stabilize protonated form of free thiols, Acetic Acid* X
''reduction activity L Lysine* X competitive inhibitor against reduction components maintain reducing components in oxidized form, CuSO4* X
enzyme inhibitor 3-day harvesting X decreased cell death, enzymatic activity SEC running buffer condition X modification of analytical conditions His formulation buffer X modification of analytical conditions Pairing the 10E8 heavy chain with the light chain of 4E10 resolves the instability issue, as shown in Figure 23, producing a functional, though less potent, antibody. This result indicates that the instability of 10E8 is due to the light chain. Various modifications of the 10E8 light chain, shown in Figures 24B-C, such as removal of a C-terminal serine, engineering a lambda-variable region and kappa-constant region chimera, engineering an additional disulfide bond between the 10E8 heavy and light chains, or genetically grafting kappa light chain regions of non-10E8 antibodies onto the 10E8 light chain do not fully resolve 10E8 instability. As shown in Figures 25 and 26A-B, the instability is likely due to a combination of the Complementarity Determining Regions ("CDRs") and the framework regions ("FWs") of 10E8. Using 10E8-HC/4E10-LC, each 10E8 light chain CDR was grafted into 4E1OLC individually or in concert, as shown in Figure 25. Addition of 10E8 LC CDR2 and CDR3 are well tolerated, but addition of 10E8 LC
CDR1 disrupts the single peak. When all 10E8 CDRs are grafted onto 4E10, the peak is broad.
Grafting 10E8 CDRs or frameworks onto its germline light chain X results in a single peak, as shown in Figure 26A, but effectiveness in MPER binding and HIV neutralization is decreased.
Table 2 summarizes the 10E8 light chain variants tested and the efficacy thereof Table 2: Generated 10E8 LC variants Modifications Expression MPER binding SEC
Neutralization XLC' ¨> AS -\/ -\/ X
XLC' ¨> KLC -\/ -\/ X
LC CDR grafting (KLC Ab1) -\/
LC CDR grafting (KLC Ab2) -\/
H-L S-S bond X X
10E8-H/4E 10-L CDR1 (10E8) X X
10E8-H/4E 10-L CDR2 (10E8) 10E8-H/4E 10-L CDR3 (10E8) 10E8-H/4E 10-L CDR123 (10E8) -\/
Variant H6L10 of 10E8 antibody was found to be active, non-autoreactive, and stable by size exclusion chromatography, as shown in Figure 27. H6L10/Pro 140 and its parental antibodies were found to have comparable pharmacokinetics profiles in mice, as shown in Figure 28.
However, as shown in Figure 29, the H6L10 variant of 10E8 (referred to as 10E8 v1.0) combined with P140 in a bispecific antibody is substantially less potent than 10E8/P140 when tested against a large panel of HIV strains. The H6L10 variant of 10E8 (referred to as 10E8 v1.0) combined with iMab in a bispecific antibody retains the same relative amount of potency as compared to 10E8/iMab when tested against a large panel of HIV strains, but 10E8v i.o/iMab possesses the same instability as 10E8/iMab as determined by size exclusion chromatography and indicated by an X in Table 3. In an embodiment, the H6L10 variant may further include a S74W mutation.
Table 3 below lists exemplary variants, their activities, size exclusion chromatography results, and pharmacokinetics ("PK") results (see also Figure 46).
Table 3: Exemplary variants that are stable while retaining anti-HIV activity Construct Activity SEC PK
10E8/P140 +++++ X
10E8,1.0/P140 ++
10E8,1.1/P140 ++++
10E8,2.0/P140 +++ X ND
10E8,3.0/P140 +++++ -\/ X
10E8/Nab +++ X X
10E8,1.0/Nab +++ X X
10E8,1.1iiMab +++ X X
10E8,2.0/Nab ++++
10E8,3.0/Nab ++++ -\/
As indicated above, 10E8vi.ois a somatic variant of 10E8 known as H6L10. As a mAb, H6L10 has a single peak by SEC but reduced activity compared to 10E8. H6L10/Pro140 CrossMab has single SEC peak and good mouse PK, but reduced anti-HIV activity. H6L10/iMab CrossMab has double SEC peaks and poor mouse PK, but its activity against HIV is roughly the same as 10E8/iMab. 10E8vi.iincludes a single point mutation in H6L10. When paired with Pro140 in a CrossMab bispecific, this construct has single SEC peak and good mouse PK. Its activity against HIV is improved as compared to 10E8V1.0/Pro140, but still slightly less than that of 10E8/Pro140. When paired with iMab in a CrossMab bispecific, this construct has double SEC
peaks and poor mouse PK, and its activity against HIV is still roughly the same as 10E8/iMab and 10E8V1.0/iMab. 10E8v2.0is a chimeric antibody variant of 10E8 in which the FW1, CDR1 and part of FW2 are from 10E8vi.o and in which the remaining part of FW2, CDR2, FW3, CDR3 and FW4 are from 10E8. When paired with Pro140 in a CrossMab bispecific, this construct has double SEC peaks and has reduced activity against HIV as compared to 10E8/Pro140. When paired with iMab in a CrossMab bispecific, this construct has a single SEC
peak, good PK, and activity against HIV that is improved over 10E8/iMab. 10E8v3.ois a somatic variant of 10E8 known as H11L1. H11L1/Pro140 CrossMab has a single SEC peak and better anti-HIV activity than any other 10E8/Pro140 construct (including the original one identified), but has poor mouse PK due to autoreactivity. H11L1/iMab CrossMab has a single SEC peak and anti-HIV activity .. that is better than the original 10E8/iMab identified and roughly equivalent activity to that observed for 10E8V2.0/iMab, but has poor mouse PK due to autoreactivity.
The variant of 10E8 that produced a single SEC peak in the context of a particular CrossMab bispecific was different when paired with Pro140 or iMab. It appears that the stability of the 10E8 arm of these CrossMab bispecific antibodies is context dependent and will vary depending of what antibody it is paired with. Thus, one variant (10E8vi.i) was identified that was stable by SEC and with good mouse PK and good anti-HIV activity when paired with Pro140.
Another variant (1 0E8v2.o) was also identified that was stable by SEC with good mouse PK and with better anti-HIV activity than the originally identified 10E8/iMab.
Table 4 below describes the autoreactivity of tested variants, where "ANA"
refers to anti-nuclear activity and "ACA" refers to anti-cardiolipin activity.
Table 4: Autoreactivity assessment in vitro Antibodies ANA ACA *Hep-2 (50pg/mL) Staining score Negative control Low positive control High positive control ++++ ++++
iMab Pro140 10E8,4.0 0 10E8,4A
10E8Q.o 10E8vIo -1+ +/++ 0.5 10E8v1.0/13140 10E82.0/iMab 10E8,4.1/P140 10E83.0/iMab 10E8v3.0/13140 -1+
Figure 30 depicts the pharmacokinetics of 10E8 and CrossMab antibodies derived from several 10E8 variants and iMab or P140 in a mouse model. As shown in Figures [[31]]
31A-D and 32A-B [[32]], 10E8vid/P140 and 1 0E8v2.o/iMab improve anti-HIV activity and stability, and have good stability when stored in PBS at 4 C. Figure 33 depicts a native mass spectroscopy analysis of 10E8v2.o/iMab (N297A). Figures 34A-C compare the activity of 10E8vid/P140 and 10E8v2.o/iMab on a HIV Clade C panel, and compares their IC50 and IC80 efficacy. Figures 35 and 36 compare the potency of 10E8vid/P140, 10E8v2.0/iMab, and various monoclonal antibodies against HIV.
Example 2. Development of HIV CrossMab Antibodies with Improved Solubility, Stability, and/or Potency Experiments were conducted to develop 10E8/iMab CrossMab antibodies with improved solubility, stability, and activity.
Initially, a number of hydrophobic residues were identified on the surface of the 10E8.2/iMab antibody (also referred to as the 10E8v2.0/iMab antibody) which may negatively affect solubility and stability of the bispecific antibody. The hydrophobic residues are presented in Table 5 below (with reference to the Kabat numbering system):
Table 5.
Location Residue L15 Leu L40 Pro L45 Ile L112 Pro H72 Leu H75 Ile H77 Phe H89 Leu H98 Tyr H100a Phe H100b Trp H100e Tyr H100f Pro H100i Pro H108 Leu H170 Leu The hydrophobic residues were mutated either alone or in combination to yield 10E8.2/iMab variants that were assessed for their functional activity against HIV and in vivo pharmacokinetic profiles (see Figures 37A-B). The amino acid sequences of the various 10E8.2/iMab variants are provided elsewhere herein.
Specifically, in vitro neutralization assays were performed to test the activity of the 10E8.2/iMab variants against HIV. Pseudoviruses were prepared as previously described in Sun et al. 2014.
JAIDS. 66, 473-483. Virus neutralization was assessed with a single cycle assay using TZM-bl cells and HIV-1 pseudoviruses as described previously (Seaman et al. 2010. J.
Virol. 84, 1439-1452). As shown in Figure 37A, some of the 10E8.2/iMab variants (e.g., 10E8.2.1/iMab, 10E8.2.2/iMab, and 10E8.2.3/iMab antibodies) retained functional activity in the in vitro HIV-1 neutralization assay as compared to the parental 10E8.2/iMab antibody.
For in vivo pharmacokinetics analysis, BALB/c mice were divided into groups of three, and mice in each group were administered intraperitoneally with 100 [tg of the indicated antibody. Blood was drawn from all animals at Days 1, 2, 4, 7 and 10 post antibody administration, and serum was isolated and analyzed for levels of antibody in individual mice. CoStar 96-Well EIA/RIA
plates were coated with 100 ng per well of goat anti-human IgG Fc-y fragment overnight at 4 C.
Plates were washed three times with PBS + Tween and blocked with PBS
containing 5% milk and 0.5% BSA for 2 hours at room temperature. Mouse serum from the treated animals, and purified antibody in PBS for the standard curves, were added to the wells in 1:2 serial dilutions in PBS containing 2% milk and 0.2% BSA and incubated for 2 hours. After washing, peroxidase-conjugated goat anti-human IgG was incubated for 1 hour at room temperature.
Samples were detected by TMB Liquid Substrate System and spectrophotometric readings were performed at 450 nm. As shown in Figure 37B, some of the 10E8.2/iMab variants (e.g., 10E8.2.1/iMab, 10E8.2.2/iMab, and 10E8.2.3/iMab antibodies) exhibited similar pharmacokinetic profiles as the parental 10E8.2/iMab antibody.
Additionally, the precipitation profiles of the variants were evaluated under thermal stress-inducing conditions. Specifically, 10E8.2/iMab variants were expressed in 293 cells, purified using a Protein A column, exchanged into a solution of PBS (pH 7.4), and concentrated to >30 mg/mL using a membrane with a nominal molecular weight limit of 50 kDa.
Samples were then incubated at 50 C and assessed visually for precipitation at the indicated time points. Results from the thermal stress analysis are shown in Figure 37C. These results indicate that the 10E8.2.3/iMab variant retained the best combination of favorable antiviral activity by in vitro neutralization, favorable in vivo pharmacokinetics, and increased solubility (decreased precipitation) under thermal stress inducing conditions.
Based on the favorable properties of 10E8.2.3/iMab in the precipitation assay, additional hydrophilic variants and combinations were created off of this bispecific antibody backbone variant. Each of these new variants was evaluated for its aggregation potential by size exclusion chromatography (SEC) after incubation in thermal stress-inducing conditions (see Figures 38A-B). In particular, SEC was used to assess the physicochemical homogeneity of the bispecific antibody variants and to resolve monomers from non-monomeric species. Results indicated that the variants 10E8.4/iMab and 10E8.2.10/iMab exhibited the least aggregation, as indicated by a decreased peak size between 7 mL and 11 mL in Figure 38B. The 10E8.4/iMab variant was advanced into additional solubility and stability studies. This variant comprises a combination of 6 hydrophobic to hydrophilic residue mutations as compared to the parental 10E8.2/iMab antibody. Figures 45A-B provide a sequence alignment of the parental 10E8.2/iMab antibody and the 10E8.4/iMab variant.
Based on the favorable functional and pharmacokinetic characteristics and the reduction in precipitation and aggregation characteristics of the 10E8.4/iMab antibody, additional studies were performed to evaluate the solubility, turbidity, thermostability and forced degradation characteristics of the 10E8.4/iMab antibody in comparison to the 10E8.2/iMab antibody.
For example, the solubility of the 10E8.4/iMab antibody at 4 C was determined.
In one set of experiments, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into target buffers and were concentrated via ultracentrifugation at 3000-5000g, 4 C. Protein concentrations at different time points were determined by absorbance at 280nm using a NanoDrop 2000 spectrophotometer. All measurements were repeated twice with 2.5 [IL sample each time and an average was taken, and then protein concentration was plotted versus time. The maximum protein concentration achieved was determined as the solubility of the protein. As shown in Figures 39A-B, at concentrations above 50mg/mL, the 10E8.4/iMab antibody showed consistently higher protein concentrations and solubility as compared to the 10E8.2/iMab antibody in buffers 1 (acetate buffer, pH 4.5) and 2 (histidine buffer, pH
5.5).
The turbidity characteristics of the 10E8.4/iMab antibody were also analyzed.
In the analysis, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into target buffers and were concentrated via ultracentrifugation at 3000-5000g, 4 C. Absorbance at 280 nm and 350 nm were measured over time using a NanoDrop 2000 spectrophotometer. All measurements were repeated twice with 2.5 [EL sample each time and an average was taken, and protein concentration (A280) was plotted versus turbidity (A350) for similar timepoints during the ultracentrifugation process. As shown in Figure 40, the turbidity of both 10E8.2/iMab and 10E8.4/iMab antibodies increased with protein concentration over time. In particular, the 10E8.2/iMab antibody showed higher turbidity than 10E8.4/iMab at the same protein concentrations over 100 mg/mL in both buffer conditions tested.
Additionally, the thermostability profile of the 10E8.4/iMab antibody was compared against the parental 10E8.2/iMab antibody using differential scanning calorimetry (DSC).
DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. Each peak in the thermogram corresponds to a heat effect associated with a specific process, such as crystallization or melting, and is an indication of the stability of a molecule as temperature is increased. To determine the thermostability of the 10E8.2/iMab and 10E8.4/iMab antibodies, each bispecific antibody was buffer exchanged into identical buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-[tm filter.
Samples were then diluted to 1 mg/mL with reference buffers. Reference buffers (400 [EL) were added into the odd-numbered wells of a 96-well plate and 400 [EL of samples were added into the even-numbered wells of the same plate. The plates were scanned from 20 C to 90 C with a rate of 200 C/hr. Analysis of thermograms was performed with MicroCal VP-Capillary DSC
Automated data analysis software. As shown in Figure 41, both 10E8.2/iMab and 10E8.4/iMab antibodies exhibited similar thermostability when evaluated by DSC.
Figure 42 shows the results from a turbidity analysis after forced degradation of the 10E8.2/iMab and 10E8.4/iMab antibodies. The 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into identical buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL
and aseptically filtered with 0.22-11m filter. Samples were then incubated at 50 C to induce forced degradation, and both pre-centrifugation and post-centrifugation samples of 10E8.2/iMab and 10E8.4/iMab were measured for development of turbidity by absorbance at 350 nm at 0 days, 3 days and 6 days after incubation began. Results indicate that the 10E8.2/iMab antibody exhibited overall higher turbidity than 10E8.4/iMab at all time points studied.
Molecule purity after forced degradation of the 10E8.2/iMab and 10E8.4/iMab antibodies was also assessed. The 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into two buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-11m filter. Samples were then incubated at 50 C to induce forced degradation, and the percentage of molecule purity by SDS-Gel Capillary Electrophoresis in non-reducing conditions was determined at 0 days, 3 days and 6 days after incubation began.
To conduct SDS-Gel Capillary Electrophoresis, a denaturing solution was prepared by mixing sample buffer, 10% SDS and 100 mM N-Ethylmaleimide at 20:1:0.7 volume ratio. Two microliters of samples and 74, denaturing solution were mixed well, incubated at 70 C for 10 mins and spun down.
35 H20 was added to the sample, then 42 [IL of the mixture was transferred into 96-well plate and centrifuged at 4000 rpm for 20 mins to remove air bubbles. After the plate was loaded, samples were sipped, stained, separated and detected in the Microchip which was filled with destaining-gel, fluorescent dye and marker. The data was then analysed with LabChip GX
Reviewer to determine the percentage of intact bispecific antibody molecule versus smaller antibody fragments in each sample. As indicated in Table 6 below, the 10E8.4/iMab antibody showed better intact molecule purity than the 10E8.2/iMab antibody in both buffers at all timepoints studied:
Table 6.
Purity (%) Formulation Timepoint 10E8.2/iMab 10E8.4/iMab Buffer #1 TO 71.6 87.6 Buffer #1 3D 77.3 87.2 Buffer #1 6D 76.2 87.0 Buffer #4 TO 74.9 89.2 Buffer #4 3D 81.9 92.6 Buffer #4 6D 89.4 94.6 An aggregation analysis was also performed. Specifically, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into two buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-[tm filter.
Samples were then incubated at 50 C to induce forced degradation, and the high molecular weight (HMW) fraction in each protein sample was determined by SE-HPLC (size exclusion chromatography) as a measurement of aggregation. Size exclusion chromatography was performed using an Agilent 1260 Infinity system and a TSKGel G3000SWXL column (300x7.8 mm, 5p,m). The mobile phase was 50mM PB, 300 mM NaCl, pH 7.0 0.2 and the flow rate was set as 1.0 mL/min.
Samples were centrifuged (-10000 rpm for 2 min at 4 C), injected and detected at 280 nm to determine the percentage of HMW in the samples. As shown in Table 7 below, although the 10E8.4/iMab antibody had a larger HMW population at TO, it showed a less rapid change in HMW percentage over time during incubation at the forced degradation-inducing conditions as compared to 10E8.2/iMab.
Table 7.
HMW peak (%) Formulation Timepoint 10E8.2/iMab 10E8.4/iMab Buffer #1 TO 7.7 13.8 Buffer #1 3D 39.2 27.2 Buffer #1 6D 49.7 35.8 Buffer #4 TO 5.9 11.0 Buffer #4 3D 37.1 25.0 Buffer #4 6D 44.9 31.3 Additionally, as shown in Figure 43, the functional activities of the 10E8.2/iMab and 10E8.4/iMab antibodies were compared in vitro. In particular, virus neutralization was assessed with a single cycle assay using TZM-bl cells and 118 HIV-1 tier-2 HIV-1 Env pseudoviruses representing diverse clades and origins as described previously (Seaman et al.
2010. J. Virol. 84, 1439-1452). Results indicate that, in addition to its improvement in solubility, decrease in turbidity and improvement in biophysical properties under thermal stress inducing conditions, the 10E8.4/iMab antibody also exhibited an approximately 2.5-fold enhancement in neutralization activity against a large panel of HIV-1 Env pseudotyped viruses as compared to 10E8.2/iMab.
The functional activities of the 10E8.2/iMab and 10E8.4/iMab antibodies were also compared in vivo. Immunodeficient NSG mice (NOD. Cg-Prkdcsc'd 112relM/SzJ) were reconstituted with human hematopoietic stem cells and infected with Tier-2 clade B HIV-1 JR-CSF
four weeks prior to the initiation of antibody treatment. Mice were then treated weekly with modified variants of 10E8.2/iMab or 10E8.4/iMab that allowed for their evaluation in humanized mice. As shown in Figure 44, a maximum mean viral load reduction of ¨1.7 log was observed in mice treated with 10E8.2/iMab, and a maximum mean viral load reduction of ¨2.4 log was observed in mice treated with 10E8.4/iMab."
The terms and expressions employed herein are used as terms and expressions of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof In addition, having described certain embodiments of the invention, it will be apparent to those of ordinary skill in the art that other embodiments incorporating the concepts disclosed herein may be used without departing from the spirit and scope of the invention. Accordingly, the described embodiments are to be considered in all respects as only illustrative and not restrictive.
In various embodiments, the present invention provides improved bispecific antibodies that demonstrate advantageous properties related to solubility, stability, and therapeutic activity. It is contemplated that such antibodies may be particularly suited for large-scale commercial production. For example, such antibodies may exhibit increased solubility, reduced aggregation, reduced precipitation, and/or enhanced stability or resistance to degradation during manufacturing.
In an exemplary embodiment, the improved bispecific antibody is a variant of the 10E8V2.0/iMab antibody (also referred to as 10E8.2/iMab antibody). In such embodiments, the variant may exhibit enhanced solubility, stability, and/or therapeutic activity (e.g., antiviral activity) compared to the parent 10E8V2.0/iMab antibody.
In some embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the heavy chain or light chain sequences of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO: 33, or SEQ ID NO:34).
In various embodiments, the bispecific antibody (e.g., HIV CrossMab antibody) may comprise a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid alterations with respect to the heavy chain or light chain sequences of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO: 33, or SEQ ID NO:34).
In various embodiments, the bispecific antibody may comprise one or more amino acid alterations in the Complementarity Determining Regions (CDRs) of the 10E8V2.0/iMab antibody (e.g., the CDR1, CDR2 or CDR3 regions). In another embodiment, the bispecific antibody may comprise one or more amino acid alterations in the framework regions (FWs) of the bispecific antibody (e.g., the FW1, FW2, FW3, or FW4 regions). In a further embodiment, the amino acid alterations may be in the joining regions (J regions) of the 10E8V2.0/iMab antibody (e.g., the J1, J2, J3, J4, J5, J6, or J7 regions).
In some embodiments, the bispecific antibody comprises a variant heavy chain derived from 10E8V2.0 (i.e., SEQ ID NO:34). In such embodiments, the bispecific antibody may include one or more mutations at positions selected from L72, 175, F77, L89, Y98, F100a, W100b, Y100e, P100f, P100g, L108, and/or L170 of the heavy chain (the mutation positions on SEQ ID NO: 34 are determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include one or more mutations at positions selected from L72, 175, F77, and/or L108. In some embodiment, the bispecific antibody may include the one or more mutations selected from L72K, I75K, F77T, and L108K. In an embodiment, the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47.
In some embodiments, the bispecific antibody comprises a variant light chain derived from 10E8V2.0 (i.e., SEQ ID NO:33). In such embodiments, the bispecific antibody may include one or more mutations at positions selected from L15, P40, 145, and P112 of the light chain (the mutation positions on SEQ ID NO: 33 are determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include one or more mutations at positions selected from P40 and 145. In some embodiments, the bispecific antibody may include one or more mutations selected from P4OT and I45K. In an embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46.
In some embodiments, the bispecific antibody comprises a variant light chain derived from MV1 (i.e., SEQ ID NO: 1). In such embodiments, the bispecific antibody may include mutations at positions 52-54 (the mutation position on SEQ ID NO: 1 is determined by the Kabat numbering system). In some embodiments, the bispecific antibody may include amino acid mutations at positions 52-54. In some embodiments, the amino acid at position 52 is mutated to Asn (N), the amino acid at position 53 is mutated to Ser (S), and the amino acid at position 54 is mutated to Thr (T). In some embodiments, the Asn mutation at position 52 is N-linked glycosylated. In an embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.
In illustrative embodiment, the bispecific antibody comprises a heavy and light chain derived from 10E8 comprising the amino acid sequence of SEQ ID NO: 47 and SEQ ID
NO:46, respectively. The bispecific antibody further comprises a heavy and light chain derived from MV1 comprising the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2, respectively.
Modification of the amino acid sequence of recombinant binding protein is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described, for example, in Sambrook et at., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.
Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein.
Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.
Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains.
Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions. Specific expression and purification conditions will vary depending upon the expression system employed.
In various embodiments, the bispecific antibodies of the present invention (e.g., HIV CrossMab antibodies) are used in therapy. For example, the bispecific antibody (e.g., HIV CrossMab antibody) can be used to neutralize HIV in a mammal (e.g., a human patient).
For example, antibodies of the invention can bind to HIV so as to partially or completely inhibit one or more biological activities of the virus. In an embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a R5-tropic HIV. In another embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a X4-tropic HIV. In a further embodiment, the bispecific antibody (e.g., HIV CrossMab antibody) neutralizes a R5X4 dual-tropic HIV. In some embodiments, use of the antibody to neutralize HIV in a mammal comprises administering to the mammal a therapeutically effective amount of the antibody.
Generally, a therapeutically effective amount of active component is in the range of, for example, about 0.1 mg/kg to about 100 mg/kg, e.g., about 1 mg/kg to about 100 mg/kg, e.g., about 1 mg/kg to about 10 mg/kg of the body weight of the patient. In various embodiments, a therapeutically effective amount of active component is in a range of about 0.01 mg/kg to about 30 mg/kg of the body weight of the patient, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg body weight, inclusive of all values and ranges there between.
In some embodiments, the therapeutically effective amount of active component is any value between about 1 to 10 mg/kg, between about 10 to 20 mg/kg, between about 20 to 30 mg/kg, between about 30 to 40 mg/kg, between about 40 to 50 mg/kg, between about 50 to 60 mg/kg, between about 60 to 70 mg/kg, between about 70 to 80 mg/kg, between about 80 to 90 mg/kg, or between about 90 to 100 mg/kg.
In some embodiments, the therapeutically effective amount of active component is about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 6 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 60 mg/kg delivered intravenously (IV).
In some embodiments, the therapeutically effective amount of active component is about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, or about 20 mg/kg delivered subcutaneously (s.c.) or intramuscularly (i.m).
The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level.
Alternatively, the initial dosage can be smaller than the optimum, and the dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from, for example, 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are more than once daily, about once per day, about twice a day, about three times a day, about four times a day, about five times a day, about every other day, about every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year. Formulation of antibody-based drugs is within ordinary skill in the art.
In various embodiments, the antibodies of the invention may be administered for a prolonged period. For example, the antibodies may be administered for at least about 1 week, at least about 4 weeks, about 8 weeks, or at least about 12 weeks. In some embodiments, the regimen is for at least about 1 month, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about years, at least about 15 years, at least about 20 years, at least about 30 years, at least about 40 years, or at least about 50 years.
For therapeutic use, an antibody may be combined with a pharmaceutically acceptable carrier.
10 As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
Pharmaceutical compositions containing antibodies, such as those disclosed herein, can be presented in a dosage unit form and can be prepared by any suitable method. A
pharmaceutical composition should be formulated to be compatible with its intended route of administration.
Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. In an embodiment, the route of administration for antibodies of the invention is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
In some embodiments, the pharmaceutical compositions are formulated as a composition adapted for oral administration. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, sprinkles, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
In some embodiments, the pharmaceutical compositions are formulated as a composition adapted for parenteral administration. Dosage forms suitable for parenteral administration (e.g.
-- intravenous, subcutaneous, intramuscular, or intraperitoneal injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use.
They may contain, for example, suspending or dispersing agents.
In some embodiments, the compositions may additionally include pharmaceutically acceptable excipients or carriers. Exemplary excipients include sodium citrate, dicalcium phosphate, etc., and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and Bakers Special Sugar, etc., b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpolypyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC), and hydroxymethyl cellulose etc., c) humectants such as glycerol, etc., d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers such as crospovidone (cross-linked polyvinylpyrrolidone), croscarmellose sodium (cross-linked sodium carboxymethylcellulose), sodium starch glycolate, -- etc., e) solution retarding agents such as paraffin, etc., f) absorption accelerators such as quaternary ammonium compounds, etc., g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, etc., h) absorbents such as kaolin and bentonite clay, etc., and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, glyceryl behenate, etc., and mixtures of such excipients. One of skill in the art will recognize that particular excipients may have two or more functions.
Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
EXAMPLES
Example 1. Construction and Characterization of HIV CrossMab Antibodies Figures [[13]] 13A-E and Figures 14A-E [[14]] demonstrate that some iMab-based CrossMabs have greater potency and breadth than parental Abs. Except otherwise stated, all iMab-based bispecific antibodies were constructed using the MV1 variant. IC80, the antibody concentration that confers 80% neutralization of viral infectivity, is one method to evaluate antibody potency against HIV. The lower the IC80 number (indicated in the y-axis of the graphs in term of antibody concentration (m/m1)), the more potent the antibody is at neutralizing a particular HIV
strain or isolate. IC50, the antibody concentration that confers 50%
neutralization of viral infectivity, is another method to evaluate antibody potency against HIV. The lower the IC50 number (indicated in the y-axis of the graphs in term of antibody concentration (m/m1)), the more potent the antibody is at neutralizing a particular HIV strain or isolate.
Various sets of antibodies were tested against a large panel of HIV-1 pseudoviruses (118 different HIV viral isolates) representative of HIV envelope diversity by geography, clade, tropism, and stage of infection. IC80 and IC50 were used to evaluate the strength of antiviral potency and breadth. Figures 13E and 14E clearly demonstrate that, as compared to the parental antibodies iMab and 10E8, the bispecific CrossMab of the two together (10E8/iMab) neutralizes almost all HIV viruses (each virus is indicated as a dot) more potently. The other antibody sets (used to make 145/iMab, 117/iMab, 128/iMab and 151/iMab) sometimes enhance HIV
potency compared to their parental components and sometimes do not.
As shown in Figures 15A-E, the antibody iMab is also relatively potent in cell-cell neutralizing assays. PGT145, 3BNC117, 10E8, PGT128 and PGT151 are relatively potent at neutralizing cell-free viral infection, but are poor in neutralizing viruses in cell-cell transmission assays.
Creating bispecific antibodies including PGT145, 3BNC117, 10E8, PGT128 and PGT151 with iMab makes these chimeric antibodies active at neutralizing viruses in a cell-cell transmission assay. It can be seen that 10E8/iMab is the most potent antibody in these comparative studies. It is also found that 10E8/iMab is most active in preventing cell-cell transmission in vitro.
As illustrated in Figure 16, the improved potency of 10E8/iMab is statistically significant.
Figure 3 shows that improved potency requires covalent linkage of the antibody, i.e., the CrossMab format (since co-administration of two parental antibodies, iMab and 10E8, provides a lower MPI than the fused and physically linked bispecific 10E8/iMab antibody).
Figures 10, 11A-E, 12A-E, 13A-E, and 14A-E provide further evidence of the improved potency of iMab-derived CrossMab antibodies over its parental antibodies.
In summary, it is found that, for the iMab-based CrossMabs (fused with PGT145, 3BNC117, PGT151, PGT128 and 10E8), 117/iMab improves breadth but not potency; 145/iMab, 151/iMab and 128/iMab improve breadth and potency; and 10E8/iMab markedly improves breadth and potency. In terms of epitope location/accessibility and potential models of neutralization, 10E8/iMab appears to exhibit pre- and post-attachment neutralization;
145/iMab, 151/iMab and 117/iMab appear to exhibit pre-attachment neutralization; and 117/iMab may show signs of steric restriction and potentially reduced potency for some viruses. 10E8/iMab also exhibits potent activity against HIV cell-to-cell transmission.
As also shown in Figures 13A-D and 14A-D, Pro 140-based CrossMab activities are sometimes weaker than their parental antibodies and corresponding iMab-based CrossMabs, as shown by the high concentrations required to reach IC80 and IC50. Anchoring of these four mAbs to the host cell receptor CCR5 via another host cell receptor-binding antibody called Pro 140 does not improve the antiviral potency or breadth (as measured by IC80 against a large panel of HIV
isolates) compared to their respective parental antibodies. These panels indicate that Pro140-based CrossMabs for these four antibodies are weaker than their corresponding iMab-based CrossMabs (IC50 and IC80 comparisons of Pro140-based vs. iMab-based CrossMabs).
As shown in Figures 13E and 14E, 10E8/P140, a fifth Pro 140-based CrossMab, is more potent than its parental antibodies and 10E8/iMab CrossMab. These panels illustrate a comparison of the potency (IC80 or IC50) of parental mAb Pro140 (right-most column of data points in Figures 13E and 14E), bispecific CrossMab 10E8/P140 (second from right column of data points in Figures 13E and 14E), and parental mAb 10E8 (center column of data points in Figures 13E and 14E) against a large panel of HIV isolates. These panels also illustrate a comparison of the potency (IC80 or IC50) of parental mAb iMab (left-most column of data points in Figures 13E
and 14E), bispecific CrossMab 10E8/iMab (second from left column of data points in Figures 13E and 14E), and parental mAb 10E8 (center column of data points in Figures 13E and 14E) against a large panel of HIV isolates. The second from left and second from right columns of data points in Figures 13E and 14E illustrate a comparison of the potency (IC80 or IC50) of the bispecific CrossMabs 10E8/iMab and 10E8/P140 against a large panel of HIV
isolates.
Pro 140 is known to not have activity against X4 HIV viruses, as X4 viruses use CXCR4 as a co-receptor for HIV-1 entry, and Pro 140 binds to CCR5. 10E8 alone has very weak activity against X4 viruses. However, the bispecific CrossMab 10E8/Pro 140 can neutralize all X4 viruses tested to date better than either of the parent antibodies. Figures 4A-J illustrate the effectiveness of 10E8, Pro 140, and 10E8/P140 bispecific CrossMab antibody in inhibition of various strains of HIV.
As shown in Figures 5A-G, 10E8/Pro140 CrossMab is a more potent inhibitor of various strains of HIV than the co-administration of the two parental antibodies, demonstrating a synergistic, not merely additive, enhancement of potency with this particular bispecific antibody.
As shown in Figures 6A-D, a CrossMab of 10E8 fused to a non-membrane bound antibody (X19) does not provide enhanced potency, as can be seen when compared to membrane bound 10E8/P140. Thus, the potency of the 10E8/P140 CrossMab appears to require anchoring of 10E8 to the cell membrane. However, membrane binding alone does not afford the enhanced potency of these CrossMabs. Figures 7A-H show that anchoring 10E8 on HER2 does not provide substantial potency enhancement as compared to anchoring 10E8 on CCR5.
Anchoring of 10E8 to a viral receptor specifically (in this case CCR5 via Pro 140 or CD4 via iMab) provides enhanced antiviral activity.
A10E8 is a mutant version of the 10E8 mAb that has a one amino acid deletion in the light chain FR3. Compared to 10E8, A10E8 has a much weaker epitope binding activity, as illustrated in Figures 8A-C. However, once the A10E8 was anchored on a cell receptor (by combining A10E8 and iMab in a CrossMab antibody - iMab specifically binds cell receptor CD4), Figures 8D-E, show that its inhibition activity is improved. These data suggest the contribution of specific cell receptor anchoring, i.e., anchoring on a viral receptor or a viral co-receptor, in enhancing the activity of this HIV antibody. Still, while A 10E8/P140 CrossMab has improved antiviral activity over A10E8, it is still not as potent as 10E8/P140 CrossMab. A10E8/P140 CrossMab is comparatively more effective in neutralizing R5 viruses than it is in neutralizing X4 viruses.
4E10 is an anti-gp41 MPER mAb known to be less potent than the anti-gp41 MPER
mAb 10E8.
Similar to the results for A10E8, Figures 20 and 21A-G show that anchoring 4E10 on co-receptor CCR5 (via Pro 140 in a CrossMab antibody) enhanced antiviral activity of 4E10 significantly.
Taken together, this suggests that the anchoring of a number of anti-gp41 MPER
Abs to either CCR5 or CD4 (via combining the MPER Abs with P140 or iMab in a CrossMab antibody bispecific) can greatly improve the potency and breadth of the respective anti-gp41 MPER Ab.
Multiple parameters contribute to enhanced activity of certain bispecific CrossMabs against HIV, including parental Ab potency, affinity, and pre- and post-attachment neutralization abilities. In particular, the 10E8/Pro140 CrossMab represents an effective combination in terms of overcoming energetic, spatial and temporal constraints, targeting sequential/interdependent steps in the entry process, epitope location/accessibility, binding affinity, pre-and post-attachment neutralization, and binding geometry. As shown in Figure 20, 4E10/Pro140 has a greater binding affinity for MPER than A10E8/Pro140 and 10E8/Pro140. Figures 9A-G show the inhibition potency of 10E8/Pro140, A10E8/Pro140 and 4E10/Pro140, and their parental antibodies 10E8, A10E8 and 4E10 against various strains of HIV. Figures 10, 13A-E, 14A-E, 15A-E, 16, and 17A-B provide additional evidence of the greater potency of CrossMab antibodies as compared to their parental antibodies individually and the parental antibodies in combination.
The enhanced antiviral coverage of 10E8/iMab and 10E8/Pro140 CrossMabs is illustrated in Figure 10, which depict the potency and breadth of several antibodies against HIV. The x-axis indicates the concentration of a particular antibody, the y-axis indicates the percent of a large panel of HIV viral isolates neutralized by a particular antibody at a specific concentration, and each line indicates a different antibody evaluated. The left-most lines along the x-axis and those that can closely approach or reach 100% on the y-axis indicate a highly potent and broad antibody against HIV. 10E8/P140 CrossMab and 10E8/iMab CrossMab are among the most effective antibodies with respect to both viral coverage and potency, and are significantly more effective than their parental antibodies.
Figures 18A-H and 19A-C show the potency of the CrossMab 10E8/515H7 antibody as compared to its parental antibodies and previously discussed antibodies. The potency of a CrossMab antibody does not appear to correlate directly with the density of cell membrane protein targets, as the density of CCR5 (the target of Pro140) is less than that of CD4 (the target of ibalizumab), yet the potency of 10E8/Pro140-derived CrossMab antibody is greater than that of 10E8/iMab-derived CrossMab antibody.
As shown in Figures 22A-B, the lack of single, sharp peaks in size exclusion chromatography indicates a type of instability indicative of multiple molecular species for 10E8 and 10E8-derived CrossMab antibodies. Table 1 recites various process and formulation modifications used to resolve the 10E8 instability. However, as indicated by the "X" in the SEC or Size Exclusion Chromatography column, the modifications were unsuccessful in providing a single, sharp peak.
Table 1: Process and formulation screen to resolve 10E8 instability Conditions SEC Purpose E DTA* X sequester metal ions, enzymatic activity stabilize protonated form of free thiols, Acetic Acid* X
''reduction activity L Lysine* X competitive inhibitor against reduction components maintain reducing components in oxidized form, CuSO4* X
enzyme inhibitor 3-day harvesting X decreased cell death, enzymatic activity SEC running buffer condition X modification of analytical conditions His formulation buffer X modification of analytical conditions Pairing the 10E8 heavy chain with the light chain of 4E10 resolves the instability issue, as shown in Figure 23, producing a functional, though less potent, antibody. This result indicates that the instability of 10E8 is due to the light chain. Various modifications of the 10E8 light chain, shown in Figures 24B-C, such as removal of a C-terminal serine, engineering a lambda-variable region and kappa-constant region chimera, engineering an additional disulfide bond between the 10E8 heavy and light chains, or genetically grafting kappa light chain regions of non-10E8 antibodies onto the 10E8 light chain do not fully resolve 10E8 instability. As shown in Figures 25 and 26A-B, the instability is likely due to a combination of the Complementarity Determining Regions ("CDRs") and the framework regions ("FWs") of 10E8. Using 10E8-HC/4E10-LC, each 10E8 light chain CDR was grafted into 4E1OLC individually or in concert, as shown in Figure 25. Addition of 10E8 LC CDR2 and CDR3 are well tolerated, but addition of 10E8 LC
CDR1 disrupts the single peak. When all 10E8 CDRs are grafted onto 4E10, the peak is broad.
Grafting 10E8 CDRs or frameworks onto its germline light chain X results in a single peak, as shown in Figure 26A, but effectiveness in MPER binding and HIV neutralization is decreased.
Table 2 summarizes the 10E8 light chain variants tested and the efficacy thereof Table 2: Generated 10E8 LC variants Modifications Expression MPER binding SEC
Neutralization XLC' ¨> AS -\/ -\/ X
XLC' ¨> KLC -\/ -\/ X
LC CDR grafting (KLC Ab1) -\/
LC CDR grafting (KLC Ab2) -\/
H-L S-S bond X X
10E8-H/4E 10-L CDR1 (10E8) X X
10E8-H/4E 10-L CDR2 (10E8) 10E8-H/4E 10-L CDR3 (10E8) 10E8-H/4E 10-L CDR123 (10E8) -\/
Variant H6L10 of 10E8 antibody was found to be active, non-autoreactive, and stable by size exclusion chromatography, as shown in Figure 27. H6L10/Pro 140 and its parental antibodies were found to have comparable pharmacokinetics profiles in mice, as shown in Figure 28.
However, as shown in Figure 29, the H6L10 variant of 10E8 (referred to as 10E8 v1.0) combined with P140 in a bispecific antibody is substantially less potent than 10E8/P140 when tested against a large panel of HIV strains. The H6L10 variant of 10E8 (referred to as 10E8 v1.0) combined with iMab in a bispecific antibody retains the same relative amount of potency as compared to 10E8/iMab when tested against a large panel of HIV strains, but 10E8v i.o/iMab possesses the same instability as 10E8/iMab as determined by size exclusion chromatography and indicated by an X in Table 3. In an embodiment, the H6L10 variant may further include a S74W mutation.
Table 3 below lists exemplary variants, their activities, size exclusion chromatography results, and pharmacokinetics ("PK") results (see also Figure 46).
Table 3: Exemplary variants that are stable while retaining anti-HIV activity Construct Activity SEC PK
10E8/P140 +++++ X
10E8,1.0/P140 ++
10E8,1.1/P140 ++++
10E8,2.0/P140 +++ X ND
10E8,3.0/P140 +++++ -\/ X
10E8/Nab +++ X X
10E8,1.0/Nab +++ X X
10E8,1.1iiMab +++ X X
10E8,2.0/Nab ++++
10E8,3.0/Nab ++++ -\/
As indicated above, 10E8vi.ois a somatic variant of 10E8 known as H6L10. As a mAb, H6L10 has a single peak by SEC but reduced activity compared to 10E8. H6L10/Pro140 CrossMab has single SEC peak and good mouse PK, but reduced anti-HIV activity. H6L10/iMab CrossMab has double SEC peaks and poor mouse PK, but its activity against HIV is roughly the same as 10E8/iMab. 10E8vi.iincludes a single point mutation in H6L10. When paired with Pro140 in a CrossMab bispecific, this construct has single SEC peak and good mouse PK. Its activity against HIV is improved as compared to 10E8V1.0/Pro140, but still slightly less than that of 10E8/Pro140. When paired with iMab in a CrossMab bispecific, this construct has double SEC
peaks and poor mouse PK, and its activity against HIV is still roughly the same as 10E8/iMab and 10E8V1.0/iMab. 10E8v2.0is a chimeric antibody variant of 10E8 in which the FW1, CDR1 and part of FW2 are from 10E8vi.o and in which the remaining part of FW2, CDR2, FW3, CDR3 and FW4 are from 10E8. When paired with Pro140 in a CrossMab bispecific, this construct has double SEC peaks and has reduced activity against HIV as compared to 10E8/Pro140. When paired with iMab in a CrossMab bispecific, this construct has a single SEC
peak, good PK, and activity against HIV that is improved over 10E8/iMab. 10E8v3.ois a somatic variant of 10E8 known as H11L1. H11L1/Pro140 CrossMab has a single SEC peak and better anti-HIV activity than any other 10E8/Pro140 construct (including the original one identified), but has poor mouse PK due to autoreactivity. H11L1/iMab CrossMab has a single SEC peak and anti-HIV activity .. that is better than the original 10E8/iMab identified and roughly equivalent activity to that observed for 10E8V2.0/iMab, but has poor mouse PK due to autoreactivity.
The variant of 10E8 that produced a single SEC peak in the context of a particular CrossMab bispecific was different when paired with Pro140 or iMab. It appears that the stability of the 10E8 arm of these CrossMab bispecific antibodies is context dependent and will vary depending of what antibody it is paired with. Thus, one variant (10E8vi.i) was identified that was stable by SEC and with good mouse PK and good anti-HIV activity when paired with Pro140.
Another variant (1 0E8v2.o) was also identified that was stable by SEC with good mouse PK and with better anti-HIV activity than the originally identified 10E8/iMab.
Table 4 below describes the autoreactivity of tested variants, where "ANA"
refers to anti-nuclear activity and "ACA" refers to anti-cardiolipin activity.
Table 4: Autoreactivity assessment in vitro Antibodies ANA ACA *Hep-2 (50pg/mL) Staining score Negative control Low positive control High positive control ++++ ++++
iMab Pro140 10E8,4.0 0 10E8,4A
10E8Q.o 10E8vIo -1+ +/++ 0.5 10E8v1.0/13140 10E82.0/iMab 10E8,4.1/P140 10E83.0/iMab 10E8v3.0/13140 -1+
Figure 30 depicts the pharmacokinetics of 10E8 and CrossMab antibodies derived from several 10E8 variants and iMab or P140 in a mouse model. As shown in Figures [[31]]
31A-D and 32A-B [[32]], 10E8vid/P140 and 1 0E8v2.o/iMab improve anti-HIV activity and stability, and have good stability when stored in PBS at 4 C. Figure 33 depicts a native mass spectroscopy analysis of 10E8v2.o/iMab (N297A). Figures 34A-C compare the activity of 10E8vid/P140 and 10E8v2.o/iMab on a HIV Clade C panel, and compares their IC50 and IC80 efficacy. Figures 35 and 36 compare the potency of 10E8vid/P140, 10E8v2.0/iMab, and various monoclonal antibodies against HIV.
Example 2. Development of HIV CrossMab Antibodies with Improved Solubility, Stability, and/or Potency Experiments were conducted to develop 10E8/iMab CrossMab antibodies with improved solubility, stability, and activity.
Initially, a number of hydrophobic residues were identified on the surface of the 10E8.2/iMab antibody (also referred to as the 10E8v2.0/iMab antibody) which may negatively affect solubility and stability of the bispecific antibody. The hydrophobic residues are presented in Table 5 below (with reference to the Kabat numbering system):
Table 5.
Location Residue L15 Leu L40 Pro L45 Ile L112 Pro H72 Leu H75 Ile H77 Phe H89 Leu H98 Tyr H100a Phe H100b Trp H100e Tyr H100f Pro H100i Pro H108 Leu H170 Leu The hydrophobic residues were mutated either alone or in combination to yield 10E8.2/iMab variants that were assessed for their functional activity against HIV and in vivo pharmacokinetic profiles (see Figures 37A-B). The amino acid sequences of the various 10E8.2/iMab variants are provided elsewhere herein.
Specifically, in vitro neutralization assays were performed to test the activity of the 10E8.2/iMab variants against HIV. Pseudoviruses were prepared as previously described in Sun et al. 2014.
JAIDS. 66, 473-483. Virus neutralization was assessed with a single cycle assay using TZM-bl cells and HIV-1 pseudoviruses as described previously (Seaman et al. 2010. J.
Virol. 84, 1439-1452). As shown in Figure 37A, some of the 10E8.2/iMab variants (e.g., 10E8.2.1/iMab, 10E8.2.2/iMab, and 10E8.2.3/iMab antibodies) retained functional activity in the in vitro HIV-1 neutralization assay as compared to the parental 10E8.2/iMab antibody.
For in vivo pharmacokinetics analysis, BALB/c mice were divided into groups of three, and mice in each group were administered intraperitoneally with 100 [tg of the indicated antibody. Blood was drawn from all animals at Days 1, 2, 4, 7 and 10 post antibody administration, and serum was isolated and analyzed for levels of antibody in individual mice. CoStar 96-Well EIA/RIA
plates were coated with 100 ng per well of goat anti-human IgG Fc-y fragment overnight at 4 C.
Plates were washed three times with PBS + Tween and blocked with PBS
containing 5% milk and 0.5% BSA for 2 hours at room temperature. Mouse serum from the treated animals, and purified antibody in PBS for the standard curves, were added to the wells in 1:2 serial dilutions in PBS containing 2% milk and 0.2% BSA and incubated for 2 hours. After washing, peroxidase-conjugated goat anti-human IgG was incubated for 1 hour at room temperature.
Samples were detected by TMB Liquid Substrate System and spectrophotometric readings were performed at 450 nm. As shown in Figure 37B, some of the 10E8.2/iMab variants (e.g., 10E8.2.1/iMab, 10E8.2.2/iMab, and 10E8.2.3/iMab antibodies) exhibited similar pharmacokinetic profiles as the parental 10E8.2/iMab antibody.
Additionally, the precipitation profiles of the variants were evaluated under thermal stress-inducing conditions. Specifically, 10E8.2/iMab variants were expressed in 293 cells, purified using a Protein A column, exchanged into a solution of PBS (pH 7.4), and concentrated to >30 mg/mL using a membrane with a nominal molecular weight limit of 50 kDa.
Samples were then incubated at 50 C and assessed visually for precipitation at the indicated time points. Results from the thermal stress analysis are shown in Figure 37C. These results indicate that the 10E8.2.3/iMab variant retained the best combination of favorable antiviral activity by in vitro neutralization, favorable in vivo pharmacokinetics, and increased solubility (decreased precipitation) under thermal stress inducing conditions.
Based on the favorable properties of 10E8.2.3/iMab in the precipitation assay, additional hydrophilic variants and combinations were created off of this bispecific antibody backbone variant. Each of these new variants was evaluated for its aggregation potential by size exclusion chromatography (SEC) after incubation in thermal stress-inducing conditions (see Figures 38A-B). In particular, SEC was used to assess the physicochemical homogeneity of the bispecific antibody variants and to resolve monomers from non-monomeric species. Results indicated that the variants 10E8.4/iMab and 10E8.2.10/iMab exhibited the least aggregation, as indicated by a decreased peak size between 7 mL and 11 mL in Figure 38B. The 10E8.4/iMab variant was advanced into additional solubility and stability studies. This variant comprises a combination of 6 hydrophobic to hydrophilic residue mutations as compared to the parental 10E8.2/iMab antibody. Figures 45A-B provide a sequence alignment of the parental 10E8.2/iMab antibody and the 10E8.4/iMab variant.
Based on the favorable functional and pharmacokinetic characteristics and the reduction in precipitation and aggregation characteristics of the 10E8.4/iMab antibody, additional studies were performed to evaluate the solubility, turbidity, thermostability and forced degradation characteristics of the 10E8.4/iMab antibody in comparison to the 10E8.2/iMab antibody.
For example, the solubility of the 10E8.4/iMab antibody at 4 C was determined.
In one set of experiments, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into target buffers and were concentrated via ultracentrifugation at 3000-5000g, 4 C. Protein concentrations at different time points were determined by absorbance at 280nm using a NanoDrop 2000 spectrophotometer. All measurements were repeated twice with 2.5 [IL sample each time and an average was taken, and then protein concentration was plotted versus time. The maximum protein concentration achieved was determined as the solubility of the protein. As shown in Figures 39A-B, at concentrations above 50mg/mL, the 10E8.4/iMab antibody showed consistently higher protein concentrations and solubility as compared to the 10E8.2/iMab antibody in buffers 1 (acetate buffer, pH 4.5) and 2 (histidine buffer, pH
5.5).
The turbidity characteristics of the 10E8.4/iMab antibody were also analyzed.
In the analysis, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into target buffers and were concentrated via ultracentrifugation at 3000-5000g, 4 C. Absorbance at 280 nm and 350 nm were measured over time using a NanoDrop 2000 spectrophotometer. All measurements were repeated twice with 2.5 [EL sample each time and an average was taken, and protein concentration (A280) was plotted versus turbidity (A350) for similar timepoints during the ultracentrifugation process. As shown in Figure 40, the turbidity of both 10E8.2/iMab and 10E8.4/iMab antibodies increased with protein concentration over time. In particular, the 10E8.2/iMab antibody showed higher turbidity than 10E8.4/iMab at the same protein concentrations over 100 mg/mL in both buffer conditions tested.
Additionally, the thermostability profile of the 10E8.4/iMab antibody was compared against the parental 10E8.2/iMab antibody using differential scanning calorimetry (DSC).
DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. Each peak in the thermogram corresponds to a heat effect associated with a specific process, such as crystallization or melting, and is an indication of the stability of a molecule as temperature is increased. To determine the thermostability of the 10E8.2/iMab and 10E8.4/iMab antibodies, each bispecific antibody was buffer exchanged into identical buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-[tm filter.
Samples were then diluted to 1 mg/mL with reference buffers. Reference buffers (400 [EL) were added into the odd-numbered wells of a 96-well plate and 400 [EL of samples were added into the even-numbered wells of the same plate. The plates were scanned from 20 C to 90 C with a rate of 200 C/hr. Analysis of thermograms was performed with MicroCal VP-Capillary DSC
Automated data analysis software. As shown in Figure 41, both 10E8.2/iMab and 10E8.4/iMab antibodies exhibited similar thermostability when evaluated by DSC.
Figure 42 shows the results from a turbidity analysis after forced degradation of the 10E8.2/iMab and 10E8.4/iMab antibodies. The 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into identical buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL
and aseptically filtered with 0.22-11m filter. Samples were then incubated at 50 C to induce forced degradation, and both pre-centrifugation and post-centrifugation samples of 10E8.2/iMab and 10E8.4/iMab were measured for development of turbidity by absorbance at 350 nm at 0 days, 3 days and 6 days after incubation began. Results indicate that the 10E8.2/iMab antibody exhibited overall higher turbidity than 10E8.4/iMab at all time points studied.
Molecule purity after forced degradation of the 10E8.2/iMab and 10E8.4/iMab antibodies was also assessed. The 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into two buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-11m filter. Samples were then incubated at 50 C to induce forced degradation, and the percentage of molecule purity by SDS-Gel Capillary Electrophoresis in non-reducing conditions was determined at 0 days, 3 days and 6 days after incubation began.
To conduct SDS-Gel Capillary Electrophoresis, a denaturing solution was prepared by mixing sample buffer, 10% SDS and 100 mM N-Ethylmaleimide at 20:1:0.7 volume ratio. Two microliters of samples and 74, denaturing solution were mixed well, incubated at 70 C for 10 mins and spun down.
35 H20 was added to the sample, then 42 [IL of the mixture was transferred into 96-well plate and centrifuged at 4000 rpm for 20 mins to remove air bubbles. After the plate was loaded, samples were sipped, stained, separated and detected in the Microchip which was filled with destaining-gel, fluorescent dye and marker. The data was then analysed with LabChip GX
Reviewer to determine the percentage of intact bispecific antibody molecule versus smaller antibody fragments in each sample. As indicated in Table 6 below, the 10E8.4/iMab antibody showed better intact molecule purity than the 10E8.2/iMab antibody in both buffers at all timepoints studied:
Table 6.
Purity (%) Formulation Timepoint 10E8.2/iMab 10E8.4/iMab Buffer #1 TO 71.6 87.6 Buffer #1 3D 77.3 87.2 Buffer #1 6D 76.2 87.0 Buffer #4 TO 74.9 89.2 Buffer #4 3D 81.9 92.6 Buffer #4 6D 89.4 94.6 An aggregation analysis was also performed. Specifically, the 10E8.2/iMab and 10E8.4/iMab antibodies were each buffer exchanged into two buffer compositions using ultra-filtration centrifugal devices under the condition of 4 C and 3000-5000g. The protein concentrations were then adjusted to ¨10 mg/mL and aseptically filtered with 0.22-[tm filter.
Samples were then incubated at 50 C to induce forced degradation, and the high molecular weight (HMW) fraction in each protein sample was determined by SE-HPLC (size exclusion chromatography) as a measurement of aggregation. Size exclusion chromatography was performed using an Agilent 1260 Infinity system and a TSKGel G3000SWXL column (300x7.8 mm, 5p,m). The mobile phase was 50mM PB, 300 mM NaCl, pH 7.0 0.2 and the flow rate was set as 1.0 mL/min.
Samples were centrifuged (-10000 rpm for 2 min at 4 C), injected and detected at 280 nm to determine the percentage of HMW in the samples. As shown in Table 7 below, although the 10E8.4/iMab antibody had a larger HMW population at TO, it showed a less rapid change in HMW percentage over time during incubation at the forced degradation-inducing conditions as compared to 10E8.2/iMab.
Table 7.
HMW peak (%) Formulation Timepoint 10E8.2/iMab 10E8.4/iMab Buffer #1 TO 7.7 13.8 Buffer #1 3D 39.2 27.2 Buffer #1 6D 49.7 35.8 Buffer #4 TO 5.9 11.0 Buffer #4 3D 37.1 25.0 Buffer #4 6D 44.9 31.3 Additionally, as shown in Figure 43, the functional activities of the 10E8.2/iMab and 10E8.4/iMab antibodies were compared in vitro. In particular, virus neutralization was assessed with a single cycle assay using TZM-bl cells and 118 HIV-1 tier-2 HIV-1 Env pseudoviruses representing diverse clades and origins as described previously (Seaman et al.
2010. J. Virol. 84, 1439-1452). Results indicate that, in addition to its improvement in solubility, decrease in turbidity and improvement in biophysical properties under thermal stress inducing conditions, the 10E8.4/iMab antibody also exhibited an approximately 2.5-fold enhancement in neutralization activity against a large panel of HIV-1 Env pseudotyped viruses as compared to 10E8.2/iMab.
The functional activities of the 10E8.2/iMab and 10E8.4/iMab antibodies were also compared in vivo. Immunodeficient NSG mice (NOD. Cg-Prkdcsc'd 112relM/SzJ) were reconstituted with human hematopoietic stem cells and infected with Tier-2 clade B HIV-1 JR-CSF
four weeks prior to the initiation of antibody treatment. Mice were then treated weekly with modified variants of 10E8.2/iMab or 10E8.4/iMab that allowed for their evaluation in humanized mice. As shown in Figure 44, a maximum mean viral load reduction of ¨1.7 log was observed in mice treated with 10E8.2/iMab, and a maximum mean viral load reduction of ¨2.4 log was observed in mice treated with 10E8.4/iMab."
The terms and expressions employed herein are used as terms and expressions of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof In addition, having described certain embodiments of the invention, it will be apparent to those of ordinary skill in the art that other embodiments incorporating the concepts disclosed herein may be used without departing from the spirit and scope of the invention. Accordingly, the described embodiments are to be considered in all respects as only illustrative and not restrictive.
Claims (11)
1 A bispecific antibody in a CrossMab format capable of neutralizing HIV, wherein the antibody comprises a light chain and heavy chain portion of a first antibody 10E8, or a variant thereof, that binds to a HIV envelope protein, and a light chain and heavy chain portion of a second antibody ibalizumab, or a variant thereof, that binds to a cell membrane receptor protein or a cell membrane co-receptor protein, wherein the light chain portion of the first antibody 10E8 comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 33, wherein SEQ ID NO: 33 incorporates 1-4 mutations and the heavy chain portion of the first antibody 10E8 comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 34, wherein SEQ ID NO: 34 incorporates 4-12 mutations; and the light chain portion of the second antibody ibalizumab comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 1, and the heavy chain portion of the second antibody ibalizumab comprises an amino acid sequence having at least 97% with SEQ ID
NO: 2; and wherein any amino acid alterations relative to SEQ ID NOS: 1, 2, 33, and 34 are within the variable regions.
NO: 2; and wherein any amino acid alterations relative to SEQ ID NOS: 1, 2, 33, and 34 are within the variable regions.
2. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO:
33 are amino acid positions selected from L15, P40, 145, and/or P112.
33 are amino acid positions selected from L15, P40, 145, and/or P112.
3. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO: 33 are amino acid positions selected from P40 and 145.
4. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO: 33 are P4OT and I45K.
5. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO:
34 are amino acid positions selected from L72, 175, F77, L89, Y98, F100a, W100b, Y100e, P100f, P100g, L108, and/or L170.
34 are amino acid positions selected from L72, 175, F77, L89, Y98, F100a, W100b, Y100e, P100f, P100g, L108, and/or L170.
6. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO:
34 are amino acid positions selected from L72, 175, F77, and/or L108.
34 are amino acid positions selected from L72, 175, F77, and/or L108.
7. The bispecific antibody of claim 1, wherein the mutations to SEQ ID NO: 34 are L72K, I75K, F77T, and/or L108K.
8. The bispecific antibody of claim 1, wherein the antibody comprises a light chain portion of an ibalizumab antibody comprising the amino acid sequence of SEQ ID NO: 1
9. The bispecific antibody of claim 1, wherein the antibody comprises a heavy chain portion of an ibalizumab antibody comprising the amino acid sequence of SEQ ID NO: 2.
10. A pharmaceutical composition comprising the bispecific antibody of claim 1, and a pharmaceutically acceptable carrier.
11. The pharmaceutical composition of claim 10, wherein the composition is formulated for oral, intranasal, pulmonary, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous delivery.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/850,832 US10308707B2 (en) | 2013-12-02 | 2017-12-21 | Bispecific HIV-1-neutralizing antibodies |
US15/850,832 | 2017-12-21 | ||
PCT/US2018/066643 WO2019135921A1 (en) | 2017-12-21 | 2018-12-20 | Bispecific hiv-1-neutralizing antibodies |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3085351A1 true CA3085351A1 (en) | 2019-07-11 |
Family
ID=67143961
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3085351A Abandoned CA3085351A1 (en) | 2017-12-21 | 2018-12-20 | Bispecific hiv-1-neutralizing antibodies |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP3728311A4 (en) |
JP (1) | JP7345861B2 (en) |
CN (1) | CN111819196B (en) |
AU (1) | AU2018399587A1 (en) |
CA (1) | CA3085351A1 (en) |
TW (1) | TWI745643B (en) |
WO (1) | WO2019135921A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10308707B2 (en) | 2013-12-02 | 2019-06-04 | Aaron Diamond Aids Research Center | Bispecific HIV-1-neutralizing antibodies |
CA3230492A1 (en) * | 2021-08-30 | 2023-03-09 | Kanglin Biotechnology (Hangzhou) Co., Ltd. | Gene sequence construct for gene therapy for hiv infection |
WO2024008177A1 (en) * | 2022-07-08 | 2024-01-11 | Nanjing Curegene Technology Co., Ltd. | Engineered cells and uses thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104955847A (en) * | 2012-12-04 | 2015-09-30 | 马里兰州大学(巴尔的摩) | HIV-1 Env-binding antibodies, fusion proteins, and methods of use |
CN106102837B (en) * | 2013-12-02 | 2020-10-13 | 艾伦戴蒙德艾滋病研究中心 | Bispecific HIV-1-neutralizing antibodies |
WO2016054053A2 (en) * | 2014-09-29 | 2016-04-07 | Duke University | Hiv-1 antibodies and uses thereof (adcc and bispecific abs) |
BR112018008011A2 (en) * | 2015-10-25 | 2018-10-30 | Sanofi | trypecific and / or trivalent binding proteins for prevention or treatment of HIV infection |
-
2018
- 2018-12-20 AU AU2018399587A patent/AU2018399587A1/en not_active Abandoned
- 2018-12-20 CN CN201880089788.4A patent/CN111819196B/en active Active
- 2018-12-20 EP EP18898849.7A patent/EP3728311A4/en active Pending
- 2018-12-20 JP JP2020535114A patent/JP7345861B2/en active Active
- 2018-12-20 TW TW107146176A patent/TWI745643B/en active
- 2018-12-20 CA CA3085351A patent/CA3085351A1/en not_active Abandoned
- 2018-12-20 WO PCT/US2018/066643 patent/WO2019135921A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
JP2021506945A (en) | 2021-02-22 |
AU2018399587A1 (en) | 2020-07-09 |
CN111819196B (en) | 2022-10-28 |
WO2019135921A1 (en) | 2019-07-11 |
TWI745643B (en) | 2021-11-11 |
JP7345861B2 (en) | 2023-09-19 |
CN111819196A (en) | 2020-10-23 |
TW201938583A (en) | 2019-10-01 |
EP3728311A1 (en) | 2020-10-28 |
EP3728311A4 (en) | 2021-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US12098187B2 (en) | Bispecific HIV-1-neutralizing antibodies | |
US9884905B2 (en) | Bispecific HIV-1 neutralizing antibodies | |
CN110234355B (en) | Monomeric human IgG1Fc and bispecific antibodies | |
US10689434B2 (en) | Antibody against hepatitis B surface antigen and use thereof | |
CA3085351A1 (en) | Bispecific hiv-1-neutralizing antibodies | |
CN114751987A (en) | Multispecific antibody for neutralizing coronavirus | |
CN114751986A (en) | Multispecific antibodies for neutralizing novel coronaviruses | |
KR20230010691A (en) | ST2 antigen binding protein | |
CN114751988A (en) | Multispecific antibodies for neutralizing coronaviruses | |
TW202409093A (en) | Fusion proteins | |
WO2024123897A1 (en) | Broadly neutralizing and potent antibodies against hiv | |
CN117320752A (en) | Modified FcRn binding fragments with improved half-life | |
CN117402237A (en) | Bispecific antibody for resisting novel coronavirus and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20200714 |
|
FZDE | Discontinued |
Effective date: 20221004 |
|
FZDE | Discontinued |
Effective date: 20221004 |