Immunoconjugates 2

Search Strategies at the Interface of Chemistry and Biology: Immunoconjugates 2

Agenda

• Overview • Immunoglobulin (antibody) nomenclature in CAS REGISTRYSM • Search examples • Classification codes

Immunoconjugates are antibodies conjugated to a toxin, radioisotope, or label • A cytotoxic drug is linked to a monoclonal antibody for delivery to a target cancer cell that recognizes the antibody mAb - linker - cytotoxin • Ideally, conjugates are stable until delivered to cancer cells and don’t release their “payloads” to healthy cells ‒ Advances in stable linkers have greatly increased efficiency of immunoconjugates • Antibodies to unique or overexpressed antigens for cancer reduces take-up by healthy cells 4

How immunoconjugates work 1. Antibody binds to cognate antigen on target tumor cell 2. Immunoconjugate complex is internalized via endosomal/lysosomal pathway 3. Depending on linker, the cytotoxic agent is released via chemical cleavage or lysosomal degradation, delivering the drug directly to the tumor cell 4. Cytotoxins are mainly two categories: DNA modifying agents, or microtubule disrupting agents

Example of an immunoconjugate complex

Brentuximab vedotin - Adcetris®

Image source: https://en.wikipedia.org/wiki/File:MMAE-MAB-conjugate_skeletal.svg. T his image is in the public domain. 6

Searching for immunoconjugates will have to account for what is available in a database

• What information is indexed? ‒ Structure ‒ Sequence ‒ Controlled terms • Pieces and parts? • Entire complex?

CAS REGISTRY record for brentuximab vedotin

RN 914088-09-8 REGISTRY ED Entered STN: 28 Nov 2006 CN Immunoglobulin G1, anti-(human CD30 (antigen)) (human-mouse monoclonal cAC10 γ1-chain), disulfide with human-mouse monoclonal cAC10 κ-chain, dimer, complex with N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1- oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy] carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2- hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1- pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L- valinamide (CA INDEX NAME) OTHER NAMES: CN Adcetris CN Brentuximab vedotin Immunoconjugates in REGISTRY receive a CN CAC10-vcMMAE CN cAC10-vcMMAE descriptive CA Index Name that includes the CN SGN 35 antibody (immunoglobulin) name and the MF Unspecified chemical attachment. CI MAN SR CA LC STN Files: ADISINSIGHT, CA, CAPLUS, CBNB, CHEMCATS, CHEMLIST, EMBASE, IMSPATENTS, IMSRESEARCH, IPA, TOXCENTER, USPAT2, USPATFULL

*** STRUCTURE DIAGRAM IS NOT AVAILABLE *** 126 REFERENCES IN FILE CA (1907 TO DATE) 6 REFERENCES TO NON-SPECIFIC DERIVATIVES IN FILE CA 132 REFERENCES IN FILE CAPLUS (1907 TO DATE) Manual Registrations in CAS REGISTRY

• MAN - Class identifier assigned to a substance which cannot be assigned a structure connection table by the CAS Registry System ‒ Substances with a complete structure, but which cannot be processed by the CAS Registry System, e.g., > 253 non-hydrogen atoms ‒ Substances with an unknown or generic structure ‒ Multicomponent substances consisting of components with complete structures and components with unknown or generic structures

Examples of immunoconjugates in DCR

AN.S DCR-728845 CN.P BRENTUXIMAB-VEDOTIN SY ADCETRIS; BRENTUXIMAB VEDOTIN; BRENTUXIMAB-VEDOTIN; CAC10-MMAE4; CAC10-MMAE8; CAC10-VAL-CIT-MONOMETHYL AURISTATIN E; CAC10-VAL-CIT-VEDOTIN; CDX-011; CR-011-VC-MMAE; CR-011-VC-VEDOTIN; GLEMBATUMUMAB VEDOTIN; GLEMBATUMUMAB-VC-MMAE; GLEMBATUMUMAB-VC-VEDOTIN; SGN-35

NO STRUCTURE DIAGRAM AVAILABLE FOR THIS ACCESSION NUMBER

CMT The peptide monomethyl auristain E conjugated to the chimeric anti-CD30 antibody, cAC10. MF Unknown

AN.S DCR-271958 CN.P CANTUZUMAB MERTANSINE SY CANTUZUMAB MERTANSINE; CANTUZUMAB-MERTANSINE; SB-408075

CMT An antibody-directed small-molecule cytotoxic agent. It is a conjugate of the humanized monoclonal antibody C242 with a semisynthetic maytansinoid DM1 MF Unknown

Search strategies for immunoconjugates will be dependent on the area of interest Immunoconjugate piece Search strategies may include

Antibody or antibody fragment Sequence Nomenclature Keywords and antigen terms Linker Structure Cleavable vs. non-cleavable Keywords Natural vs. non-natural Spacers, stretchers, connectors, attachments Cytotoxin Structure Nomenclature Class of substances Whole immunoconjugate Nomenclature Class of substances via keywords

A comprehensive search might encompass all of these approaches. 11

Antibodies (immunoglobulins) have a Y-shaped structure

Antigen binding site Antigen binding site

CAS indexes CDR = complementarity determining immunoglobulins in region

REGISTRY by name and VL = variable region - light chain by sequence, if known. VH = variable region - heavy chain SM In CAplus they are CL = constant region - light chain indexed as Controlled CH = constant region - heavy chain Terms. 12

What gets a CAS Registry Number® (RN)?

• CAS registers the immunoglobulin, with a descriptive CA Index Name and sequence, if disclosed • CAS registers discrete chemical substances that can be part of the synthesis of a mAB conjugate, e.g., the starting materials and final products up to the point of attachment to the mAb if the reference discloses the synthesis • The final immunoconjugate will receive a RN, with a CA Index Name and usually the USAN name • CAplus indexing may include the final RN, or it may be indexed as a derivative of the drug or a derivative of the immunoglobulin 13

Vedotin is the cytotoxin, linker and attachment RN 646502-53-6 REGISTRY ED Entered STN: 05 Feb 2004 CN L-Valinamide, N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1- oxohexyl]-L-valyl-N5-(aminocarbonyl)-L- ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4- [(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1- methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1- methylpropyl]-4-oxobutyl]-N-methyl- (CA INDEX NAME) OTHER NAMES: CN 36: PN: WO2007140371 PAGE: 273 claimed protein CN Maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E CN mc-vc-PAB-MMAE CN VcMMAE CN Vedotin FS PROTEIN SEQUENCE; STEREOSEARCH MF C68 H105 N11 O15 SR CA LC STN Files: CA, CAPLUS, CASREACT, CHEMCATS, EMBASE, TOXCENTER, USPAT2, USPATFULL **RELATED SEQUENCES AVAILABLE WITH SEQLINK**

Immunoglobulin nomenclature used by USAN and World Health Organization (WHO) • Names follow the general format: ‒ Prefix - target - species of origin - mab

TARGET SPECIES OF ORIGIN Example -t(u)- tumor -o- mouse Ibritumomab

-l(i)- Immuno- -zu- humanized Daclizumab modulating

-c(i)- cardiovascular -xi- chimeric Abciximab

-t(u) tumor -u- human Olaratumab 15

CAS immunoglobulin nomenclature is descriptive

Format Immunoglobulin _____, anti-(_____) fragment (animal source monoclonal heavy chain), disulfide with animal source monoclonal light chain, dimer

Example

CN Immunoglobulin G1, anti-(human interleukin 2 receptor) (human- γ mouse monoclonal clone 1H4 1-chain), disulfide with human-mouse monoclonal clone 1H4 light chain, dimer (CA INDEX NAME) CN Daclizumab

CAS Index Name identifies the immunoglobulin class (Immunoglobulin G1) and the cognate antigen (human interleukin 2 receptor). Human-mouse indicates a humanized antibody. 16

Immunoconjugate nomenclature combines the descriptive immunoglobulin name with the chemical name of the organic attachment

CN Immunoglobulin G1, anti-(human p185neu receptor) (human-mouse monoclonal rhuMab HER2 γ1-chain), disulfide with human-mouse monoclonal rhuMab HER2 light chain, dimer, tetraamide with N2′-[3- [[1-[(4-carboxycyclohexyl)methyl]-2,5-dioxo-3-pyrrolidinyl]thio]- 1-oxopropyl]-N2′-deacetylmaytansine OTHER NAMES: CN Kadcyla Green text is the immunoglobulin nomenclature, CN PRO 132365 purple text is the organic linker and cytotoxin. CN T-DM 1 CN Trastuzumab emtansine CN Trastuzumab-MCC-DM 1 MF Unspecified CI COM, MAN SR CAS Client Services LC STN Files: ADISINSIGHT, CA, CAPLUS, CBNB, CHEMCATS, CHEMLIST, EMBASE, IMSPATENTS, IMSRESEARCH, IPA, TOXCENTER, USPATFULL

*** STRUCTURE DIAGRAM IS NOT AVAILABLE ***

Use immunoglobulin nomenclature to collect a set of antibodies in REGISTRY

=> FILE REGISTRY

=> S (IMMUNOGLOBULIN (L) ANTI (L) CD44)/CNS L1 307 (IMMUNOGLOBULIN (L) ANTI (L) CD44)/CNS

Nomenclature searches in the Chemical Name Segment field (CNS) or Basic Index are a quick way to find sets of named antibodies in REGISTRY. Use sequence searches for more comprehensive searching.

=> S (IMMUNOGLOBULIN (L) HUMAN (L) .KAPPA. (L) DNA (L) SYNTHETIC) L2 1209 (IMMUNOGLOBULIN (L) HUMAN (L) .KAPPA. (L) DNA (L) SYNTHETIC)

=> S L2 AND P/DT AND CLAIMED L3 1191 L2 AND P/DT AND CLAIMED

Search for the cytotoxin

Search Question Find relevant patent references for immunoconjugates that have maytansinoid cytotoxins.

Maytansine 19

Search Strategy

1. Search the cytotoxin substructure in REGISTRY 2. Crossover to HCAplus and refine 3. Search cytotoxin substructure in MARPAT® and WPINDEX, find bibliographic references 4. Use DUP IDE and FSORT to group by patent family 5. Use the Patent Family Manager to extract one record from each family 6. Display answers

Run the substructure search in REGISTRY

=> FILE REGISTRY

=> Uploading . . . L1 STRUCTURE UPLOADED

=> D L1 HAS NO ANSWERS L1 STR

The epoxide ring was left => S L1 SSS SAM off in the substructure FULL FILE PROJECTIONS: ONLINE **COMPLETE** search, since de-epoxy L2 50 SEA SSS SAM L1 derivatives do not include the ring. => S L1 SSS FULL L3 1007 SEA SSS FUL L1

=> SAVE TEMP MAYTANSIN/A ANSWER SET L3 HAS BEEN SAVED AS 'MAYTANSIN/A' 21

Evaluate answers with D SCAN

=> D SCAN

IN INDEX NAME NOT YET ASSIGNED MF C60 H80 Br Cl N8 O20

IN Maytansine, 2'-de(acetylmethylamino)-4,5-deepoxy-4,5-didehydro- 20-O-demethyl-2'-methyl-, (4E)- MF C31 H41 Cl N2 O8

Crossover to HCAplus and EXPAND in the CA Lexicon

=> FILE HCAPLUS Antibody Drug Conjugate is not a => E ANTIBODY DRUG CONJUGATE controlled term, but Antibody Conjugates E# FREQUENCY AT TERM has two associated terms. ------E1 0 2 ANTIBODY CONJUGATES/CT E2 671 8 ANTIBODY DIVERSITY/CT E3 0 --> ANTIBODY DRUG CONJUGATE/CT E4 0 2 ANTIBODY FRAGMENTS/CT E5 0 2 ANTIBODY FUSION PRODUCTS/CT E6 0 2 ANTIBODY FUSION PROTEINS/CT . . .

=> E E1+ALL E13 0 --> Antibody conjugates/CT E14 4587 USE Immunoconjugates/CT ********** END ********** Immunoconjugates is the current preferred controlled terminology in HCAplus.

EXPAND immunoconjugates in the CA Lexicon

=> E E14+ALL . . . E28 222332 BT3 Drug delivery systems/CT E29 0 BT2 Pharmaceutical dosage forms (non-CA heading)/CT E30 28782 BT1 Pharmaceutical carriers/CT E31 4587 --> Immunoconjugates/CT HNTE Valid heading during volume 146 (2007) to present. E32 OLD Antibodies (L) conjugates/CT E33 OLD Antibodies and Immunoglobulins (L) IgG, conjugates/CT E34 OLD Antibodies and Immunoglobulins (L) IgG, monoclonal, conjugates/CT E35 OLD Antibodies and Immunoglobulins (L) IgG1, monoclonal, conjugates/CT E36 OLD Antibodies and Immunoglobulins (L) IgG2a, monoclonal, conjugates/CT E37 OLD Antibodies and Immunoglobulins (L) conjugates/CT E38 OLD Drug delivery systems (L) immunoconjugates/CT . . . 24

Immunoconjugates has a number of OLD and UF terms

. . . E39 OLD Immunoglobulins (L) G, conjugates/CT E40 OLD Immunoglobulins (L) G, monoclonal, conjugates/CT E41 OLD Immunoglobulins (L) G1, monoclonal, conjugates/CT E42 OLD Immunoglobulins (L) G2a, monoclonal, conjugates/CT E43 OLD Immunoglobulins (L) conjugates/CT E44 OLD Pharmaceutical dosage forms (L) immunoconjugates/CT E45 UF Antibody conjugates/CT E46 UF Conjugates antibodies/CT E47 UF Ig conjugates/CT E48 UF Immunoconjugate drugs/CT E49 UF Immunoconjugate pharmaceuticals/CT E50 UF Immunoglobulin conjugates/CT E51 UF Pharmaceutical immunoconjugates/CT E52 1404 NT1 Immunotoxins/CT ********** END **********

Combine structure answers with synonyms

=> S L3 AND (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN) Postings are included here to remind 1012 L3 you how important plurals and 357032 CONJUGAT? 6931 IMMUNOCONJUGAT? abbreviations are! 447721 ANTIBODY Plurals, abbreviations and spellings 531508 ANTIBODIES are ON permanently for all searches. 702162 ANTIBODY (ANTIBODY OR ANTIBODIES) 106645 IMMUNOGLOBULIN 251395 IMMUNOGLOBULINS 320891 IMMUNOGLOBULIN (IMMUNOGLOBULIN OR IMMUNOGLOBULINS) 85400 IG 21674 IGS 97780 IG (IG OR IGS) 361355 IMMUNOGLOBULIN (IMMUNOGLOBULIN OR IG) L4 532 L3 AND (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN) 26

Option: Use the (L) operator for more focused results

=> S L3 (L) (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN) L5 396 L3 (L) (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN)

=> D SCAN TI Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection IT 64987-85-5 139504-50-0, DM1 745017-94-1, Monomethylauristatin F RL: BSU (Biological study, unclassified); PAC (Pharmacological activity); THU (Therapeutic use); BIOL (Biological study); USES (conjugate with antibody; antibody-drug conjugates for treatment of non-Hodgkin's lymphoma)

TI TAT (Tumor-associated Antigenic Target) polypeptides and methods for diagnosis and treatment of tumors of glial origin IT 57103-68-1D, Maytansinol, conjugates 113440-58-7D, Calicheamicin, conjugates RL: THU (Therapeutic use); BIOL (Biological study); USES (Uses) (TAT (Tumor-associated Antigenic Target) polypeptides and methods for diagnosis and treatment of tumors of glial origin)

Many RNs have D or DP appended, for derivatized

TI Preparation of maytansinoid antibody conjugates by a one-step process IT 799840-96-3DP, antibody conjugates, crosslinked; RL: PRP (Properties); SPN (Synthetic preparation); THU (Therapeutic use); BIOL (Biological study); PREP (Preparation); USES (Uses) (Maytansinoid DM4; one-step process for preparation of antibody conjugates with maytansinoid and bifunctional crosslinker for treatment of cancer and other diseases) IT 139504-50-0DP, Maytansinoid DM1, antibody conjugates, crosslinked RL: PRP (Properties); SPN (Synthetic preparation); THU (Therapeutic use); BIOL (Biological study); PREP (Preparation); USES (Uses) (one-step process for preparation of antibody conjugates with maytansinoid and bifunctional crosslinker for treatment of cancer and other diseases) 28

Compare AND vs. (L) operator

=> S L4 NOT L5 L6 136 L4 NOT L5

=> D SCAN TI HITIND TI Radially expandable catheter balloon with textile sheath IT Monoclonal antibodies RL: THU (Therapeutic use); BIOL (Biological study); USES (Uses) (muscle cell proliferation inhibitors; radially expandable catheter balloon with textile sheath) IT 98530-12-2, Interferon .alpha.-2b RL: THU (Therapeutic use); BIOL (Biological study); USES (Uses) (conjugates with PEG; radially expandable catheter balloon with textile sheath) IT 35457-80-8, Midecamycin 35607-66-0, Cefoxitin 35846-53-8, Maytansine 35963-37-2, Inotodiol 36011-19-5, Cytochalasin E 36150-14-8, Usambarensine 36150-15-9, Dihydrousambarensine RL: THU (Therapeutic use); BIOL (Biological study); USES (Uses) (radially expandable catheter balloon with textile sheath) The terms are all present in the record, but the reference is not relevant to immunoconjugates. 29

AND retrieves more answers, some potentially good

TI cDNA and protein sequences for human cancer-linked genes and the use of the genes as target for chemotherapy IT Drug delivery systems (immunoconjugates; cDNA and protein sequences for human cancer-linked genes and use of genes as target for chemotherapy) IT Antibodies and Immunoglobulins RL: ARG (Analytical reagent use); BSU (Biological study, unclassified); DGN (Diagnostic use); THU (Therapeutic use); ANST (Analytical study); BIOL(Biological study); USES (Uses) (recombinant, synthetic; cDNA and protein sequences for human cancer-linked genes and use of genes as target for chemotherapy) IT 33069-62-4, Taxol 75037-46-6, Gelonin 108212-75-5 108212- 76-6D, di-Me hydrazide 113440-58-7, Calicheamicin 114977-28- 5, Taxotere 138148-68-2 155233-45-7 RL: BSU (Biological study, unclassified); THU (Therapeutic use); BIOL (Biological study); USES (Uses) (cDNA and protein sequences for human cancer-linked genes and use of genes as target for chemotherapy) Some of references are potentially relevant.

Separate patent and non-patent records, extend the search to MARPAT

=> S L4 AND P/DT Separate HCAplus answers into patent L7 457 L4 AND P/DT and non-patent references to make => S L4 NOT L7 patent family grouping easier. L8 75 L4 NOT L7

=> FILE MARPAT

=> S L3 SSS SAM FULL FILE PROJECTIONS: ONLINE **COMPLETE** L9 3 SEA SSS SAM L1 Search the final answer set L-number => S L3 SSS FULL from REGISTRY in MARPAT for cost- effectiveness. L10 62 SEA SSS FUL L1

Search MARPAT results in HCAplus; refine with text

=> FILE HCAPLUS

=> S L10 AND (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN) L11 29 L10 AND (CONJUGAT? OR IMMUNOCONJUGAT? OR ANTIBODY OR IMMUNOGLOBULIN) Add search terms in HCAplus to => S L11 NOT L4 MARPAT records, then NOT out L12 3 L11 NOT L4 previously found HCAplus records to isolate the unique MARPAT records.

Return to MARPAT and combine with

=> FILE MARPAT the original query to restore hit structure highlighting in MARPAT. => S L10 AND L12 3 L12 L13 3 L10 AND L12

Extend structure search to WPINDEX and retrieve bibliographic references

=> FILE WPINDEX

=> S L1 SSS SAM FULL FILE PROJECTIONS: ONLINE **COMPLETE** L14 10 SEA SSS SAM L1

=> S L1 SSS FULL L15 132 SEA SSS FUL L1

=> S L15/DCR L16 220 L15/DCR

=> D HIS . . .

Merge answers and group by invention

=> DUP IDE L7 L13 L17 L18 625 DUP IDE L7 L13 L17 (INCLUDES 103 SETS OF DUPLICATES) ANSWERS '1-457' FROM FILE HCAPLUS ANSWERS '458-460' FROM FILE MARPAT ANSWERS '461-625' FROM FILE WPINDEX DUP IDE merges answers into a single L- => SAVE L18 MAYTANSINADC/A number for FSORT. ANSWER SET L18 HAS BEEN SAVED SAVE the original answer set for future

=> FSORT L18 retrieval. . . . FAMILY SORT (FSORT) groups records by L19 625 FSO L18 patent families across databases.

131 Multi-record Families Answers 1-340 Family 1 Answers 1-2 Family 2 Answers 3-4 Family 3 Answers 5-6 . . . Family 130 Answers 337-338 Family 131 Answers 339-340 285 Individual Records Answers 341-625 0 Non-patent Records 34

Use the Patent Family Manager to extract one member from each family

Click the L-number and select Patent Family Manager from the pop-up menu. 35

STN Express® runs a script to extract the first member of each patent family, plus all of the individual records => FIL HCAPLUS, MARPAT, WPINDEX

=> FSORT L19 L20 625 FSO L19 131 Multi-record Families Answers 1-340 Family 1 Answers 1-2 Family 2 Answers 3-4 Family 3 Answers 5-6 Family 4 Answers 7-8 . . . => SORT L20 PY 1 3 5 7 9 11 . . . L21 81 SORT L20 1 3 5 7 9 11 13 15 17 19 ... PY . . . => SORT L20 PY 341-625 The commands shown are all run L23 285 SORT L20 341-625 PY automatically to give a final answer => DUP REM L23; DEL L23 Y set of 285 patent references. PROCESSING COMPLETED FOR L23 L24 285 DUP REM L23 (0 DUPLICATES REMOVED) ANSWERS '1-247' FROM FILE HCAPLUS ANSWERS '248-249' FROM FILE MARPAT ANSWERS '250-285' FROM FILE WPINDEX 36

Display answers in your favorite format

=> D BIB ABS HITSTR FQHITEXG 1 250 248

AN 2014:274791 HCAPLUS Full-text TI Conjugates including an antibody moiety, a polypeptide that traverses the blood-brain barrier, and a cytotoxin IN Castaigne, Jean-Paul; Yang, Gaoqiang; Che, Christian; Demeule, Michel PA AngioChem Inc., Can. PATENT NO. KIND DATE APPLICATION NO. DATE ------WO 2014026286 A1 20140220 WO 2013-CA50625 20130814 PRAI US 2012-61682991 P 20120814 US 2013-61865071 P 20130812 AB The present invention relates to antibody-polypeptide-cytotoxin conjugates and methods of making, packaging, and using the conjugates. The polypeptide can be a Kunitz-type protease inhibitor or a derivative thereof that facilitates transport of the conjugate across the blood-brain barrier and/or into cancer cells outside the CNS, and the antibody moiety selectively binds a target within the CNS or in peripheral tumors to direct the cytotoxic agent to that target (e.g., a tumor or cancer cell). . .

HITSTR display shows structure that retrieved answer

IT 139504-50-0 1228105-53-0 RL: PAC (Pharmacological activity); RCT (Reactant); THU (Therapeutic use); BIOL (Biological study); RACT (Reactant or reagent); USES (Uses) (conjugates including antibody moiety, polypeptide that traverses blood-brain barrier, and cytotoxin) CN Maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- 38

WPINDEX record

AN 2013-C35742 [201318] WPINDEX Full-text TI Reducing immunogenicity of monoclonal antibody upon administration to a vertebrate, comprises mixing monoclonal antibody with agent (alkyl glycoside surfactant e.g. dodecyl maltoside) and administering the obtained composition to vertebrate DC A11; A96; B04; B07; D16 IN MAGGIO E T PA (AEGI-N) AEGIS THERAPERUTICS LLC PI US 20130039851 A1 20130214 (201318)* EN 43[15] . . . AB US 20130039851 A1 UPAB: 20130314 NOVELTY - Method (M1) for reducing immunogenicity of a monoclonal antibody upon administration to a vertebrate, comprises: mixing the monoclonal antibody with an agent to form a composition, where the agent is at least one alkyl glycoside surfactant; and administering the composition to a vertebrate, thus reducing immunogenicity of the monoclonal antibody upon administration. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for: (1) a method (M2) for reducing aggregation of a monoclonal. . . 39

HITSTR display from WPINDEX

AN.S DCR-271958 CN.P CANTUZUMAB MERTANSINE SDCN RB7BXW

Unique MARPAT record

AN 144:348897 MARPAT Full-text TI Immunoglobulins containing free cysteine thiols and their use as carriers for the targeted delivery of therapeutic agents IN Eigenbrot, Charles W.; Junutula, Jagath Reddy; Lowman, Henry; Raab, Helga E.; Vandlen, Richard PA Genentech, Inc., USA PATENT NO. KIND DATE APPLICATION NO. DATE ------WO 2006034488 A2 20060330 WO 2005-US34353 20050922 WO 2006034488 A3 20060928 AU 2005286607 A1 20060330 AU 2005-286607 20050922 AU 2005286607 B2 20110127 CA 2580141 A1 20060330 CA 2005-2580141 20050922 CA 2580141 C 20131210 KR 2007054682 A 20070529 KR 2007-7006617 20050922 KR 1270829 B1 20130607 EP 1791565 A2 20070606 EP 2005-811919 20050922 CN 101065151 A 20071031 CN 2005-80040207 20050922 JP 2008516896 T 20080522 JP 2007-533692 20050922 JP 4948413 B2 20120606 . . . 41

MARPAT FQHITEXG format

AB Antibodies are modified by replacing one or more amino acids of a parent antibody with a cysteine that is not involved in a disulfide bridge and therefore presenting a free thiol group available for conjugation. Similarly, this may be applied to the Fab fragment of the antibody to give a ThioFab antibody fragments. . .

Additional displayed G-groups: G4 = G5 / carbocycle (opt. substd.) / 25-3 26-9 / arylene (opt. substd.) / . . .

G19 = R <"antibody residue"> Patent location: claim 67 Note G19 is an R node “antibody residue” which can only be found if the node is set to match level ANY or if the node is not included in the substructure query. 42

Antibody sequence search for the immunoglobulin

Search Question Find relevant patent references for immunoconjugates that have similar sequences to the light chain of anti - (CTLA-4) Immunoglobulin G1, that are being conjugated to maytansinoid cytotoxins.

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY 51 GAFSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG 101 QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK 151 VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ 201 GLSSPVTKSF NRGEC

Resources for sequence searching: http://www.stn-international.com/biosequence_searching.html 43

Search strategy

1. Run BLAST® searches in USGENE®, PCTGEN and DGENE 2. Run CAS REGISTRY BLAST search 3. Transfer patent numbers from USGENE, PCTGEN and DGENE into HCAplus 4. Combine sequence results with structure results 44

Run a USGENE BLAST search

=> FILE REGISTRY Maytansinoid answers were activated in REGISTRY to begin. => ACTIVATE MAYTANSINE/A L3 1008 SEA SSS FUL L1

=> FILE USGENE These commands are automatically run by the STN Express Sequence Query Upload wizard. => UPL R BLAST Uploading ...STN Express 8.5\Queries\anti CTL4 light chain.txt

UPLOAD SUCCESSFULLY COMPLETED L4 GENERATED

=> D LQUE L4 L4 ANSWER 1 USGENE COPYRIGHT 2014 SEQUENCEBASE CORP on STN LQUE EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTVerify that the UPLOAD was successful with D KSFNRGEC LQUE. Use the L-number to run the BLAST search. => RUN BLAST L4 /SQP -F F

Run BLAST search in USGENE

ENTER EITHER THE NUMBER OF ANSWERS YOU WISH TO KEEP OR ENTER MINIMUM PERCENT OF SELF SCORE FOLLOWED BY % (BEST ANSWER PERCENTAGE OF SELF SCORE IS 100%) ENTER (ALL) OR ? :95% L5 RUN STATEMENT CREATED L5 361 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIY GAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC/SQP.-F F

=> SOR SCORE D PROCESSING COMPLETED FOR L5 L6 361 SOR L5 SCORE D

=> D TRIAL SCORE ALIGN 1- Save answers temporarily so you can come back to results if you wish. => SAVE TEMP USG1/A ENTER L#, L# RANGE, ALL, OR (END):L6 ANSWER SET L6 HAS BEEN SAVED AS 'USG1/A'

Repeat search in PCTGEN and DGENE

=> FILE PCTGEN => RUN BLAST L4 /SQP -F F 95% identity was chosen as the cutoff.

ENTER (ALL) OR ? :95% L7 RUN STATEMENT CREATED L7 114 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPG . . . => SOR SCORE D; D TRIAL SCORE ALIGN 1- L8 . . . => SAVE TEMP L8 PCT1/A ANSWER SET L8 HAS BEEN SAVED AS 'PCT1/A'

=> FILE DGENE => RUN BLAST L4 /SQP -F F

ENTER (ALL) OR ? :95% L9 RUN STATEMENT CREATED L9 245 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPG. . . => SOR SCORE D; D TRIAL SCORE ALIGN 1- L10 . . . => SAVE TEMP L0 DGEN1/A ANSWER SET L10 HAS BEEN SAVED AS 'DGEN1/A' 47

Merge answers from USGENE, PCTGEN and DGENE, TRANSFER patent numbers to HCAplus

=> DUP IDE L6 L8 L10 L11 720 DUP IDE L6 L8 L10 (INCLUDES 0 SETS OF DUPLICATES) ANSWERS '1-361' FROM FILE USGENE ANSWERS '362-475' FROM FILE PCTGEN ANSWERS '476-720' FROM FILE DGENE DUPLICATE IDENTIFY (DUP IDE) is used to create a single multifile L-number (L11).

=> FILE HCAPLUS Use TRANSFER (TRA) to extract and search patent numbers in HCAplus. => TRA PN 1- L11 L12 TRANSFER L11 1- PN : 305 TERMS L13 130 L12 ALL TERMS IN L12 RETRIEVED.

Run a CAS REGISTRY BLAST search

1. Launch BLAST 2. Search the sequence 3. Examine and evaluate alignment/relevance of sequence answers 4. Retrieve REGISTRY records on STN® 49

BLAST sequence answers are retrieved automatically

=> FIL REGISTRY

=> QUE (1480891-75-5 OR 1415165-41-1 OR 1333363-57-7 OR 912689-11-3 OR 911525-14-9 OR 477202-00-9 OR 477202-00-9 OR 1427128-72-0 OR 1266910-84-2 OR 1163827-35-7 OR 1334145-04-8 OR 1334145-04-8 OR 1453140-42-5)/RN

L14 QUE (1480891-75-5 OR 1415165-41-1 OR 1333363-57-7 OR 912689-11-3 . . .

=> S L121 OR L122 OR L123 OR L124 OR L125 OR L126

L127 1343 L121 OR L122 OR L123 OR L124 OR L125 OR L126

REGISTRY BLAST automatically executes the commands and gives a final answer set of 1343 sequences in REGISTRY. All answers were 95% identity or higher. 50

Crossover the HCAplus and refine

=> FILE HCAPLUS

=> S L127 AND P/DT L128 981 L127 AND P/DT L13 = Patent references transferred from => S L13 OR L128 USGENE, PCTGEN, and DGENE. L129 1000 L13 OR L128 L128 = Patent references from REGISTRY BLAST search.

=> S L129 AND (L3 OR (MAYT!NSIN? OR MYT!NSIN? OR MERT!ANSIN?)) L132 27 L128 AND (L3 OR (MAYT!NSIN? OR MYT!NSIN? OR MERT!ANSIN?)) L129 was combined with the structure search from REGISTRY, as well as alternative keywords for maytansinoids.

=> D BIB ABS HITSTR 1-27

Post-processed report with HIT structures and BLAST alignments 52

DGENE abstract and indexing focuses on the sequence information BBE41633 peptide DGENE TI Adjuvant therapy method comprises administering immunoconjugate and second anti-epidermal growth factor receptor 2 antibody to subject having human epidermal growth factor receptor 2 positive cancer, preferably breast cancer. IN Bryant J L PA (GETH) GENENTECH INC. PI US-20140086940 A1 20140327 58 . . . PSL Disclosure; Page 26 DED 22 MAY 2014 (first entry) DESC Ab-MC-vc-PAB-MMAF peptide. KW MMAF; Monomethylauristatin F; antibody therapy; breast tumor; cancer; chemotherapy; cytostatic; immunoconjugate; surgery; therapeutic. ORGN Synthetic. AB The present invention relates to a novel method of adjuvant therapy. The method involves administering to a human subject having HER2 positive cancer, following definitive surgery, anthracycline cyclophosphamide (AC) based chemotherapy, followed by administration of (a) an immunoconjugate comprising an anti-ErbB2 antibody capable of binding to HER2 domain IV bound by trastuzumab (HERCEPTIN), wherein the anti-ErbB2 antibody is conjugated to a maytansinoid, and (b) a second anti-ErbB2 antibody, wherein the antibody is an HER2 dimerization inhibitor. (cont’d) 53

DGENE feature table lists modification sites, if known

The HER2 positive cancer can be HER2 positive breast cancer, preferably non metastatic HER2 positive breast cancer. The present sequence represents a monomethylauristatin F (MMAF) peptide containing construct, which can be used in preparing the immunoconjugate, for performing the adjuvant therapy for treating HER2 positive cancer. AA 0 A; 0 R; 0 N; 0 D; 0 B; 0 C; 0 Q; 0 E; 0 Z; 0 G; 0 H; 0 I; 0 L; 0 K; 0 M; 1 F; 0 P; 0 S; 0 T; 0 W; 0 Y; 3 V; 4 Others SQL 8 SEQ 1 vxxvvxxf

Keyword search in HCAplus

Search Question

Find references for immunoconjugates that incorporate non-natural amino acids into the final product, using “click” chemistry. 55

Search for immunoconjugates incorporating non-natural amino acids => FILE HCAPLUS

=> S ((ANTIBODY OR IMMUNOGLOBULIN) (L) CONJUGAT?) OR IMMUNOCONJUGAT? L1 52144 ((ANTIBODY OR IMMUNOGLOBULIN) (L) CONJUGAT?) OR IMMUNOCONJUGAT?

=> S SPAAC OR CUAAC OR STRAIN PROMOTED OR AZIDE ALKYNE OR HUISGEN OR CLICK L2 14500 SPAAC OR CUAAC OR STRAIN PROMOTED OR AZIDE ALKYNE OR HUISGEN OR CLICK

=> S L1 AND L2 L3 154 L1 AND L2

=> S L3 AND (NON-NATURAL OR NONATURAL OR NONNATURAL OR UNNATURAL OR UNATURAL OR UN-NATURAL OR "NOT" NATURAL) L4 15 L3 AND (NON-NATURAL OR NONATURAL OR NONNATURAL OR UNNATURAL OR UNATURAL OR UN-NATURAL OR "NOT" NATURAL)

Evaluate and display answers

=> D TI 1-15

TI Facile Method for the Site-Specific, Covalent Attachment of Full-Length IgG onto Nanoparticles

TI Production of Site-Specific Antibody-Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System

TI Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells

TI Orthogonal Assembly of a Designed Ankyrin Repeat Protein- Cytotoxin Conjugate with a Clickable Serum Albumin Module for Half-Life Extension

TI Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry ● ● ●

=> D BIB ABS HITIND 1-15

Identify useful CPC classifications in HCAplus

=> FILE HCAPLUS

=> S IMMUNOCONJUGATES/CT AND P/DT L1 3468 IMMUNOCONJUGATES/CT AND P/DT A representative set of patents were => ANA CPC IPC 1-2000 analyzed for CPC and IPC codes

=> D TOP20 CPC TERM # # OCC # DOC % DOC CPC IPC ------4 4989 675 33.75 A61K2039-505 11 3211 480 24.00 C07K2317-92 Look for 4-5 digits after the . . . dash- these are likely CPC 33 1633 245 12.25 C07K0016-30 specific definitions. 34 1917 239 11.95 C07K2316-96 . . . 43 1461 198 9.90 C07K2317-732 2000 series codes, e.g. 44 1458 195 9.75 C07K2317-55 C07K2317-NNNNN are 47 1260 191 9.55 C07K0016-18 additional codes only, not 51 939 179 8.95 A61K0045-06 invention level. 57 1137 162 8.10 A61K0047-48561 63 1077 151 7.55 C07K0016-2863

Useful CPC classifications for immunoconjugates

• A61K2039-505+HIE/CPC Medicinal preparations containing antigens or antibodies; comprising antibodies (~10000 documents) • A61K0047-48+HIE/CPC Medicinal preparations where the a non-active ingredient is chemically bound to an active ingredient ( a number of relevant classes here) • A61K0051-10+HIE/CPC radioactive substances conjugated to antibodies (new in 2013) • C07K2317-00+HIE/CPC Immunoglobulins specific features • C07K0016-18+HIE/CPC Immunoglobulins (IGs), e.g. monoclonal or polyclonal antibodies against material from animals or humans (New in 2013: many antibodies against specific antigens have their own CPC classification) • Chemical codes for the organic cytotoxin This list is exemplary, not exhaustive. Use +HIE to see the entire classification hierarchy.

IPC codes

• A61P0035-00/IPC Antineoplastic agents • A61K0039-395/IPC Medicinal preparation containing antibodies • C12N0015-09 Recombinant DNA technology • C07K0016-00 Immunoglobulins, e.g. monoclonal or polyclonal antibodies

This list is exemplary, not exhaustive.

Derwent Manual Code classifications

=> FILE WPIX

=> E B04-Q01+ALL/MC E1 814578 BT2 B04/MC DEF NATURAL PRODUCTS (OR GENETICALLY ENGINEERED), POLYMERS E2 32 BT1 B04-Q/MC DEF DRUG CONJUGATES GENERAL HNTE (2013- ) E3 21 --> B04-Q01/MC DEF ANTIBODY-DRUG CONJUGATES HNTE (2013- ) ********** END ********** Derwent added a new manual code for antibody drug conjugates, but it is too new to be very useful for retrospective searching. It will be very good going forward. 61

Derwent Manual Code classifications

=> E B04-G01+ALL/MC E8 6 BT1 B04-G/MC DEF ANTIBODY DEFINED IN TERMS OF ANTIGEN E9 59165 --> B04-G01/MC DEF ANTIBODY DEFINED IN TERMS OF ANTIGEN GENERAL AND OTHER HNTE (1994- ) E10 17581 RT B04-B04C/MC DEF ANTIGENS, GENERAL ANTIBODY (PRE-94) HNTE (1965- ) E11 948 NT1 B04-G01A/MC DEF CHIMERIC ANTIBODY HNTE (2005- ) SM E12 886 NT1 B04-G01B/MC Most DWPI records for DEF HUMAN ANTIBODY immunoconjugates are HNTE (2005- ) indexed at the antibody level, E13 2117 NT1 B04-G01C/MC along with codes for cancer DEF HUMANIZED ANTIBODY and therapeutic use. HNTE (2005- ) E14 130 NT1 B04-G01D/MC DEF MURINE ANTIBODY HNTE (2005- ) For more information …

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