A Journey into the Antibody Jungle: Detours, Traps, Shortcuts, and Unknowns · 2013. 6. 24. · • ECLA codes are not searchable in REGISTER PLUS and EPO Boards ofAppeals • EPO

A Journey into the Antibody Jungle:

Detours, Traps, Shortcuts, and Unknowns

L. FALCIOLA

RiboVax Biotechnologies SA

Petit-Lancy, Geneva, Switzerland

L. Falciola © - PIUG 2007: Session on Biotechnology (Costa Mesa, CA; May 8th 2007) 2

• Antibodies are secreted glycoproteins that: – Represent the principal effectors of adaptive immune system

– Bind target molecules (antigens) with high affinity and specificity

• Antibodies can neutralize/eliminate pathogenic agents by interacting with cells and blood components and triggering specific biological functions

• Best characterized and widely used antibodies are monoclonal antibodies:– Secreted by cells clonally derived from a single cell

– Having a specific protein sequence

– Providing reproducible biological activities in diverse clinical settings

– Tunable for pharmacokinetics, affinity, and activity by chemical/sequence modification

– Produced using different recombinant DNA and cell culture technologies

• Due to their binding/functional activities and adaptability, antibodies are possibly the molecular tools more extensively used in modern biology

An Introduction to Antibodies:

Basic Information


Whole, Natural IgG

(150 kDa)


Structural Variants

Modified from Laffly,

Hum Antibodies 2005

CH2

CH3

CH1

VH

CL

VL

CDRH C

DRL

HEAVY CHAIN

LIG

HT CHAIN

FV

Fab

Fc

ANTIGEN

BINDING • A basic distinction can be made between:

– The sequences of the heavy and of the light chain

– The variable regions within the heavy (VH) and light (VL)

chains that

• Are linked to the respective constant regions (CH and CL)

• Contain the Complementarity Determining Regions (CDRs)

• Many alternative antibody formats have been

developed for different scopes as:

– Antibody fragments with single or double antigen binding

domains, and comprising

• only VH,

• isolated CDRs, or

• VH and VL, combined or not with CH/CL sequences (e.g. Fv,

Fab, scFv, etc.)

– Recombinant proteins that contain

• Multiple antigen binding domains, or

• Linked to functional groups (e.g. PEG, toxins, etc.)



Discovery & Production Technologies

• Many technologies have been developed for isolating, screening, producing, and optimizing antibodies based on:– Hybridomas, immortalized B cells, transgenic animals, phage display

– Chimerization / humanization / randomization of sequences

• Recombinant DNA & cell culture technologies make possible to obtain large amount of purified antibodies to be used in different contexts:– In vitro

• Against solubilized antigens (e.g. immunopurification, ELISA, Western blot)

• Against antigens present in intact cells/tissues (e.g. cell-based assays, FACS, immunofluorescence)

– In vivo (e.g. immunoimaging, immunotherapy, cell depletion)

• For additional information on antibodies (activities, structures, commercial

and medical importance, R&D technologies) see some recent reviews

Carter, Nat Rev Imm 2006 Laffly, Hum Antib 2005 Kim, Mol Cells 2005

Schrama, Nat Rev Drug Disc 2006 Lipman, ILAR J 2006


Searching for Antibodies:

Multiple Fields of Activities and Analysis

PRODUCT STRUCTURE

- Antibody Sequence

- Antibody Format

R&D TECHNOLOGIES

- Discovery, screening platforms

- Cloning, expression systems

ANTIBODY- RELATED

SUBJECT MATTER

PRODUCT ACTIVITY

- Antigen, Epitope Binding

- Functional, Biological Activity

COMMERCIAL USES

- Medical or other uses

- Administration, formulations


• Keyword-based searching strategies should take into consideration the different scientific definitions that are commonly used:– The terms defining antibodies

• Anticor* (FR), antikor* (DE)

• Immunoglobulins, in general and specific types (IgG, IgM) or formats (e.g. minibody, diabody)

• Phrase searching for types of antibodies (“monoclonal antibody”, “human antibody”, “polyclonal antibody”, “recombinant antibody”, etc.)

• Names of specific antibodies (codes for clones and cell lines, official/commercial name)

• Generic acronyms (mabs, fabs, hmabs, scfv, etc.)

– The terms defining the antigen or the biological activity• Antibodies are often named after the antigen (e.g. for TNF: anti-TNF, antiTNF, α-TNF)

• Antibody activity (e.g. antitumoral, antiviral)

• Using proximity operators to link general antibody-related keywords to target-/activity-/origin-related keywords (e.g. “antigen binding”)

• Sequence searching is important not only to determine the patentability of an antibody sequence, but (when available) also for identifying the antigen, in particular: – Alternative synonyms for a protein, and/or

– In the case of large proteins, specific subsequences recognized by antibodies (epitopes)

Searching for Antibodies:

General Criteria


• The complexity of antibody-based biological systems allows many alternatives for defining antibody-related patentable matters (see Webber, Nat Rev Drug Disc 2006)

• In fact, many different “descriptors” have been used for defining an antibody in a claim, often making difficult to search such claims:– Antibody sequence (minimal or longer sequences)

– Binding a specific (non-)biological antigen, with or without a reference to protein sequence that is recognized

– The type and/or the format of the antibody

– (Non) competition with a known antibody for the binding to an antigen / epitope

– Presence (or absence) of a biological activity, often with reference to specific concentration and/or to the use of specific assays

– Binding all / specific variants of an epitope/antigen

– Process-related features (e.g. isolated and/or produced using a specific process)

– Generic wording (e.g. claiming a ligand, a receptor, an heterodimer, an agonist, or an antagonist, and then indicating that it can be an antibody in a dependent claim)

Information on Antibodies in Patent Documents:

Claiming Antibodies


• A large variety of IPC codes have been used for classifying antibody-related invention, but the most relevant and frequently used codes are:– Immunoglobulins, as a class of proteins (C07K 16/*)

– Medicinal preparations containing antibodies (A61K39/395, but see also /40, /42)

– Immunoassays (the very general G01N 33/53, but see also /53*-/57*)

– Preparation of monoclonal antibodies (C12P 21/08)

– Recombinant DNA technologies related to antibody genes (C12N 15/13)

– Methods for screening antibody libraries (C40B 30/04)

– Diagnostic preparations containing antibodies (A61K 49/16, A61K 51/10)

• ECLA gives some additional help by providing some useful subcodes, in particular under C07K 16/* and A61K 39/395, for specific matters:– Origin of the antigen (for non-human antigens)

– Type of protein (for human antigens, as purified proteins or cell types)

– Source of the antibody (purified from phage libraries or by biological fluids)

• See Foglia (World Pat Inf 2007) and Scott (World Pat Inf 2007) on IPC/ECLA searching


Classifying Antibody-Related Inventions


• Keyword- and IPC-based searches may provide different outlook on patent production

• The number of PCT applications claiming different antibody-related subject matters can be analyzed according to the specific IPCs


Trends in PCT Filing on Antibodies

2.5

5.0

7.5

10.0

12.5

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004PCT Filing

Year

Records (x100)

15.0

17.5

20.0

22.5

25.0

Antibod* or Anticor* in abstract

A61K 39/395

A61K 39/395 and C07K 16/*

C07K 16/*

C07K 16/* and G01N 33/53

G01N 33/53



0.5

1.0

1.5

2.0

3.0

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004PCT Filing

Year

Records (x1000


Comparing General Trends in PCT Filing

• The trends in PCT filing can be compared using:

– Antibody-related IPCs

– Keyword-based search

– More generic IPCs still related to human health and proteins

(see also the data provided in Tansey, World Pat Inf 2005)

4.0

5.0

6.0C07K 16/* or A61K 39/395 or

G01N 33/53


C07K */* (peptides and proteins)

A61K 39/* (medicinal

preparations containing antigens

or antibodies)

G01N 33/* (investigating or

analyzing materials by specific

methods )


• EPO website gives free access to the information related to EP patent proceedings through distinct databases– ESP@CENET, INPADOC (covering also other patent authorities)

– REGISTER PLUS, EPO Board of Appeals (covering only EP proceedings)

• REGISTER PLUS, together with INPADOC and EPO Board of Appeals, provides information on both:– Status of the proceedings

– Actions and arguments made by EPO and applicants during the proceedings

• However, the databases differ considerably in terms of both:– Updated information amongst databases

– The actual “searchability” of databases, for example:

• ECLA codes are not searchable in REGISTER PLUS and EPO Boards of Appeals

• EPO Boards of Appeals has a search window, without codes for limiting the search to fields

• ESP@CENET and REGISTER PLUS has a full range of limitations, (e.g. the impossibility to distinguish between EP-A and EP-B documents)


Searching for EP Information using EPO Databases


2.5

5.0

7.5

10.0

12.5

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004EP Filing

Year

20.0

Records(x100) 15.0

17.5

Antibod*/AB

EP-A only

EP-B (as Mar. 07)

C07K 16/*

EP-A only

EP-B (as Mar. 07)

A61K 39/395

EP-A only

EP-B (as Mar. 07)

G01N 33/53

EP-A only

EP-B (as Mar. 07)

• Specific trends in patent filing and granting at EPO can be observed by applying keyword- or IPC-based search strategies

• However it is hard to establish how much the trends are affected by:– The choices in research objectives by applicants,

– The drafting style of patent attorneys,

– The attitude of EPO examiners (when assigning ECLA and examining claims)


Trends in Filing and Granting at EPO



Trends in Post-Grant Proceedings at EPO

• The analysis of REGISTER PLUS can provide an overview of the outcome of opposition proceedings, for example:– Approx. 150 EP patents having at least one of the main IPCs for antibodies (C07K 16/*,

A61K 39/395, G01N 33/53) and filed between 1995 and 2004 have been opposed

– Opposition is still pending for 62% and Appeal is pending for another 16%

– Patent was revoked for 9% or maintained (amended or non amended) in another 9%

– Opposition was withdrawn for 4%

• Recent decisions of EPO Tech. Board of Appeals points to the technical aspects relevant to how antibody is defined in the patent application, see:– T898/05 (correspondence of the claimed antibody with the deposited biological material)

– T255/05 (process defining the antibody)

– T1300/05 (activity defining the antibody), wherein such a claim is defined as being clear, concise, and sufficient

“A monoclonal antibody generated against a cell line susceptible to infection by macrophage-tropic HIV-1 isolates, and derived from the HuT 78 T lymphoblastoid cell line, wherein said antibody inhibits HIV-1 envelope glycoprotein mediated membrane fusion between HeLa-envJR-FL and said cell line, but does not inhibit HIV-1 envelope glycoprotein mediated membrane fusion between HeLa-envLAI and Sup-T1 cells or between HeLa-envLAI and HeLa-CD4+ cells”


%

Series3

KEYWORD

0 50 100 150 200

Granted in 1997-2001

Granted in 2002-2006

Patents on antibodies (against

animal/human proteins, C07K16/18-36)


Comparing Trends in Patent Granting at EPO and USPTO

Specific trends in patent filing and granting at EPO and USPTO can be observed by limiting the search to specific periods and specific IPCs:– both in relative and absolute terms

– not uniformly amongst IPCs

050100150200

Patents on antibodies (against

microorganisms, C07K 16/12,/14,/20)

Patents on antibodies (against virus,

C07K 16/08, /10)

Patents on antibodies

(Generic IPC, C07K 16/00)

Patents on antibodies

(Any IPC, C07K 16/*)

Patents on proteins

(C07K */*)

Total number of granted patents

US PATENTS EP PATENTS

%%

341288

2751

1880

364221

1.2k1.2k

4.3k3.4k

17k

13k

823k909k

5131149

57162

81

146

135272

8371832

2.9k5.8k

177k 316k


• The analysis of scientific literature for patent-related matters should take into consideration the specific features of literature related to:– Publisher’s policies

• Printed vs electronic publication date and content, submission of sequences to databases

– Provider’s policies• Type of indexing, choice of journals, frequency of update

– Author’s attitude • Anticipating the information on the web or at a congress, choice of terms in title/abstract

• Simply by taking MEDLINE as an example: – Indexing considers several variants on the antibody theme combining:

• Antibodies, Immunoglobulin, with

• Variable region, heavy chain, light chain, viral, monoclonal, fab, etc.

– There are two main indexing categories (MeSH term/MH or substance name/RN) but:• Only 10% of records having “antibodies” as MH have also “antibodies” as RN

• 99% of records having “antibodies, monoclonal” as MH have also “antibodies, monoclonal”as RN

• See Amaniadiou (TIBTECH 2006) on text mining in MEDLINE and scientific literature

Information on Antibodies in the Scientific Literature:

General Considerations


Series3

KEYWORD

30 90 150 210 270 330

MEDLINE

“antibody” in title/abstract “monoclonal antibody” in title/abstract “antibody” in full-text only

“antibody” in indexing only “monoclonal antibody” in indexing only “monoclonal antibody” in full-text only

BIOSIS

SCISEARCH

EMBASE

CAPLUS-lit

HIGHWIRE

Records

(x 1000)60 120 180 240 300 360 390 450 510420 480 540 600570 630

The content of literature databases clearly differs:- quantitatively, in absolute terms (due to journal selection/coverage), but also

- qualitatively, in relative terms (due to type of indexing)


Overview of Scientific Production (1976-2006)


2

4

6

8

10

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

16

18

20

22

24

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006


Content of Databases (Antibod*)

Records (x 1000)

TITLE/ABSTRACT

INDEXING ONLY

12

14

The potentially relevant content differs:

- Among databases

- Throughout time

BIOSIS

CAPLUS-lit

EMBASE

MEDLINE

SCISEARCH

Publication Year


5

10

15

20

25

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

40

45

50

55

60

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006


Content of Databases (“Monoclonal Antibod*”)

Records (x 100)

TITLE/ABSTRACT

INDEXING ONLY

BIOSIS

CAPLUS-lit

EMBASE

MEDLINE

SCISEARCH30

35

65

The outcome of more detailed search strategies may differ due to indexing

Publication Year


• Even though antibodies are large proteins, the protein sequencesactually involved in antigen binding are restricted to the variable region of heavy and light chains (90-150 amino acids) containing the CDRs (each one containing 3-20 amino acids)

• The scientific literature shows that specific CDRs (in particular CDRH3) can be isolated and transferred on different (non-)antibody protein frames, still maintaining the original antigen binding properties

• However, different approaches in claiming antibody sequences areapplied, having as minimal sequence:– CDRH3, alone or combined with other CDRs

– Variable region of the heavy chain, alone or combined with the variable region of the light chain

Searching Antibody Sequences:

General Considerations

VL

VH

CDRL1 CDRL2 CDRL3

CDRH1 CDRH2 CDRH3


• The dedicated databases:– are in limited number and

– reflect the disclosure of DNA / protein sequences in patent documents differently

• Two Examples DGENE (protein sequence numbers, limited GENOMEQUEST (number of US patents and

by antibody-related keywords and sequence length) applications, limited to IPC C07K 16/*)

By Publication Year By Earlier Priority Year

PATENT RECORDS

100 200 300 400 500 600 700

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006


Databases of Antibody Sequences Within Patent DocumentsPROTEIN SEQUENCES RECORDS

12000 9000 6000 3000 1500 1000 500


• The protein sequence is essential for defining an antibody, in general and for its patentability when compared to prior art

• However, when considering the sequence of an antibody originallydisclosed in an article, it can be a challenging task:– To understand if the article actually contains DNA/protein sequences without reading it

– To define exactly by which date the sequences are actually searchable in a public database by sequence homology

• For example, by sampling 100 human immunoglobulins heavy/light chains in Pubprot:– In 35%, the entry year in the sequence database is the same of the publication

– In 30%, the sequence has been entered before the year of publication

– In 35%, a sequence database entry is not associated to an article, leaving to different scenarios

• No article publication

• No link to a subsequent or previous article (maybe listing the antibody under a different name and/or with a different authors’ list)


The Disclosure of Antibody Sequences in the Literature


• As sequence searching can be useful for searching antigen-defined antibodies, keywords searching can be useful to identify documents including antibodies for which the sequence is, for whatever reason, not searchable by sequence homology

• In patent databases, relevant subsets of records can be identified by combining searches for:– Antibody-related and target-related keywords in abstract/claims, and

– “Seq id” in claims

Possibly combining also to specific ECLA, IPCs

• In literature databases, appropriate combinations of indexes can allow to restrict to a subset of records, for example in MEDLINE using MEsH indexing related to:– Molecular cloning, sequence data

– Forms and elements of Immunoglobulins (fabs, heavy chain, light chain, variable region, monoclonal)

– The desired functional and/or antigen binding activity


The Keyword-/IPC-based Search of Antibody Sequences


• This overview of searchable resources for antibody information shows:– The importance of a structured approach to searching that takes into

account the different degrees of usefulness for each search criteria (see Nijhof, World Pat Inf 2007)• Keywords

• Indexing/classification

• Sequence

– But also how heavily search results may be affected, qualitatively and quantitatively, by differences in:• The type and the amount of information that is actually searchable using criteria

when applied a sufficiently elaborated manner

• The evolution of the database organization and content throughout time

• Exploratory, general searches (e.g. phrase searching, IPC/ECLA codes, sequence searching, limitation to indexed and not indexed information) may provide an insight to hard-to-anticipate, “deep” biasing effects due to databases that may considerably affect time and outcome of the search

CONCLUSIONS


THANKS FOR YOUR ATTENTION

[email protected]

The opinions expressed in this presentation are the personal opinions of the author

and are not to be considered opinions of Ribovax Biotechnologies