Tissue Cross-Reactivity Screening Services

2011

Version 10.25.2010.v1

Table Of Contents

Protein Therapeutics Screening Services …………………………………………………………. page 1

Therapeutic Clone Screening Services …………………………………………………………. page 1

Tissue Cross-Reactivity Screening (using Frozen Tissue Arrays) ………...…….….…. page 2

Tissue Cross-Reactivity Screening (using Full Frozen Specimens) ……….……….……. page 3

Other LifeSpan Products and Services …………………………………………………………. page 4

For more than 15 years LifeSpan has been producing and characterizing antibodies for use in immunohistochemistry.

Working with all classes of antibodies; polyclonal, monoclonal, fusion, humanized, human, and otherwise engineered,

LifeSpan understands how each antibody type can behave when used to immunolabel fixed and frozen tissues.

Experience differentiating specific from non-specific signal, and adjusting assay conditions in order to produce the

most specific signal, makes LifeSpan well qualified to provide its clients with guidance when selecting protein

therapeutics for continued development, or to produce critical Tissue Cross-Reactivity data for internal research

purposes, or submission with IND applications.

The following services specifically apply to Protein Therapeutic testing:

Therapeutic Clone Screening Service

Tissue Cross-Reactivity Screening (using Frozen Tissue Arrays)

Tissue Cross-Reactivity Screening (using Frozen Full Specimen) (IND quality)

Each service is conducted on a fee-for-service basis and can be customized to address the clients specific needs.

Therapeutic Clone Screening Service During the development of therapeutic antibodies, it is important to quickly and effectively screen interim clones for

those that are most specific to the target antigen and exhibit the least non-specific binding to non-target tissues.

LifeSpan has the experience and resources needed to provide this service, helping investigators quickly advance

effective clones and terminate those that are less optimal early in the process.

Protocol: Either individually or in groups, LifeSpan will characterize clones

supplied by the client in Immunohistochemistry against frozen positive and

negative controls, plus a standard panel of normal frozen human tissue

types. The results are interpreted by a LifeSpan Pathologist and the results

summarized for the client.

Antibodies: LifeSpan will work with the client in order to determine the

most appropriate signaling system for the detection of the therapeutic

clones. In some cases it may be necessary to conjugate the clones with FITC

in order to use an anti-FITC secondary antibody. An appropriate isotype

control antibody should be included to control for potential non-specific

staining due to Fc mediated binding.

Controls and Tissues: Client supplied controls, such as xenografts and

transfected cell lines, can be used in combination with frozen human tissue

controls supplied by LifeSpan.

Immunohistochemistry: A 4-step dilution series of each clone and the

isotype control will be run against the selected control tissues and standard

normal panel.

Analysis and reporting: The results will be interpreted by an experienced

LifeSpan Pathologist and a report generated summarizing the findings.

To learn more about these services contact: Sales@LSBio.com

or call (206) 374-1103.

Protein Therapeutics Screening Services

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Tissue Cross-Reactivity Screening (using Frozen Tissue Arrays)

Using frozen tissue arrays (TMAs) is a quick, cost-effective approach to determining the

binding specificity of a protein therapeutic. LifeSpan offers immunohistochemistry

(IHC) screening of its mouse, rat, and human frozen TMAs, each containing a broad

panel of normal tissue types in triplicate. The results of each study are interpreted by a

LifeSpan Pathologist, and an online report that includes an executive summary, tables

listing the IHC scores for each tissue and major cell types, and digital images if

requested, is delivered to the client. Using frozen tissue arrays is not recommended for

producing Tissue Cross-Reactivity (TCR) data intended for submission with IND

applications. Small tissue elements, such as those that make up TMAs have more

limited histology than full specimens and are at greater risk of falling off the slide

during processing.

Tissue Cross-Reactivity Screens (using Frozen Tissue Arrays)

Tissues: LifeSpan’s FDA Standard Tissue Arrays

Immunohistochemistry: A 4-step dilution series of the 1 experimental and 1 isotype

control antibody will be run against positive and negative control tissues to determine

the optimal concentration. A single concentration of each will then be run against the

selected FDA Standard Tissue Array.

Analysis and Reporting: The results of each study are interpreted by a LifeSpan Pathologist, and an

online report, that includes an executive summary, tables listing the IHC scores for each tissue and major

cell types, and digital images if requested, is delivered to the client.

MouseMouseMouseMouse CR0028DCR0028DCR0028DCR0028D

RatRatRatRat CR0028ECR0028ECR0028ECR0028E

HumanHumanHumanHuman CR0028FCR0028FCR0028FCR0028F

Service CR0028D: Mouse TCR Screen

Service CR0028E: Rat TCR Screen

Service CR0028F: Human TCR Screen

All studies can be customized to suit

the specific needs of the client and

are conducted under R&D conditions.

To learn more about these services

contact: Sales@LSBio.com

or call (206) 374-1103.

* Human only ** Human and Rat only

Adrenal Liver Skin

Bone Marrow* Lung Small Intestine

Brain, cerebellum Mesothelium ** Spleen

Brain, cerebrum Nerve Stomach

Brain, pituitary* Ovary Testis

Breast Pancreas Thymus

Colon Placenta* Thyroid*

Esophagus Prostate** Tonsil*

Heart Salivary gland Uterus

Kidney Skeletal muscle Uterus, cervix

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Tissue Cross-Reactivity Screening (using Full Frozen Specimens)

Tissue cross-reactivity (TCR) screens are intended to provide information about the binding of protein therapeutics

across the normal human body. Strictly based upon the FDA’s recommendations outlined in the "Points to Consider in

the Manufacture and Testing of Monoclonal Antibody Products for Human Use”, this study plan includes

characterization of the therapeutic antibody and an appropriate control antibody, a comprehensive IHC screen across

full-section frozen tissues, interpretation by a Pathologist, and duplicate executed hard copies of the final report.

Studies are conducted under GLP-like conditions and the tissue list can be expanded to include Cynomolgus in

addition to human, or modified to meet European Medicines Agency (EMEA) requirements.

Antibody Preparation: Depending upon the composition of the protein therapeutic, FITC conjugation may be required.

In these cases an anti-FITC secondary antibody conjugated with Alkaline phosphatase or HRP is used as the colorimetric

signaling system for detection.

Phase I - Antibody Characterization and Optimization: A 4-step dilution series of the experimental antibody and the

appropriate isotype control antibody will be run in IHC against positive and negative control tissues, and a small panel of

standardized normal tissues, to determine their optimal concentrations. Antibody optimization steps will then be taken as

necessary and the results reported to the client before moving forward.

Phase II - Preliminary TCR Screen: Once antibody characterization is complete, the client can elect to have a

preliminary screen of the FDA tissue list run in order to identify potential cross-reactivity issues prior to conducting the full

screen. This preliminary screen includes immunolabeling (1) specimen each of the 33 frozen normal tissue types with a

single dilution of the therapeutic and control antibodies. The resulting slides are interpreted by an experienced LifeSpan

Pathologist and the results delivered to the client in the form of an online tabular report with images if requested.

Phase III - Comprehensive TCR Screen: Both the experimental and isotype control antibodies are used at 2

concentrations to immunolabel (3) individual specimens each of the 33 frozen normal tissue types recommended by the

FDA. The resulting slides are interpreted by a LifeSpan Pathologist and the results delivered in the form of duplicate

bound reports. The report layout is based upon the FDA’s recommendations and includes a signature page, executive

summary, methods, materials, and the scoring table results.

The list of 33 tissues recommended by the FDA and screened as part of this service:

Timelines: LifeSpan makes best efforts to complete each study in a timely manner. A typical TCR study takes between 6

and 12 weeks to complete, depending upon its complexity.

R&D Study Format: Because this service is conducted under R&D rather than GLP conditions, LifeSpan can offer its

clients cost effective data without the associated overhead costs of a GLP laboratory. LifeSpan has conducted TCR studies

for a number of clients that have successfully submitted the results to the FDA as part of IND submissions.

To learn more about these services contact: Sales@LSBio.com or call (206) 374-1103.

Adrenal Eye Pancreas

Bladder Fallopian tube Parathyroid

Blood cells Heart Pituitary

Bone Marrow Kidney, medulla & cortex Placenta

Brain, Cerebellum Liver Prostate

Brain, Cortex Lung Skeletal muscle

Breast Lymph node Skin

Colon Ovary Small intestine

Endothelium

Spinal cord

Spleen

Testis

Thymus

Thyroid

Ureter

Uterus, cervix

Uterus, endometrium

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Other LifeSpan Products and Services

Immunohistochemistry Validated Antibodies

LifeSpan’s goal is to provide high-quality antibodies that target every therapeutically relevant protein target in the human body.

We currently offer one of the industry's largest catalogs of monoclonal and polyclonal antibodies covering virtually every protein

class and all applications. Many antibodies have been characterized for use in immunohistochemistry. Visit www.LSBio.com for a

complete list.

Antibody Production Services (rabbit polyclonal)

LifeSpan offers full service production of rabbit polyclonal antibodies from peptide design to the characterization of the final

product. Fully customizable, clients can specify the type of immunogen to use, modify the immunization protocol, purification

method, and exactly how they would like the resulting antibodies characterized. Methods such as ELISA, Western Blot, and frozen

and fixed IHC are available. With a decade of antibody production experience and more than 3,750 antibodies synthesized,

LifeSpan can help you develop the reagent you need.

Contract Research Services

LifeSpan provides high-quality, confidential, protein localization data through its immunohistochemistry services. Researchers can

outsource the entire localization process without having to worry about finding and characterizing target specific antibodies,

sourcing and validating difficult-to-find tissues, and having the ability to interpret the resulting immunostaining in relation to

complex human pathologies. Researchers can choose from a variety of standardized IHC services that are commonly requested

(Service Packets), or work with LifeSpan to design a custom study plan that will specifically address their particular biological

question. All studies are conducted under R&D conditions, are confidential, and the results are proprietary to the client.

Standard Immunohistochemistry Service Packets

LifeSpan has designed more than 50 Standard IHC Service Packets enabling customers to order commonly requested contract

research study plans quickly and easily. Each study includes the characterization of an antibody for use in IHC in fixed human

tissues, the immunolabeling of a defined panel of tissues supplied by LifeSpan, and a Pathologist’s interpretation of the results.

Each Service Packet can be customized to include multiple antibodies, to combine tissue lists, or to specify the level of

interpretation. A complete list of Service Packets is available at www.LSBio.com.

Histology and Pathology Services

LifeSpan's histology core and staff of experienced IHC and diagnostic pathologists are available on a fee-for-service basis to

provide timely and cost effective histopathology services. Available services include: tissue dissection, grossing, mapping,

decalcification, processing, embedding, sectioning (fixed and frozen), routine and special stains such as H&E, Masson's Trichrome

(Collagen), Verhoeff-Van Gieson (Elastin) and others, pathology interpretation, Staging and Grading, analysis of IHC and ISH

stained slides, digital imaging and analysis.

Protein Localization Data (off-the-shelf IHC reports from the DrugTarget Database)

The DrugTarget Database contains comprehensive protein localization Gene Reports for more than 600 protein targets. Each

report includes expression data mined from the public domain and proprietary IHC protein localization data across 2 to 3

specimens each of 22 normal peripheral tissues, 11 normal brain tissues, and 29 disease types. All results are interpreted by a

LifeSpan Pathologist, and more than 300 images illustrate the staining. IHC allows for the correlation of a proteins expression with

histopathology, valuable data for the identification and prioritization of potential drug targets. Visit www.LSBio.com to view a

complete list of available Gene Reports.

To learn more about these services contact: Sales@LSBio.com or call (206) 374-1103.

Tissue Arrays LifeSpan offers more than 1,000 high-quality formalin-fixed paraffin-embedded (FFPE) and frozen tissue arrays containing normal

and diseased human tissues, as well as tissue from commonly studied non-human species. Tissue arrays are a cost effective

resource for producing protein localization data across many tissue specimens simultaneously. All LifeSpan TMAs are available for

use in LifeSpan's Contract Research Services, and many are also available for purchase as unstained slides. A complete list of

TMAs is available at www.LSBio.com.

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2401 Fourth Avenue, Suite 900

Seattle, WA 98121

www.LSBio.com

antibodies@LSBio.com

Antibody Hotline: 206-374-1102

Toll Free: 866-819-4732

Fax: 206-464-1723