“Rather than the conventional route of interrogating the tumor at the genomic and proteomic level in order to reveal disease-associated targets, why not ask the patient? The readout for differences in self and non-self antigens manifests itself in the form of antibodies, and our approach assumes that the answers to our drug and target discovery questions are contained within this response.”

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“Given the ongoing struggle to find new therapeutic cancer drug targets, I have long believed that a more rapid approach for discovery would consist of reversing the question,” stated Stine. “Rather than the conventional route of interrogating the tumor at the genomic and proteomic level in order to reveal disease-associated targets, why not ask the patient? The readout for differences in self and non-self antigens manifests itself in the form of antibodies, and our approach assumes that the answers to our drug and target discovery questions are contained within this response.” - J.T. Stine, Seattle Times, 2005

Applications in Humans

The statement above was the founding question addressed by Spaltudaq (today known as Theraclone Sciences).  The result of sampling a cancer patient’s immune repertoire led to the discovery of a broadly reactive and specific IgG antibody using the iSTAR method (a precursor to BLAST).

Discovery:

Screen normal/exposed and disease individuals for

  1. Neutralizing anti-infective antibodies (viral, bacterial)

  2. Tumor specific antibodies/epitopes from host B-cells


Immune monitoring

  1. Vaccine evaluations prior or during clinical trials - associate epitopes with efficacy

  2. Monitor immune response during tolerance block therapeutics such as anti-CTLA-4 or anti-PD1


Auto-immune patient B-cell screening

  1. Discover antibodies in auto-immune patient repertoire which can be therapeutic in other diseases where the eliciting target exists.

Applications in other mammals:

Discovery:

Immunize with tumor lines or clinical isolates

  1. Screen for novel tumor-specific or -selective epitopes or targets for naked antibody targeted ADCC

  2. Screen for novel internalization targets for antibody/drug conjugates

  3. Screen for functional antibodies that have a deleterious effect on tumor growth


Immunize with viral or bacterial targets

  1. Upfront functional screens can reveal novel or additional antibody epitope pairs for neutralization

  2. Use neutralization data to engineer more efficacious vaccines


Target extracellular domains of 7TM’s/GPCR’s

  1. The most “drugable” target is easily targeted with BLAST

  2. Discover antibodies that are agonists, antagonists or neutral

  3. Discover a more specific cell set to deplete due to the selective expression of GPCR’s on various cell types

An example of the use of BLAST in Type 1 diabetes (T!D) to interrogate the immunological history of their disease in order to determine the most relevant first-response cell surface proteins which were targeted on pancreatic islets cells by T-cells or other effector mechanisms.  A discovery here may lead to another use of BLAST which would consist of understanding the TCR’s responsible for this detection and selectively eliminating them with anti-TCR-specific antibodies.

Whole-cell immunization & BLAST

  1. Antibody generation in a massive scale

  2. Below is an example of the fraction of antibodies created en masse

BLAST

Functional Screen

Hit Selection

IgG Memory B-cells

Hits

Molecular

Reconstitution

V-gene

sequence

matching

  1. Reveals:

  2. v-gene epitope bias and overall immune response to target

  3. alternative target-specific hypermutated related sequences that may have other desirable properties that weren’t revealed in the screen and can now be synthesized

Peripheral Blood Sample

7 days
V-gene family clustering by sequence

Both physical and digital capture of an immune response:  Deep sequencing coupled with BLAST

Now the data set generated by next generation deep sequencing can be more than just a list of V-genes.  Hit sequences from BLAST can reveal relevant V-gene families in clustered alignments which expose other important target sequences that high throughput screens may have missed.

North Coast has developed massive high throughput microplate internalization assays that interrogate millions of B-cells at one time for identifying cell surface proteins that internalize rapidly and are selectively expressed by tumor

Toxin Conjugate HTS’s

BLAST Applications