National Research Council of Canada

Cornell Research Foundation Inc.

Canadian Patents & Development Ltd.

Research, Incorporated

Cold Spring Harbor Laboratory

Saran A Narang

Jamshid Tanha

Danica Stanimirovic

Ray J Wu

Arumugam Muruganandam

Wing L Sung

Joycelyn Entwistle

John Goodchild

Patrick T Wong

Michael D Dan

Colin Roger Mackenzie

Ginette Dubuc

Ahmad I Bukhari, Deceased

Jasmid Tanha

Jacek Stawinski

Howard Kaplan

Chander P Bahl

A phage-displayed library of llama single heavy domain antibodies (sdAbs) was enriched for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). From the enriched library, two sdAbs were selected, sequenced, subcloned, and expressed as fusion proteins with c-myc-Histags (Hisis SEQ ID NO:101). Similarly as phage-displayed sdAbs, these soluble tagged sdAbs were shown to selectively bind to HCEC and to transmigrate across in vitro human blood-brain barrier (BBB) model. In contrast to an unrelated llama sdAb, these sdAbs were also detected in the brain after i.v. injection into mice. These small (&#x2dc;13 kDa) antibody fragments have essential characteristics of brain-specific delivery vectors and can be used to facilitate drug transport across the BBB.

Single domain brain-targeting antibody fragments derived from llama antibodies

Phage display libraries are taught in which the recombinant phage population displays a plurality of potential binding fragments having preferred characteristics of solubility and/or intermolecular interaction. Also taught are methods of biasing display libraries to produce variants which more closely approximate the preferred characteristics of the parental binding fragment.

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A phage display library of variable heavy domain (VH or VH) fragments (sdAb fragments) derived from the antibody repertoire of a non-immunized llama is disclosed. The sdAb fragments of the library are characterized by the absence of cysteine residues in complementarity determining regions (CDRs) and a very low presence of residues of glutamic acid, arginine and glycine at positions 44, 45 and 47, respectively, of the VL interface of the variable heavy domain VH. The large size of the library (in the order of 10) makes it a source of antigen-binding fragments having high affinity to almost any antigen of interest. The library is preferably generated using a modified fd-tet phage growing in plaques in the absence of a tetracycline.

Single-domain antigen-binding antibody fragments derived from llama antibodies

A phage-displayed library of llama single heavy domain antibodies (sdAbs) was enriched for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). From the enriched library, two sdAbs were selected, sequenced, subcloned, and expressed as fusion proteins with c-myc-Histags. Similarly as phage-displayed sdAbs, these soluble tagged sdAbs were shown to selectively bind to HCEC and to transmigrate across in vitro human blood-brain barrier (BBB) model. In contrast to an unrelated llama sdAb, these sdAbs were also detected in the brain after i.v. injection into mice. These small (&#x2dc;13 kDa) antibody fragments have essential characteristics of brain-specific delivery vectors and can be used to facilitate drug transport across the BBB.

Single-domain brain-targeting antibody fragments derived from llama antibodies

A process is described for monitoring the production of recombinant proteins. It is based on the discovery that infrared spectra of E. coli strains and transformants which overproduce recombinant proteins can be measured as a function of pressure, and a pressure-induced distinct shifting pattern can be observed in specific spectral parameters of transformants. In particular, the difference in the pressure-induced shift of the amide III band, between the E. coli cells and the transformants over-producing the recombinant proteins provides an efficient and non-intrusive technique for on-line monitoring of the production of recombinant proteins in E. coli.

Non-intrusive process for monitoring the production of recombinant

Transposable linkers are DNA sequences recognized and employed by transposition proteins, including transposases, in the insertion of genetic information into the genetic architecture of an organism and in the movement of genetic information from one location to another in the genetic architecture of an organism. Such linkers, comprising the extreme ends of transposons, often referred to as left and right attachment sites, have been isolated from the intervening material in transposons to produce a basic building block comprising simply the extreme ends of the transposons fused together. Using restriction endonuclease recognition sites outside and between the left and right attachment sites it is possible to introduce desired genes directly or via cloning vehicles, into the genetic material of an organism. A transposable linker may also be used to introduce a desired gene, that has been placed in a cloning vehicle but is not, of itself, part of a normally transposable sequence, into the genetic material of an organism.

Transposable linkers to transfer genetic information

A human-like proinsulin gene and its analogs, have been synthesized by a combination of chemical and enzymatic methods. A number of different human-like proinsulin gene analogs with altered C-chains have also been designed and can be readily constructed as described. As a part of the strategy, an adaptor for trimming DNA has been designed, synthesized and used to recover the A-chain insulin gene with the desired sequence from a hybrid plasmid; a related adaptor for trimming DNA has been designed to shorten the C-chain gene or any gene. The synthetic proinsulin gene has been joined to a replicable cloning vehicle and the hybrid DNA transferred to a host cell. The transformed host cell has been shown to contain the desired human-like proinsulin gene.

Adaptors, and synthesis and cloning of proinsulin genes

Adaptor molecules have been prepared to comprise either start or stop signals for protein synthesis, in addition to recognition sites for restriction endonucleases. Separate adaptors may be used in a symmetrical duplex form. The start adaptor may include nucleotide base inserts to provide the correct reading frame of the triplet code in a DNA sequence with inappropriate reading frame. Insulin A-chain and B-chain genes of the human type, have been synthesized with the appropriate adaptor molecules provided on each end. The adapted DNA genes have been joined to replicable cloning vehicles and the hybrid DNA transferred to a host cell. The transformed host cell has been shown to contain the desired insulin gene.

Adaptor molecules for DNA and their application to synthesis of

Synthetic oligonucleotides have been designed and prepared which are useful in the molecular cloning of a variety of DNA molecules. By means of such oligonucleotides, genetic informational material, e.g., DNA, can be joined to a cloning vehicle and transferred into host cells by transformation. Additionally, a method for determining whether genetic informational material has been transferred into transformed host cells has been developed.

Oligonucleotides useful as adaptors in DNA cloning, adapted DNA

Arylsulfonyl tetrazoles of the formula ##STR1## where R.sub.1, R.sub.2 and R.sub.3 are selected from hydrogen, lower alkyl and lower alkoxy groups, and their preparation are described. These compounds have been found to be advantageous condensing or coupling agents via phosphoester formation in polynucleotide synthesis.

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Ottawa, Canada

Saran A Narang