JP2003516745A - Brain specific binding members - Google Patents

Abstract

  (57) [Summary] The present invention relates to specific binding members that include specific binding members capable of crossing the blood-brain barrier and are directed to the brain, ie, to brain inflammatory or blood-brain barrier disrupted moieties. Panels and mixtures of antibodies are provided, as well as individual antibody molecules and VH and VL regions, along with methods of obtaining specific binding members by selection, for example, using libraries displayed on the surface of filamentous bacteriophage. .

Description

Detailed Description of the Invention

[0001] The present invention relates to specific binding members that include specific binding members capable of crossing the blood-brain barrier and are aimed at the brain, ie at sites of cerebral inflammation or blood-brain barrier disruption. Panels and mixtures of antibodies are provided, as well as individual antibody molecules and VH and VL regions, as well as methods for obtaining specific binding members by selection with libraries displayed on the surface of filamentous bacteriophage.

[0002] The transport of many hematogenous molecules into the brain is restricted compared to other tissues in the body.
This leads to the idea of the blood-brain barrier (BBB) that isolates the brain from surrounding physiological changes (Bradbury MWB (1993) Experimental).
Physiology, 78: 453-472). Endothelial cells, which line the capillaries of the brain, have been shown to be joined by numerous tight junctions that limit the pericellular movement of water-soluble molecules. These endothelial cells also dispatch on their surface certain carrier molecules that selectively allow passage of molecules that are essential for brain function, such as glucose, amino acids, transferrin.

BBB represents a substantial impediment to the delivery of many drugs, especially antibodies and proteins such as cytokines or growth factors into the central nervous system. 400-60
With the exception of lipid-soluble molecules with a molecular weight of 0 Da, virtually all drugs resulting from either biotechnology or conventional small molecule pharmacology undergo negligible transport through the intact BBB. Identification of novel carrier molecules or receptors that associate with the BBB enhances uptake of drugs into the CNS by, for example, conjugating those receptors to their natural ligands or by selecting antibodies or drugs that bind directly to the receptor. Show possible goals for

Central nervous system (CNS) diseases that can be treated in this manner include sleep disorders (eg, narcolepsy), affective disorders, schizophrenia, regenerative disorders such as Alzheimer's disease, Parkinson's disease, frontal lobe. Temporal dementia, Huntington's chorea, demyelinating disease (eg multiple sclerosis), spinal cord injury, brain tumors (primary and secondary), viral encephalopathy (eg rabies and brain HIV), motor neuron disease, prions Symptoms include illness, hydrocephalus, cerebral infarction (primary bleeding and ischemia), epilepsy, obsessive-compulsive disorder, anxiety and phobia, substance abuse, alcohol and other substance abuse, and distress. In addition, consciousness can be adjusted as in general anesthesia.

The present invention relates to the selection and characterization of human antibodies from phage-displayed antibody libraries that have been demonstrated to cross the BBB or are restricted to only inflamed areas in brain tissue. Such antibodies can be used as agents to enhance drug transport across the blood-brain barrier, and to identify novel transport or other blood-brain barriers, or inflammatory tissue-tagged molecules that may themselves be targets for therapeutic intervention. It has value as a tool and therapeutic potential.

It is already known that some molecules on brain capillary endothelial cells are specific receptors for circulating or plasma proteins. Human microvessels have been used to characterize the human BBB receptor for insulin, insulin-like growth factor-I, insulin-like growth factor-II, transferrin and leptin (W Pardridge (1997) Journal of C.
erebral Blood Flow and Metabolism, 17
: 713-731). The BBB insulin, insulin-like growth factor, and transferrin receptor system have been shown to mediate peptide transcytosis through the BBB in vivo (eg, Du.
ffy and Pardridge, 1987, Brain Res. 420
: 32-38). Observing that peptide receptors are present on brain capillary endothelial cells, and that some of them mediate peptide transcytosis through the BBB, usually follows receptor-mediated transcytosis through the BBB. This led to the idea of a non-transporting drug that is covalently bound to a protein or peptide that is present. Initial experiments were performed with cationized albumin that undergoes absorptive-mediated transcytosis through the BBB (Kumagai et al.
． al. , 1987, J Biol. Chem 262: 15214-1521.
9). Next, the BBB-permeable rat transferrin receptor (Frid).
en et. al. , 1993, Science 259: 373-377) and mouse mAbs to the human insulin receptor have been developed (Pardrid).
ge et. al. , 1995, Pharm Res 12: 807-816).
.

The anti-human insulin receptor mAb 83-14 has been shown to couple with streptavidin and allow biotinylated compounds to be transported therewith, retaining its ability to be transported (Wu et. al, 1997, J Cli
n Invest, 100: 1804-12).

Little is known about the up or down regulation of the transport system in the BBB in disease states, but changes in expression levels occur, eg ICAM-1 expression, then ICAM-2 in the inflamed area. There is an indication that it is upregulated (Osborn, L. Cell 1990, 623-6). Lymphocytes bound to BBB increased three-fold after stimulation with lipopolysaccharide and interleukin-1 and -6, with specific m for VLA4, CD18 and CD11a.
Could be blocked by Ab (de Vries et. Al., JN)
euroimmunol 1994, 52; 1-8). Thus, the disease state is
Identifying an up-regulated transport system in the BBB can generate important guidance towards establishing selectivity in BBB transport. A scheme that emerges from in vitro and in vivo studies in animal mode is that intracellular signalling results in cross-linking of cell surface adhesion molecules leading to altered cytoskeletons, degradation of junctional complexes and ultimately increased vascular permeability. It means to start the chemical cascade.

In one aspect, the invention provides a mixture or panel of at least 5, preferably at least 10 different specific binding members, each member being an antibody VH variable region disclosed herein. G65 (SEQ ID N
O. 2), G67 (SEQ ID NO. 6), G73 (SEQ ID NO.
10), G76 (SEQ ID NO. 14), G77 (SEQ ID NO. 14).
18), G78 (SEQ ID NO. 22), G79 (SEQ ID NO.
24), G81 (SEQ ID NO. 30), G83 (SEQ ID NO.
34), G85 (SEQ ID NO. 38), G88 (SEQ ID NO.
42), G92 (SEQ ID NO. 46), G93 (SEQ ID NO. 46).
50), G95 (SEQ ID NO.54), G101 (SEQ ID NO.
． 58), G102 (SEQ ID NO. 62), G110 (SEQ ID NO.
NO. 66) and G112 (SEQ ID NO.70) VH region sequences, and / or antibody VL variable regions having the amino acid sequence selected from the group G65 (SEQ ID NO.4) disclosed herein. , G67 (SEQ
ID NO. 8), G73 (SEQ ID NO.12), G76 (SEQ
ID NO. 16), G77 (SEQ ID NO.20), G78 (SEQ
ID NO. 24), G79 (SEQ ID NO. 28), G81 (SEQ
ID NO. 32), G83 (SEQ ID NO.36), G85 (SEQ
ID NO. 40), G88 (SEQ ID NO.44), G92 (SEQ
ID NO. 48), G93 (SEQ ID NO. 52), G95 (SEQ
ID NO. 56), G101 (SEQ ID NO.60), G102 (SE
Q ID NO. 64), G110 (SEQ ID NO.68) and G11.
2 (SEQ ID NO.72) VL region sequence, having an amino acid sequence selected from the group consisting of the antibody VH variable region and / or the antibody VL variable region, each specific binding member of which is a filamentous bacteriophage particle. When displayed on a surface, the mammalian blood-brain barrier, preferably a human or rodent (eg,
Rat) can cross the blood-brain barrier. The blood-brain barrier is described in Example 1 herein.
Can be an in vitro barrier as produced by

The mixture or panel of specific binding members can include at least 11, 12, 13, 14, 15, 16, 17 or 18 of the VH or VL regions. In such mixtures, the VH or VL regions are preferably paired according to the corresponding nomenclature used herein (eg G65VH (S
EQ ID NO. 2) and G65VL (SEQ ID NO.4), G81VH
(SEQ ID NO.30) and G81VL (SEQ ID NO.32), etc.).

The panel or mixture of specific binding members provided by the present invention is useful for the selection of specific binding members across the blood brain barrier. In addition, the panels or mixtures can be used together to provide various molecular or agonist functions.

The one or more specific binding members can be selected from a panel or mixture, for example by binding with an antigen which can be on endothelial cells. If the specific binding member is displayed on a filamentous bacteriophage, this allows the nucleic acid encoding the selected specific binding member, or at least the component of this member, to be readily isolated. The encoded nucleic acid can then be manipulated and used by recombinant expression to produce more specific binding member or components thereof.

In a more general aspect, the invention provides for contacting a library of specific binding members displayed on the surface of filamentous bacteriophage particles, and mammalian cells when displayed on filamentous bacteriophage particles. Providing a method of obtaining one or more specific binding members comprising selecting one or more specific binding members of a library capable of crossing the blood-brain barrier of a. Following selection for their ability to cross the blood-brain barrier, the ability to bind brain or CNS cell types, such as endothelial cells, or meninges, parenchyma, choroid plexus, cerebrum, cerebellum, spinal cord cells or microglial cells. It is possible to make one or more further selections for.

When displayed on filamentous bacteriophage particles, a population or mixture of specific binding members capable of crossing the mammalian blood-brain barrier is selected by selection from a phage display library as disclosed. Obtainable. Such a population or mixture can include at least 10 ⁶ various specific binding members. About 10,000 specific binding members, 1,000, 10
It is possible to use one or more additional rounds of selection to provide 0, 10 to 20 or less various specific binding members, and subpopulations of individual specific binding members.

The invention further provides a plurality of various antibody VH variable regions obtained from a mixture, panel, population or library as disclosed, and further obtained from such a mixture, panel, population or library. A plurality of various antibody VL variable regions is provided.

The individual VH and VL regions whose sequences are shown herein each represent a separate aspect of the invention. VH whose sequence is disclosed herein
The regions can be combined with the VL regions whose sequences are disclosed herein or other VL regions to provide VH / VL pairs that represent the antigen binding site of an antibody. Similarly, a VL region whose sequence is disclosed herein can be combined with a VH region whose sequence is disclosed herein or another VH region. Pairing of VH and VL regions represents a further aspect of the invention. Preferred pairings are referred to herein by corresponding names, eg G65VH (SE
Q ID NO. 2) and G65VL (SEQ ID NO.4), G81VH (
SEQ ID NO. 30) and G81VL (SEQ ID NO. 32), etc.

Accordingly, individual aspects of the invention include G65, G67, G73, G76, G77,
G78, G79, G81, G83, G85, G88, G92, G93, G95,
A specific binding member comprising a VH / VL pairing selected from the group consisting of G101, G102, G110 and G112 (SEQ ID NO. As described above) is provided. Each VH region and each VL region of each of these pairs represents a further aspect of the invention. Among the VH / VL pairs disclosed herein, G67, G7
3, G76, G78, G79, G83, G85, G88, G92, G93, G9
5 and G110 represent preferred embodiments because they are cross-reactive with human tissue.

These actively cross the blood-brain barrier as phage antibodies and specifically recognize brain endothelial cells, as shown by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Among them, G93 (VH SEQ ID NO.50; VL SEQ I
D NO. 52) and G73 (VH SEQ ID NO. 10; VL SE
Q ID NO. 12) is a particularly preferred embodiment of the invention; they exhibit high levels of transport across the blood-brain barrier and cross-react with antigens on human endothelial cells.

As mentioned above, additional VH and VL regions and their pairs can be obtained from filamentous bacteriophage libraries with selectivity for their ability to cross the blood-brain barrier.

A further aspect of the invention is D5VH (SEQ ID NO.74) and VL (SEQ ID NO.7) whose amino acid sequences are disclosed herein.
6) Provide the area. D5VH / VL is directed against human serum amyloid protein (SAP) that crosses the blood-brain barrier. The experimental evidence contained below is D
5 is actively transported across the blood-brain barrier.

According to the present invention, one or more CDRs are picked up from any VH or VL,
It can be incorporated into a suitable framework. This is discussed further below.

VH and VL regions and CDRs whose sequences have been set up herein and which can be used for specific binding members for brain and / or endothelial cell antigens.
Variants of s can be obtained by the method of sequence alteration or mutation and screening. Such methods are also provided by the present invention.

In addition to antibody sequences, specific binding members may, for example, form peptides or polypeptides such as folding regions or confer additional functional characteristics to the molecule in addition to its ability to bind antigen. , Can contain other amino acids. Specific binding members of the invention can carry a detectable label or be conjugated to a toxin or enzyme (eg, via a peptidyl bond or linker). Thus, the VH or VL region according to the invention can be provided in the form of a fusion with additional amino acids.

A further aspect of the invention provides a method for obtaining one or more specific binding members with desirable properties, which method comprises contacting a library or panel of specific binding members with one or more particular binding members. Including selecting one. Such methods can use phage display technology in which specific binding members in a library or panel are displayed on the surface of bacteriophage particles, each of which has a specific binding member or component thereof (eg, VH region). ) Is included in the nucleic acid. The nucleic acid can be taken from a bacteriophage particle containing a nucleic acid encoding a selected specific binding member or component thereof, the nucleic acid having the sequence of nucleic acids from the particle being the encoded product, or It can be used to provide additional nucleic acids with sequences, or variants or derivatives (by recombinant techniques).

In a further aspect, the present invention provides an isolated nucleic acid comprising a sequence encoding a specific binding member as described above, and expressing said nucleic acid under conditions to effect expression of said binding member. , And a method of preparing a particular binding member of the invention, comprising recovering the binding member.

A specific binding member according to the invention may be a human, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention. It can be used in a method for treating or diagnosing the animal body. The specific binding member can be unconjugated or conjugated to the active agent. Diseases treatable by the present invention include neurological diseases including Alzheimer's disease, prion diseases, AIDS-related dementia, and any disease associated with inflammation occurring in the brain or CNS.

[0027]   These and other aspects of the invention are described in further detail below.

The term specific binding member This describes a member of a pair of molecules that have binding specificities for one another. The members of a specific binding pair can be naturally derived or wholly or partially synthetically produced. One member of a molecule pair has a portion or cavity on its surface that specifically binds to and thus complements the particular spatial and polar machinery of the other molecule pair member. Thus, the members of the pair have the property of specifically binding to each other. Examples of specific binding pair types include antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application relates to antigen-antibody type reactions.

Antibodies This describes immunoglobulins, whether naturally derived or wholly or partially synthetically produced. These are derived from natural sources, or
They can be partly or wholly synthetically produced. Examples of antibodies include
Immunoglobulin isotypes and their isomorphic subclasses; Fab, scF
a fragment containing an antigen-binding region such as v, Fv, dAb, or Fd; and diabodies (
diabodies).

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can include introducing DNA encoding the immunoglobulin variable or complementarity determining regions (CDRs) of the antibody into the constant region or constant region plus framework regions of various immunoglobulins. For example, EP-
See A-184187, GB2188638A or EP-A-239400. Hybridomas or other cells that produce antibodies may be susceptible to genetic mutations or other changes that may or may not alter the binding specificity of the antibodies produced.

Since antibodies can be modified in many ways, the term “antibody” should be construed as covering any particular binding member or substance which has a binding region with the required specificity. is there. Thus, the term covers antibody fragments, antibody derivatives, functional equivalents and homologues, whether natural or wholly or partially synthetic, including any polypeptide containing an immunoglobulin binding region. Thus, a chimeric molecule comprising an immunoglobulin binding region or equivalent fused to another polypeptide is included. Cloning and expression of chimeric antibody
P-A-0120694 and EP-A-0125023.

It has been shown that fragments of whole antibodies can serve the function of binding antigen. Examples of binding fragments include (i) Fab fragment consisting of VL, VH, CL and CH1 regions, (
ii) Fd fragment consisting of VH and CH1 regions, (iii) Fv fragment consisting of VL and VH regions of a single antibody, (iv) dAb fragment consisting of VH region (Ward
, E. S. et. al. , Nature 341, 544-546 (1989).
), (V) isolated CDR region, (vi) F (ab ′) 2 fragment, 2 linked Fab
A single-valent Fv molecule (scFv) (Bird) in which a bivalent fragment containing fragment, (vii) VH and VL regions are linked by a peptide linker that allows the two regions to join to form an antigen binding site. et al, Science, 24
2,423-426,1988; Huston et al, PNAS USA.
, 85, 5879-5883, 1988), (viii) bispecific single chain Fv
Dimer (PCT / US92 / 09965) and (ix) "Diabody",
Multivalent or multispecific fragments constructed by gene fusion (WO94 / 1380
No. 4; Holliger et al, Proc. Natl. Acad. S
ci. USA 90 6444-6448, 1993). Fv, s
The cFv or diabodies molecules can be stabilized by the incorporation of disulfide bridges connecting the VH and VL regions (Y. Reiter et al.
al, Nature Biotech, 14, 1239-1245, 1996).
. It is also possible to create minibodies containing scFv bound to the CH3 region (S. Hu et al, Cancer Res., 56, 3).
055-3061, 1996).

Diabodies are multimers of polypeptides, each polypeptide comprising a first region containing the binding region of an immunoglobulin light chain and a second region containing the binding region of an immunoglobulin heavy chain, these two regions being Although bound (eg, by a peptide linker), they cannot bind to each other to form an antigen binding site: an antigen binding site is one polypeptide first region within a multimer and another polypeptide within the multimer. It is formed by bonding with the second region (WO94 / 13804).

If bispecific antibodies are to be used, they can be prepared in various ways, for example chemically or from hybrid hybridomas (Hollinger, P.
． and Winter G.M. Current Opinion Biotec
hnol. 4, 446-449 (1993)) or any of the bispecific antibody fragments described above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed using only the variable region and no Fc region, potentially reducing the effects of anti-idiotypic responses.

Bispecific diabodies to bispecific whole antibodies may also be particularly useful because they can be easily constructed and expressed in E. coli. Diabodies of appropriate binding specificity (and many other polypeptides such as antibody fragments) can be readily selected using phage display from libraries (WO94 / 1380).
No. 4). If one arm of the body is kept in a constant state, for example with a specificity directed against antigen X, then the other arm is altered and an antibody of the appropriate specificity is selected. You can create a library. Bispecific whole antibodies can be generated by knobs-into-holes engineering (JBB Ridgeway et al, Protein Eng.
, 9, 616-621, 1996).

Antigen-binding region This describes the part of an antibody that comprises the part that specifically binds and is complementary to part or all of the antigen. When the antigen is large, the antibody can only bind to a specific portion of the antigen, the portion of which is called the epitope. The antigen binding region can be provided by one or more antibody variable regions (eg so-called Fd antibody fragments consisting of VH regions). Preferably, the antigen binding region comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

Specific This means that one member of a specific binding pair has its specific binding partner (s).
It can be used to refer to situations in which the molecule does not show any significant binding. The term is specific to, for example, a particular epitope whose antigen-binding region is carried by many antigens, in which case the specific binding member carrying the antigen-binding region can bind to various antigens carrying the epitope. It is also applicable in some cases.

Comprise This is generally used in the sense of include, ie, allowing the presence of one or more additional features or ingredients. Isolated This refers to the state in which a particular binding member of the invention or a nucleic acid encoding such a binding member is present according to the invention. Members and nucleic acids are found together in their natural environment or in the environment in which they are produced (eg, cell culture) when such preparations are performed in vitro or in vivo by recombinant DNA technology. Free or substantially free of materials with which they are naturally associated, such as the polypeptides or nucleic acids of. The member and nucleic acid can be formulated with diluents or adjuvants and still be practically isolated-for example,
Members are usually mixed with gelatin or other carriers when used to coat microtiter plates for immunoassay applications, or pharmaceutically acceptable carriers or dilutions when used for diagnostics or therapeutics. Mixed with the agent.
Specific binding members may be natural or heterologous eukaryotic cells (eg CHO or NS
0 (ECACC85110503) cells) or they can be non-glycosylated (eg, when produced by expression in prokaryotic cells).

“Substantially as configured” refers to the relevant CDR or VH or V of the present invention.
It is meant that the L region is either identical or highly similar to the particular region with the sequences set out herein. “Highly similar” means 1 to
It means that 5, preferably 1 to 4 such as 1 to 3 or 1 or 2, or 3 or 4 substitutions can be made in the CDR and / or VH or VL regions.

The CDR-bearing structures of the present invention will generally have a C at the position corresponding to the CDRs of the naturally occurring VH and VL antibody variable regions encoded by the transfer immunoglobulin gene.
It consists of an antibody heavy or light chain sequence or a substantial portion thereof, in which the DR is located. The structure and location of immunoglobulin variable regions is described in (Kabat, EA et.
al, Sequences of Proteins of Immunolo
musical Interest. 4th Edition. US Department
ment of Health and Human Services. 19
87, and the latest version, now on the Internet (http://immuno.bm
e. nwu. edu) available)).

Preferably, the CDR amino acid sequence substantially as set out herein is carried as a CDR in a human variable region or substantially in that portion. VLCDR3 and especially VHCDR3 as substantially set herein
The sequences (see, eg, Table 4) depict preferred embodiments of the invention, each of which is carried as a VLCDR3 or VHCDR3, respectively, in the variable region of, or a substantial portion of, a human light or heavy chain, respectively. Preferably.

The variable regions used in the present invention can be derived from any germline or metastatic human variable region or can be synthetic variable regions based on the consensus sequence of known human variable regions. CDR-inducing sequences of the invention (eg, CDR
3) can be introduced into the repertoire of variable regions lacking CDRs (eg CDR3) using recombinant DNA technology.

For example, Marks et al. (Bio / Technology, 199)
2, 10: 779-783) is a VH variable lacking CDR3 in which a common primer directed directly or close to the 5'end of the variable region region is used in conjunction with a common primer for the third framework of the human VH gene. Provide a repertoire of areas,
A method of producing a repertoire of antibody variable regions is described. Marks et al. Further describe how this repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, the CDR3-derivatized sequences of the invention can be mixed with a repertoire of VH or VL regions lacking CDR3,
The mixed complete VH or VL region can be mixed with the cognate VH or VL region to provide a specific binding member of the invention. The repertoire can then be displayed in a suitable host system, such as the phage display system of WO92 / 01047, so that suitable specific binding members can be selected. The repertoire is any individual member above 10 ⁴
For example, it can consist of 10 ⁶ to 10 ⁸ or 10 ¹⁰ members.

Similar mixing or combining techniques are also described by Stemmer (N
ature, 1994, 370: 389-391) and describes techniques for the β-lactamase gene while observing that this method can also be used for the production of antibodies.

A further alternative is to use the random mutagenesis of one or more selected VH and / or VL genes to generate mutations within the complete variable region, and CDR-derivatization of the invention. To generate a new VH or VL region carrying the sequence. Such a technique is based on the use of mutant PCR in Gram et al. (1992).
, Proc. Natl. Acad. Sci. , USA, 89: 3576-358.
0).

Another method that can be used is to direct mutagenesis to the CDR regions of the VH or VL genes. These technologies are available from Barbas et al.
1994, Proc. Natl. Acad. Sci. , USA, 91: 3809.
~ 3813) and Schier et al. (1996, J. Mol. Bio).
l. 263: 551-567).

All of the above-mentioned techniques are known in the art as such and do not themselves form part of the invention. One of ordinary skill in the art can use such techniques to provide the specific binding members of the invention using methodologies conventional in the art. A further aspect of the invention provides a method for obtaining antibody-antigen binding regions with desirable properties, such as the ability to cross the blood-brain barrier and / or specificity for brain and / or endothelial cell antigens, the method comprising: A VH region, which is an amino acid sequence variant of the VH region, is provided by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the VH region set in the present specification. In combination with one or more VL regions, desirable properties, such as ability to cross the blood-brain barrier and / or specificity for an antigen, and optionally one or more favorable properties, such as an inflamed part of the brain, blood brain Testing VH / VL combinations, or multiple combinations, for identifying antibody-antigen binding regions that have the ability to bind a disrupted portion of the barrier Including the door.

One or more sequence variants of the VL region disclosed herein include one or more V
Similar methods combined with H regions can be used.

A further aspect of the invention provides a method of producing a specific binding member with desirable properties, which method comprises: (a) containing the CDR3 to be replaced or lacking the CDR3 coding region. Providing a starting repertoire of nucleic acids encoding any VH region, (b) combining said repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for VHCDR3. , So that the donor nucleic acid is inserted into the CDR3 region in the repertoire to provide a production repertoire of nucleic acids encoding the VH region, (c) expressing the nucleic acid of the production repertoire, and (d) desirable Selecting a specific binding member having characteristics, and (e) recovering the specific binding member or the nucleic acid encoding it. Including the.

Again, a similar method combined with a repertoire of nucleic acids encoding the VL region, in which the VLCDR3 of the invention either contains the CDR3 to be replaced in or lacks the CDR3 coding region, is It can be used.

Similarly, one or more, or all, of the 3 CDRs can be grafted into the repertoire of VH or VL regions, which in turn are tailored for specific binding members or specific binding members with desirable properties. Be sorted.

As mentioned above, the desired properties are to cross the blood-brain barrier, bind endothelial cells or other brain cell antigens, bind inflammatory or blood-brain barrier disrupting parts of the brain, and ICAM.
Can be any one or more of the ability to bind or bind the transferrin receptor.

A substantial portion of the immunoglobulin variable region comprises at least 3 CDR regions together with their intervening framework regions. Preferably, the parts are also first and fourth
Includes at least about 50% of either or both framework regions, the 50% being the first
50% C-terminal of the framework region and 50% N-terminal of the fourth framework region. The additional residues at the N-terminus or C-terminus of a substantial portion of the variable region can be those not normally associated with naturally occurring variable range regions. For example, recombinant D
Construction of specific binding members of the invention made by NA technology results in the introduction of N- or C-terminal residues encoded by a linker introduced to facilitate cloning or other engineering steps. It is possible. In other engineering steps, the variable regions of the invention may be linked to immunoglobulin heavy chains, other variable regions (eg, in the construction of diabodies), or additional protein sequences including protein labels as discussed in more detail below. Introducing a linker for conjugation.

In preferred embodiments of the invention, specific binding members comprising a pair of VH and VL regions are preferred, although a single binding region based on either VH or VL region sequences forms a further aspect of the invention. . It is known that a single immunoglobulin region, especially the VH region, can bind the target antigen in a specific way.

In the case of any of the single-stranded specific binding regions, these regions are used to select complementary regions capable of forming 2-region specific binding members with the desired properties. Is possible.

This is the case where individual colonies containing either H or L chain clones were used to infect a complete library of clones encoding the other chain (L or H) and the resulting 2 chain What is achieved by a phage display selection method using the so-called hierarchical double combinatorial method as disclosed in WO 92/01047, wherein the specific binding members are selected by phage display techniques such as those described in that patent. It is possible. This technique is also disclosed in Marks et al., Supra.

Specific binding members of the invention can further include antibody constant regions or portions thereof. For example, the VL region can be attached at its C-terminal portion to an antibody light chain constant region that comprises a human Cκ or Cλ chain, preferably a Cλ chain. Similarly, specific binding members based on the VH region can be attached to all or part of an immunoglobulin heavy chain derived from any antibody isotype, such as IgG, IgA, IgE and IgM and any isotype sub-class, particularly IgG1 and IgG4. In contrast, it is possible to attach at its C-terminal end.
IgG4 is preferred.

The antibodies of the present invention can be labeled with a detectable or functional label. Detectable labels include radioisotope labeling, such as ¹³¹ I or ⁹⁹ Tc, which can be attached to the antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels also include certain cognate detectable moieties, eg, chemical moieties such as biotin that are detectable via binding to labeled avidin.

The antibodies of the present invention are designed for use in methods of diagnosis or treatment of human or animal subjects, preferably humans.

Accordingly, a further aspect of the present invention is a method of treatment comprising administration of a specific binding member as provided, a pharmaceutical composition comprising such a specific binding member, and the manufacture of a medicament for administration, eg Provided is the use of such specific binding members in a method of making a medicament or other composition comprising formulating the specific binding member with at least one additional component such as a pharmaceutically acceptable excipient.

The composition provided by the present invention can be administered to an individual. Administration is
Preferably, in a "therapeutically effective amount", sufficient to show benefit to the patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the subject being treated. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody are well known in the art; Ledermann J. et al. A. et al. (1991)
Int J. Cancer 47: 659-664; Bagshawe K .; D
． et al. (1991) Antibody, Immunoconjugat
es and Radiopharmaceuticals 4: 915-92
See 2.

The compositions may be administered either alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

The antibodies of the invention may be administered via any suitable route, usually via intravenous injection into the bloodstream.
It can be administered to patients in need of treatment. The exact dose will depend on whether the antibody is diagnostic or therapeutic, the size and location of the area to be treated (eg trauma), the exact type of antibody (eg whole antibody, fragments or diabodies). ),
And a number of factors, including the type of detectable label or other molecule attached to the antibody. Typical antibody dose is 0.5m for systemic administration
g to 100 g, and 10 μg to 1 mg for local administration. Generally, the antibody is a whole antibody, preferably the IgG4 subclass. This is a monotherapy dose for adult patients and should be adjusted proportionally for children and infants and for other antibody formats in proportion to molecular weight and pharmacokinetics. Is possible. Treatment can be by continuous infusion or can be repeated as appropriate daily, 2 days a week, weekly or monthly. The treatment can be repeated within the same day for the treatment of short term problems such as epilepsy and traumatic brain injury.

Whole antibodies of the IgG4 subclass are used for systemic and local administration, but
It is presently preferred that scFv antibodies may be of particular value for topical administration.

The specific binding member of the invention is usually administered in the form of a pharmaceutical composition which can include at least one component in addition to the specific binding member.

Accordingly, the pharmaceutical composition according to the invention and for use according to the invention comprises, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or well-known to the person skilled in the art. Other materials can be included. Such materials are preferably non-toxic and do not interfere with the efficacy of the active ingredient. The exact type of carrier or other material will depend on the route of administration, which may be oral, or by injection, eg intravenous.

Pharmaceutical compositions for oral administration may take the form of tablets, capsules, powders or liquids. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

For injection, or injection at the site of pain, the active ingredient is p
H, in the form of a parenterally acceptable aqueous solution with isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringers Injection, or Lactated Ringers Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Other treatments may include administration of suitable doses of analgesics or anti-emetics.

The present invention provides a method comprising causing or allowing binding of a specific binding member provided herein to an antigen. As mentioned above, such binding can occur in vivo, for example after administration of the specific binding member or a nucleic acid encoding the specific binding member, or it can occur in vitro. is there.

The amount of the specific binding member bound to the antigen can be measured. Weighing
It is possible to relate to the amount of antigen in the test sample, which may be of diagnostic interest.

The reactivity of the antibody on the sample can be measured by any suitable means. Radioimmunoassay (RIA) is one possibility. The radiolabeled antigen is mixed with unlabeled antigen (test sample) and left to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody is measured. The more antigen in the test sample, the less radioactive antigen will be bound to the antibody. Binding protein competition assays can also be used with non-radioactive antigens, using the antigen or analog linked to a reporter molecule. The reporter molecule can be a fluorescent dye, a phosphor or a dye laser with spectrally isolated absorption or emission properties. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin and Texas red. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include polymeric colloidal particles or particulate materials, such as colored, magnetic or paramagnetic latex beads, and directly or indirectly by visually observable or electronically detectable signals. And biologically or chemically active agents that may cause it to be detected or otherwise recorded in. These molecules can be, for example, enzymes that catalyze reactions that produce or change color or produce changes in electrical properties. They can be molecularly excitable, so that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They can include chemicals used with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems can be used.

The signal generated by an individual antibody-reporter conjugate can be used to derive quantifiable absolute or relative data of related antibody binding in a sample (normal and test).

The present invention also provides for the use of a specific binding member as described above for measuring antigen levels in a competition assay, ie by using the specific binding member provided by the invention in a competition assay. A method of measuring the level of an antigen therein is provided. This may be the case when there is no need to physically separate the bound from the unbound antigen. It is one possibility to attach the reporter molecule to a specific binding member such that upon binding a physical or optical change occurs. The reporter molecule is capable of producing a signal that is directly or indirectly detectable and preferably measurable. The attachment of the reporter molecule can be direct or indirect, eg covalently via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

The invention also provides for the direct measurement of antigen levels, for example by using a specific binding member according to the invention in a biosensor system.

The manner in which binding is measured is not a feature of the invention, and those of skill in the art can select the manner that is more suitable to their taste and general knowledge.

The invention further provides a V region comprising a CDR that binds an antigen and that has amino acids substantially as set out herein or an amino acid sequence substantially as set up herein. To any specific binding member that includes a V region with a specific binding member that competes for binding to an antigen. Competition between binding members can be achieved, for example, by tagging a specific reporter molecule to one binding member that can be detected in the presence of other untagged binding member (s) to bind the same epitope or By allowing the identification of specific binding members that bind overlapping epitopes, it is possible to easily measure in vitro. Competition can be measured, for example, using an ELISA, such as a whole cell ELISA using immobilized brain endothelial cells that seeks suppression of the signal when the cells are pre-incubated with a potential competing antibody.

In a test for competition, peptide fragments of the antigen can be used,
In particular the peptide comprises the epitope of interest. Peptides can have and can be used with the epitope sequence plus one or more amino acids at either end. Such peptides may be said to "consist essentially of" a particular sequence. Specific binding members according to the present invention may be such that their binding to their antigen is suppressed by peptides having or including the given sequence.
In tests against this, it is possible to use peptides with either the sequence plus one or more amino acids.

Specific binding members that bind a specific peptide can be isolated from a phage display library by, for example, picking up the peptide (s).

The invention further provides an isolated nucleic acid encoding a specific binding member of the invention. Nucleic acid includes DNA and RNA. In a preferred embodiment, the invention provides a nucleic acid encoding a CDR or VH or VL region of the invention as defined above.

The present invention also provides constructs in the form of plasmids, vectors, transcription traits or expression cassettes containing at least one of the above-mentioned polynucleotides.

The present invention also provides a recombinant host cell that comprises one or more constructs as described above.
Nucleic acids encoding any provided CDR, VH or VL regions, or specific binding members may, as such, include methods of producing the encoded product, including expression from the encoding nucleic acid. , Themselves form an aspect of the invention. Expression can be conveniently achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, the VH or VL region, or specific binding member, can be isolated and / or purified using any suitable technique and then used as appropriate.

Specific binding members, VH and / or VL regions, and encoding nucleic acid molecules and vectors according to the present invention are, for example, isolated from their natural environment and / or
Or in a purified and substantially pure or homogeneous form, or in the form of no or substantially no nucleic acid or genetic system other than the sequence encoding the polypeptide having the required function in the case of nucleic acids. Is possible. Nucleic acids according to the present invention include DNA or RNA and may be synthetic in whole or in part.
References to nucleotide sequences set out herein refer to D having a particular sequence.
It includes NA molecules, and RNA molecules having a particular sequence in which U is substituted for T unless otherwise required.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli. A suitable host in which the coding sequence contains a TAG suppressive stop codon is a suppressor strain such as the sup44 strain of TG1. In such lines, the TAG codon is read as the encoded glutamine. Embodiments of the invention in which the suppressor strain can be used to produce the encoded polypeptide by expression from an encoded nucleic acid containing the TAG codon include any SEQ ID NO. 37, 43 and 69 (G85VH SE respectively
Q ID NO. 38, G88VL SEQ ID NO. 44, and G11
2VH SEQ ID NO. (Encoding 70) is used.

Expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For review, see, for example, Pluckthun, A .; Bio / Techn
LOGY 9: 545-551 (1991). Expression of eukaryotic cells in culture is also available to those skilled in the art as an option for the production of specific binding members, and as a result of recent studies, see, for example, Ref, M. et al. E. (1993) Cur
r. Opinion Biotech. 4: 573-576; Trill J. et al.
J. et al. (1995) Curr. Opinion Biotech 6
: 553-560.

Suitable vectors contain appropriate regulatory sequences and can be selected or constructed, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other suitable sequences. . The vector can be a plasmid, a suitable viral eg phage, or a phagemid. More specifically, for example, Molecular Cloning: a L
Laboratory Manual: 2nd edition, Sambrook
k et al. , 1989, Cold Spring Harbor Lab
See oratory Press. For example, many known techniques and procedures for nucleic acid manipulation in the production of nucleic acid constructs, mutagenesis, linkage, introduction of DNA into cells and gene expression, and protein analysis are described in Short Pr.
otocols in Molecular Biology, Second
Edition, Ausubel et al. eds. , John Wile
y & Sons, 1992. Sambrook et
al. And Ausubel et al. The disclosure of which is incorporated herein by reference.

Thus, a further aspect of the invention provides a host cell containing the nucleic acids disclosed herein. Yet a further aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction can use any possible technology. Suitable techniques for eukaryotic cells include calcium phosphate nucleic acid transfer, DE
AE-dextran, electroporation, liposome-mediated nucleic acid transfer and retrovirus or other viruses such as transduction with baculovirus against vaccinia or insect cells. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation and nucleic acid transfer using bacteriophage.

Introduction can then cause or allow expression from nucleic acids, for example by culturing the host cells under conditions for gene expression. In one embodiment, the nucleic acid of the invention is integrated into the genome (eg chromosome) of the host cell. Integration can be facilitated by inclusion of sequences that facilitate recombination with the genome according to standard techniques.

The present invention also provides a method that includes using the above-described construct in an expression system to express a specific binding member or polypeptide as described above.

EXAMPLES Aspects and embodiments of the invention will now be described through the following experimentally related examples.

[0092] Abbreviation Immunocytochemistry (ICC) Interleukin 1-β (IL-1β) Enzyme-linked immunosorbent assay (ELISA) Serum amyloid protein (SAP) Bovine serum albumin (BSA) Blood-brain barrier (BBB)

List of Examples Example 1: Formation of an in vitro blood-brain barrier cell culture test vessel. Example 2: Selection of antibody-expressing phage across the in vitro blood-brain barrier. Example 3: Characterization of selected antibodies by endothelial cell ELISA and sequencing. Example 4: Demonstration of clonal phage transport. Example 5: Rat brain immunocytochemistry of endothelial cell binding antibody clones. Example 6: Testing of rat endothelial cell-binding antibodies for ICC cross-reactivity with a human tissue panel. Example 7: Selection of SAP-binding antibodies. Example 8: Characterization of SAP-binding antibodies based on sequence and ability to cross the blood-brain barrier in the presence or absence of SAP.

Example 1-Formation of in vitro blood-brain barrier cell culture chamber (a) Production of primary cell culture to form in vitro blood-brain barrier (i) Astrocyte culture from rat brain tissue Preparation of Astrocytes The protocol used for the preparation of individual cultures of astrocytes is described in K. D. McC
arty and J. De Vellis (J. Cell. Biol., 1
980, vol. 85,890-902). Using a similar method, the resulting astrocyte cultures are reported to be> 98% pure by immunocytochemistry with antibodies to glial fibrillary acidic protein (GFAP). A summary of this protocol for astrocyte cultures has been published (GMA Wells., 1996, in Glia, vol.
． 18, p. 332 340). From the use of 12-24 hour old rat pups, the absence of viable neurons in the cell suspension obtained from dissociation of the cerebral cortex and the ease of cell sieving by mechanical sieving techniques than using trypsinization Dissociation was confirmed. The length of the initial culture period was maintained between 7 and 9 days to ensure stratification of astrocytes, microglia and oligodendrocytes. The procedure for isolation of individual glial cell cultures was carried out when the primary cell cultures were exposed to sheer forces in shaking the cultures at 37 ° C. in a ring incubator overnight (about 16 hours). It depends on the selective detachment of other cells from the tightly attached type 1 astrocyte layer. Separated astrocyte cultures can be maintained for several weeks, allowing investigation of this cell type.

Fifteen 12-24 hour old rat pups were placed in a chamber containing halothane for approximately 5 minutes or until there was no movement from the animal. The neck of each animal was broken by tightening tightly under the head. Three to four baby rats were decapitated and their heads were placed vertically on a pad of sterile filter paper so that blood from the cut was absorbed. The neck was smeared on filter paper to remove excess blood, then the top was turned down and the skin at the base of the neck was grasped with a finger. The skin from the back of the neck to just above the eyes was cut using dissection scissors and the skin was folded and removed from the skull. Next, both sides of the skull were cut from the back side of the neck to just above the eyes and peeled off with tweezers. The optic nerve was cut with a spatula, the cerebral hemisphere was removed from the cavity with the same spatula (without the cerebellum), and 20 ml DMEM (Lif was used.
-4.5 g / l glucose and 110 m from eTechnologies
g / l containing sodium pyruvate) + L glutamine (Life Tech)
Noologies) + 20% fetal bovine serum (FBS) (Life Techno)
Logistics) + Penicillin / Streptomycin (Life Technology)
The tubes were placed in sterile 50 ml tubes.

Cerebral tissue in DMEM was poured from a 50 ml tube into sterile petri dishes and each individual brain tissue was removed with forceps and transferred onto a pad of sterile filter paper. The cerebral hemispheres of each brain were divided and individually rolled over the length of fresh filter paper to remove meninges and blood vessels. They were then placed in another Petri dish containing 20 ml of fresh DMEM containing 20% FBS. The brain tissue was dipped into DMEM in a Petri dish while chopping the brain tissue into tiny pieces using a single-edged razor blade. 23 in the cup-shaped container
A 0 μm reusable metal sieve was placed on top of a Sterilin pot through which 20 ml of fresh DMEM containing 20% FBS was taken. Then 20 ml of DMEM containing chopped brain tissue was taken from the Petri dish into a 25 ml pipette and pipetted onto the sieve. The chopped tissue was forcibly passed through a sieve using a glass fiber cloth. The filtered cell suspension was equally divided into two 50 ml centrifuge tubes and centrifuged at 1000 rpm for 10 minutes. The supernatant is removed and the cell pellet is gently resuspended by pipetting to a final 20
The volume was 20 ml DMEM containing% FBS.

The cell suspension was then passed through a 100 μm disposable sieve placed on top of a 50 ml tube and washed through 10 ml DMEM containing 20% FBS. The filtered cell suspension was then passed through a 70 μm disposable sieve placed on top of a new 50 ml tube and washed through 10 ml DMEM containing 20% FBS. The resulting filtered cell suspension was pipetted into a 75 cm tissue culture flask and sufficient D containing 20% FBS was added so that the final volume was 20 ml per brain used.
MEM was added. The cell suspensions were mixed and divided into appropriate numbers of 75 cm flasks so that each flask contained 20 ml of cell suspension. The tip of each flask was loosened and the flasks were placed in a 37 ° C incubator with a 5% CO atmosphere.

The flask was left undisturbed for 4 days and then inspected. Old media and tissue debris were removed and cells were fed with 20 ml fresh DMEM containing 20% FBS and left in the incubator for an additional 3 days. On day 7 of primary culture, all cells in the flask were examined for good coverage (by this stage astrocytes are preferably close to confluence). 15 ml DMEM containing 10% FBS after removing the medium
Replaced by. The flask with the loosened tip was returned to the incubator and left for a further 24-48 hours. Astrocytes were subcultured with DMEM containing 10% FBS as standard growth medium almost weekly (or when confluence was reached) with a 1: 4 dilution of cells for a period of several weeks.

(Ii) Preparation of Rat Brain Endothelial Cell Cultures Attempts to establish particularly dense cultures of brain endothelial cells (EC) from several species have resulted in disruption of cerebral tissue (usually mechanical). And a combination of enzymatic means)
, Followed by purification of microvessels by density centrifugation and subsequent plate-out on tissue culture plastic coated with extracellular interstitial proteins (for review) , J. Neurochem,
vol. 58, p. F. 117. Joo, 1992). C. was used to establish EC cultures from microvessels derived from rat cerebral tissue. C. W.
Hughes and P.H. L. The protocol was adapted from the method published by Lantos. The use of the only dissected and removed brain miso reduces the amount of myelin in the enzymatic digestion of tissues. Using this protocol, dissected, digested, and purified tissues from two Wistar rats yielded primary cultures of microvessels set up in two 6-well dishes and individual colonies of endothelial cells were cultured. After 7 days, it can be trypsinized and separated.

Cerebral tissue in DMEM was poured into sterile petri dishes and each piece of tissue was removed onto a pad of sterile filter paper. The cerebral hemispheres were separated, the outer layer of tissue was stripped, and the meninges and large blood vessels were removed by rolling the tissue round along the length of the filter paper. Brain miso was dissected from the white matter, collected together and placed in a new petri dish containing sterile serum-free DMEM containing penicillin and streptomycin. This was then chopped into fine pieces using a single-edged razor. The minced tissue was then placed in a sterile 50 ml centrifuge tube and pelleted at 200 g for 10 minutes at room temperature. The supernatant was removed and penicillin and streptomycin plus 1 mg /
ml collagen (Boehringer Mannheim, cat. no. 2)
69 638) and 10 μg / ml DNase I (Sigma Type).
II, cat. no. The tissue was resuspended in 10 ml serum free DMEM containing D4263). It was then placed on a shaking incubator at 37 ° C. for 90 minutes. The tissue digest was then pelleted at room temperature for 10 minutes at 200 g and 25% (w / v) bovine serum albumin (BSA, Fraction V fr.
om Sigma, cat. no. A9647) was resuspended in 40 ml of DMEM and ground until it had a cream colored dough. It was then spun at 1000 g for 20 minutes at room temperature to pellet capillary fragments (heavier) from myelin, astrocytes, neurons and other single cell impurities (lighter). The top layer was carefully removed to ensure that all traces such as myelin were removed. The capillary pellet was resuspended in 10 ml serum free DMEM and repelleted at 600 g at room temperature. The resulting capillary pellet was then resuspended in 10 ml DMEM containing penicillin and streptomycin plus 1 mg / ml collagenase / disease and incubated at 37 ° C. for 3 hours with shaking. The digest was then pelleted at 600 g for 5 minutes at room temperature, 2 mM L-glutamine (Sigma), 100 units / ml penicillin plus 100 μg / ml streptomycin (Sigma), 20% fetal calf serum-derived 20% plasma (First Link (Sigma)). UK) Ltd., cat.no.
60-00-850) and 75 μg / ml endothelial cell growth supplement (ECGS S
igma, cat. no. E2759) and DM containing 4.5 g / l glucose and 110 mg / ml sodium pyruvate (Sigma)
It was resuspended in 10 ml of endothelial cell growth medium (ECGM) consisting of EM with trituration. The suspension was then passed through a series of sterile 230, 100 and 40 μm sieves and passed through each sieve with a gentle wash of 10 ml of fresh ECGM. The resulting screened suspension was treated with type 1 collagen (Becton Dickin) from the rat tail.
son, cat. no. 40236) 6-hole plate (3 per hole)
Plated out in ~ 4 ml). The 6-well plate and all flasks used below were 100 μg / ml for 1 hour at room temperature with gentle shaking.
Type 1 collagen (diluted from stock solution concentration in sterile 0.02N acetic acid) of Example 1 was used to remove the collagen and rinse with PBS. Sufficient diluted collagen was added to cover the total surface area (> 5 μg / cm ² ) of tissue culture plastic.
This creates a primary enriched microvascular culture from which endothelial cells are further enriched from the possible additives described below.

The medium was replaced after 4 days with fresh ECGM on the primary culture. Large colonies of cells with endothelial-like morphology that grew spirally and radially from microvessel fragments attached to tissue culture plastic were observed after 7 days in culture. Aseptic transfer of suspected endothelial cell colonies to fresh 6-well plates (coated with type 1 collagen) for several days using filter paper soaked in a few small trypsin / EDTA. did. After a short contact with the filter paper, the trypsinized EC sticks to the filter paper after peeling from the holes,
It can be transferred to another plate. These transferred cells are 6 for 3-4 days
As soon as the colonies that had grown to cover the entire surface area of the well were formed (passage 1 cells), the wells were trypsinized and placed in a collagen-coated T ₂₅ flask (passage 2) for a further 3
Cultivated for ~ 4 days, then passaged through collagen-coated _T75 flasks (passage 3). Once the T ₇₅ flasks reach the confluence, they are either frozen in liquid nitrogen or grown in two 1: 5 passages to produce large flask stocks that replace liquid nitrogen stock preparations. Met.

(B) Preparation of In Vitro Blood-Brain Barrier Model in Tissue Culture Inserts Brain ECs can be successfully cultured on commercially available tissue culture inserts on permeable filters separating the upper chamber from the lower chamber. Has been found. Because they are available in a multi-hole format, many conditions can be tested simultaneously to measure barrier integrity or to investigate specific transport mechanisms. The electrical resistance measured across a monolayer of cells adhered to each other by tight junctions is a sensitive indicator of paracellular permeability to ions. Therefore, the quality of the EC barrier can be easily recorded to provide assurance for the model. Type 1 astrocytes, which normally project to brain capillaries ECs, induce extraordinarily high electrical resistance tight junction formation,
It is believed that some inductive effect can be provided in making ECs less leaky. Various combinations of astrocytes and endothelial cells have been tried in an attempt to establish an in vitro model of BBB. For good and reproducible results, culturing ECs in the upper chamber on the diaphragm and astrocytes in the lower chamber on the base of an associated 24-well plate generates a barrier with high endothelial cell cross-resistance. I have proved that. This was the arrangement of cells used as described in the protocol below.

Cultures of rat astrocytes and endothelial cells (EC) were prepared in advance for several days / week from frozen stocks stored in liquid nitrogen. They were preserved at the lowest passage number possible (Astrocyte passage <6, endothelial cell passage <10) to preserve the normal characteristics of primary cells. After thawing glass bottles from liquid nitrogen stock, 2 mM L-glutamine (
Sigma), 10% fetal bovine serum (FBS) (Life Technology)
ies), supplemented with 100 units / ml penicillin plus 100 μg / ml streptomycin (Sigma), 4.5 g / l glucose and 110 mg
Astrocytes were cultured in astrocyte growth medium (AGM) consisting of DMEM containing / ml sodium pyruvate (Life Technologies). Similarly, 2 mM L-glutamine (Sigma), 100 units / ml penicillin plus 100 μg / ml streptomycin (Sigma), 20% plasma-induced fetal bovine serum (First Link (UK) Ltd., cat. No. 60-00).
-850) and 75 μg / ml endothelial cell proliferation supplement (ECGS-Sigma,
cat. no. E2759), 4.5 g / l glucose and 1
Endothelial cells were cultured in endothelial cell growth medium (ECGM) as described in Section B consisting of DMEM containing 10 mg / ml sodium pyruvate (Sigma).

In setting the barrier in tissue culture, astrocytes were first placed in the lower chamber to attach and then the tissue culture insert was placed in the plate and endothelial cells were added to the upper chamber. . Astrocyte T ₁₇₅ flasks were trypsinized and cell numbers were calculated on a hemocytometer. These were then diluted to 1.0 × 10 ⁵ cells / ml in AGM,
1 ml of cell suspension was added to the wells of a 24-well bottom plate. Leave the astrocytes in C
It was allowed to attach to the surface of the lower chamber for at least 2 hours in an O ₂ incubator. While allowing the astrocytes to settle, 100 μl of the appropriate number of 8 μm inserts should be added before adding the endothelial cells.
The rat tail was coated with μg / ml type 1 collagen. The insert was placed in another lower plate and 1 ml of diluted collagen was added to the lower chamber and 0.5 ml of collagen was added to the upper chamber. The inserts were then left at room temperature in a class II tissue culture store for at least 1 hour. The collagen was then removed and the insert rinsed with PBS. Immediately after the inserts were coated with collagen and rinsed with PBS, EC T ₁₇₅ flasks were trypsinized and cell numbers were calculated on a hemocytometer. These were then diluted to 2.0 × 10 ⁵ cells / ml in AGM. The lower plate containing the culture of astrocytes was removed from the incubator and the medium on the astrocytes was replaced with 1 ml of fresh AGM. Then 70% of the coated insert
0.5 ml of 2.0 × 10 ⁵ EC / ml suspension was added to the upper chamber as well as being placed in the lower plate containing the astrocyte culture using forceps sterilized in ethanol. The plates were placed back into the CO ₂ incubator and allowed to sit for at least 24 hours.

The next day, barrier cultures were examined under a microscope. ECs were well formed to form a close-packed coating of the upper chamber, as were stellate cells of the lower chamber, proliferating over most of the surface area of the lower chamber. At this stage, the electrical resistance of the EC barrier can be measured, but it takes several days before the resistance rises when cells need to reach confluence and then form tight junctions. The medium in both the upper and lower chambers was replaced with fresh AGM every 7 days and the electrical resistance was monitored over time. High levels of electrical resistance are obtained at least 2 weeks after culture. These cultures were maintained for up to 8 weeks until any reduction in electrical resistance was observed.

(C) Measurement of Endothelial Cell Barrier Electrical Resistance Measurement of electrical resistance between tissue culture insert membranes was performed under sterile conditions to culture endothelial cells in a good model formation of the blood brain barrier in the insert. The progress of things can be monitored. Use of an Endomem chamber electrode for a 24-well insert with its concentric electrodes located above and just below the membrane reduces background resistance between 20 and 30 Ω for an empty 8 μm tissue culture insert. This background value was measured in each case and subtracted from the actual reading obtained with the insert containing EC.
Before measuring any resistance, the endome electrode was sterilized with 70% ethanol. The cup-shaped electrode was filled with 70% ethanol, and the upper electrode was returned to a predetermined position. The ethanol was then tilted to empty and drained from the electrode. Next, 70%
Rinse in sterile Astrocyte Growth Medium (AGM) 2-3 times to remove any final traces of ethanol. 1 ml of fresh AGM was added to the lower chamber of the electrode and the electrode was ready for resistance measurement of any inserts. The resistance value of the empty insert was measured at the same time as the EC containing insert and was subtracted from all barrier insert resistance values. To obtain a standardized resistance measurement, the resistance value recorded on the volt / ohmmeter was divided by the surface area of the membrane (this was 0.3 cm ² for the 24-well size insert used herein). ), And the value measured in ohms / cm ² . The resistance value was generally in the range of 500 to 600 ohm / cm ² .

Example-Selection of antibody-expressing phage across the in vitro blood-brain barrier Antibody repertoire A large single chain Fv library derived from lymphoid tissues including tonsils, bone marrow and peripheral blood lymphocytes was used. .

Polyadenylated RNA was labeled with “Quickprep mRN”.
A kit ”(Pharmacia) was used to prepare B-cells of various lymphoid tissues of 43 non-immunized donors. First line cDNA was synthesized from mRNA using the "First Line cDNA Synthesis" kit (Pharmacia) which uses random hexamers for the major synthesis. As previously described (Marks et al.,
(1991) J. Mol. Biol. 222: 581-597) V-genes are amplified using line specific primers for the VHVλ and Vκ genes and then:
It was recombined with (Gly ₄ , Ser) ₃ scFv by PCR assembly. The VH-linker-VL antibody construct was added to the phagemid vector, SFi of pCantab6.
It was cloned into the I and NotI sites. Ligation, electroporation and cell plating out were as previously described (Marks, supra).
et al,). By increasing the amount of vector and insert used and performing the multi-electroporation method, a library was generated that was approximately 1000-fold larger than previously described. It is calculated to have approximately 1.3 × ^{10 10} individual recombinants shown to be highly diversified by the BstNI fingerprint sc.
An Fv repertoire was produced.

Induction of Phage Antibody Libraries The phage antibody repertoire described above was selected for antibodies that cross the blood brain barrier. The scFv repertoire was processed as follows to release the phagemid particles. 500 ml preheated (37 ° C) 2YTAG (100 μg in a 2 l Erlenmeyer flask)
/ Ml ampicillin and 2% glucose in 2YT medium) was inoculated with approximately 3x10 ¹⁰ cells from a glycerol-preserved (-70 ° C) culture of the library. The culture was aerated at 37 ° C with good aeration until the OD _600m reached 0.7 (approximately 2
Time) allowed to grow. M13K07 helper phage (gene layer)
moi) was added to the culture up to about 10 (assuming _{600 m of} 1 corresponds to 5 × 10 ⁸ cells per ml of culture). The culture was rested at 37 ° C. for 15 minutes and then incubated at the same temperature with light aeration (200 rpm). The culture was centrifuged and the supernatant was drained from the cell pellet. 500 ml 2YTAK (10
2Y supplemented with 0 μg / ml ampicillin and 50 μg / ml kanamycin
T medium) and the cultures were grown overnight at 30 ° C. with good aeration (300 rpm).

Phage particles were purified and concentrated by a single polyethylene glycol (PEG) precipitation method (Sambrook, J., Fritsch, EF, & M.
aniatis, T .; (1990) Molecular Cloning-A
Laboratory Manual Cold Spring Harbor
, New York) 10 mM Tris containing 1 mM EDTA (Tri).
s) resuspended in 9 ml (TE). 4.0 g of CsCl was added to the phage stock and mixed gently to dissolve. A 11.5 ml ultracentrifuge tube was filled with phage and centrifuged at 40,000 rpm for 24 hours at 25 ° C. The ultracentrifugation was stopped without braking and the clear opalescent phages were collected using a Pasteur pipette. Phages were dialyzed overnight at 4 ° C against 1 liter of TE2 exchange, titrated and stored at 4 ° C.

Phage Selection from Large Phage Libraries Crossing the In Vitro Blood-Brain Barrier Set in Test Chamber Slides The in vitro BBB test tank is a means for functionally selecting phage antibodies that cross the in vitro barrier. Provide a valuable system that can be used. The phage antibody library can be placed in the top chamber of the in vitro barrier, then the phage that can cross the barrier and reach the bottom reservoir can be collected and the process repeated. This is the basis for making the following choices.

(I) First round selection 1 × 10 ¹² library phage that do not bind rat endothelial cells in 50 μl TE, ie control phage, are grown as a monolayer in the presence of astrocytes as described in Example 1. Added to the top reservoir of an endothelial culture test tank containing rat brain endothelial cells. The test vessels were cultivated either in a CO ₂ incubator at 37 ° C or on a bench on ice. Collect the medium existing in the lower storage and place the library phage in the top chamber (
Fresh media was replaced at the appropriate temperature 30, 60 and 120 minutes after addition to wells 1a, b, c and 7a, b, c). Transport to other rooms is 30, 60 or 120
Allowed for consecutive minutes, media was collected only at the final time point. The number of phage present in the medium in the lower chamber (ie the number of phage transported) was titrated. 0.5 ml of medium recovered from the bottom chamber (out of 1 ml total) was added to a 5 ml culture of E. coli TG1 growing exponentially with light aeration in 2TY culture for 1 hour at 37 ° C. By doing so, titration was achieved. Infected T
G1s was plated on 2TYAG medium in a 243 mm x 243 mm dish. Infected TG1s dilutions were also plated out and incubated overnight at 30 ° C. The number of colonies gives the phage output titer.

Table 1 shows the results of the titration experiment from the first selection. The average initial resistance value was 606 Ωcm ² .

[0114]   The phage population from hole 7a was selected as the input population for the second round of selection.

(Ii) Second Selection The colonies were scraped from a 7a243mm x 243mm plate into 3ml of 2TY culture and 15% (v / v) glycerol was added for storage at -70 ° C.
The glycerol stock solution from the first round of selection was released with helper phage to induce phagemid particles for the second round of selection. 250 μl of 7a glycerol stock
Was used to inoculate 50 ml of 2YTAG broth and incubated in a 250 ml Erlenmeyer flask (about 2 hours) at 37 ° C. until OD ₆₀₀ nM reached 0.7 with good aeration. M13K07 helper phage (moi = 10) was added to the culture, which was then incubated gently at 37 ° C. for 15 minutes, then at the same temperature for 45 minutes with light aeration (200 rpm). The culture was centrifuged and the supernatant removed from the cell pellet. Resuspend cells in 50 ml preheated 2YTAK,
The culture was grown overnight at 30 ° C. with good aeration. Phage particles were purified by PEG precipitation (Sambrook et al., 1990), concentrated, resuspended in TE to 10 ¹³ tu / ml, and then cesium grouped as described in Example 2.

Library phage, pCantab6 phage (this is the same library except that it has only fungi and its fungal tag is fused with the gene III protein and does not contain an scFv) and 2nd round 7a. 50μ of phage
1 × 10 ¹² phage per chamber in 1TE was added to the top reservoir of the in vitro blood brain barrier culture test tank. The test tank was incubated at 37 ° C. in a CO ₂ incubator. The lower reservoir was removed in 30 minutes and 500 μl of this was used to replace the top reservoir of fresh barrier culture. After a further 30 minutes, the lower reservoir of this barrier culture was removed and medium 50
0 μl was used to replace the top reservoir of new barrier culture. After another 30 minutes, this was repeated as the final dose.

The colonies were plated out and titrated as above. The results of the titration experiment from the second selection are shown in Table 2.

[0118]   Population 24 was selected as the starting population for the third round of selection.

(Iii) Third Selection The population 24 from the second selection plan was released and cesium clustered as described above for the 7a second population. Library phage, pCANTAB6 phage, population 7a and population 24 phage, 1 × 1 per chamber in 50 μl TE.
0 ¹² phage was added to the top reservoir of the pre-cold barrier culture. The test tank was incubated on ice for 30 minutes and washed 3 times with 0.5 ml of ice-cold medium. The insert and liquid in the upper chamber were then transferred to a new chamber well containing 1 ml of medium at 37 ° C and the test tank was incubated at 37 ° C in a CO ₂ incubator. The lower reservoir was removed in 30 minutes and 500 μl of this was used to replace the top reservoir of fresh barrier culture. 3 more
After 0 minutes, the lower reservoir of this barrier culture was removed and 500 μl of medium was used to replace the top reservoir of a new barrier culture. This was repeated after another 30 minutes as the last one and the barrier culture was incubated at 37 ° C. for 30 minutes.

[0120]   Colonies were plated out and titrated as above (Table 3).

Propagation of Single Selected Clones for Immunoassay 100 with individual colonies from the third round selection (test tanks 25, 26 and 29).
μl2YTAG was inoculated into individual wells of a 96-well tissue culture plate (Corning). The plates were incubated overnight at 30 ° C. with gentle shaking (200 rpm). Up to 15% glycerol was added to each well and these master plates were stored until ready for analysis.

Example 3-Characterization of Selected Antibodies by Endothelial Cell ELISA and Sequencing Endothelial Cell Phage ELISA Phage ELISA analyzed selected phages for their ability to bind cultured rat endothelial cells. Phage ELISAs were performed as follows:
Individual clones were picked into 96-well tissue culture plates containing 100 μl 2YTAG. The plates were incubated at 37 ° C for 6 hours. M13K07 helper phage was added to each well up to moi 10 and incubated at 37 ° C. for 45 minutes with gentle shaking. The plate was centrifuged at 2000 rpm for 10 minutes and the supernatant was removed. Cell pellet with kanamycin (50 μg / ml) 1
Resuspended in 00 μl 2TYA and incubated at 30 ° C. overnight. Each plate was centrifuged at 2000 rpm and 100 μl phage containing supernatant from each well was collected and gently blocked in 20 μl 18% Marvel containing 6xPBS for 1 hour at room temperature. On the other hand, 96-well tissue culture plates containing either 1 × 10 ⁵ endothelial cells per well or control uncoated plates were gently blocked in PBS containing 3% Marvel (3MPBS) for 2 hours at room temperature. The plates were then washed 3 times with PBS and 50 μl of preblocking phage was added to each well. The plate was incubated gently at room temperature for 1 hour, then the phage solution was flushed out and discarded. The plates were washed by incubation for 2 minutes in 3 changes of PBS at room temperature.

To the ELISA plate wells, add 100 μl of anti-gene 8-HRP conjugate (Pharmacia) in 3 MPBS at a ratio of 1 to 5000 dilutions and gently plate at room temperature for 1 hour. Cultured. Each plate was washed as described. Next, 100 μl of TMB substrate is added to each well, and about 30 at room temperature.
Incubation was carried out for a minute, after which the color reaction was stopped by the addition of 50 μl of 1 MH ₂ SO ₄ . The absorption signal generated by each clone was evaluated by measuring the optical density at 405 nm using a microtiter plate reader.
Clones were selected for further analysis if the ELISA signal generated on the cell-coated plates was at least twice that of the uncoated plates. Twenty of the 96 clones selected from the third round of selection were positive by the endothelial cell ELISA.

Sequencing of Anti-Endothelial Cell ScFv Antibodies The nucleotide sequence of antibody-binding rat endothelial cells was determined by first amplifying the insert DNA from each clone with vector specific primers.
Cells from individual colonies on 2YTAG agar plates were treated with the polymerase chain reaction (P) of the inserted DNA using primers pUC19 reverse and fdtsetseq.
CR) used as a template for amplification. Amplification conditions: 94 ° C for 1 minute, 55 ° C
It consisted of 30 cycles of 1 minute and 2 minutes at 72 ° C, followed by 10 minutes at 72 ° C. The PCR product was purified using the PCR Cleanup Kit (Promega) and placed in a final volume of 50 μl H ₂ O. Tuck each insert preparation between 2-5 μl
Die-terminator cycle sequencing system (Taq Dye
-Terminator cycle sequencing system)
Used as a template for sequencing using (Applied Biosystems). The primers pUC19 reverse and fdtetseq were used to sequence the heavy and light chains respectively of each clone. CDR3s and germline diversity are shown in Table 4. Detailed sequence data is included below.

Example 4-Demonstration of clonal phage transport A 4-phage antibody clone giving positive endothelial cell ELISA results and specific staining of endothelium by ICC on rat brain is crossed as an clonal preparation across the in vitro blood-brain barrier. Tested for their ability. Phages were prepared by release and cesium grouping as described in Example 2. 1 × 10 ⁸ phage in 50 μl TE was added to the upper reservoir of in vitro blood-brain barrier test tank culture pre-chilled on ice. The phages were left to bind to the endothelial cell layer on ice for 30 minutes, then the cell layer was washed 3 times with 1 ml ice-cold medium. The test tank inserts were transferred to medium preheated to 37 ° C. and incubated at 37 ° C. in a CO ₂ incubator to allow transport to proceed. After 30 minutes on ice and then 30 minutes at 37 ° C, phage across the barrier were titrated as described above (Table 5).

In the case of 4 test clones (G65, G73, G77 and G93), 4
After 30 minutes at 37 ° C, the number of phages actively crossing the barrier was greatly increased compared to the number of phages nonspecifically crossing at 30 ° C. G65 shows a 12-fold increase over background transport (3
Number of phages passed at 7 ° C / number of phages passed at 4 ° C), G73 increased 2.6 times,
G77 showed a 12.8-fold increase and G93 a 4.3-fold increase. Surface scFv
The control phage pCantab6, which does not express at all, is 37 ° C against transport at 4 ° C.
No increase in shipping was seen.

Example 5 Rat Brain Immunocytochemistry of Endothelial Cell-Binding Antibody Clones Identified as Binding to Activated Rat Endothelial Cells by Cellular ELISA 20
Clones were also tested by immunocytochemistry (ICC) for their ability to bind to inflamed rat brain parts.

Preparation of Rat Brain Part 3 week old Lewis rats were anesthetized with avertein (1 ml / 100 g) and placed in a stereotaxic framework. 1 μl of interleukin-1β (10 units) was injected into the striatum. The scalp was incised to expose the bone and a 2 mm diameter scored hole was drilled through the skull to allow the insertion of finely calibrated glass capillary tips. Rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 100 ml of saline followed by 200 ml of Karnofsky fixative (1.25% glutaraldehyde and 1.25% paraformaldehyde in phosphate buffer). The animals were killed after waking up from anesthesia. Brain removed and 3 at 4 ° C overnight
After freezing in 0% sucrose, Tissue-Tek (Miles In
c, Elkhart, USA) and snap frozen in liquid nitrogen.

Preparation of Phages for ICC Phage clones were inoculated into 1 ml 2TYGA in deep well microtiter plates and grown at 37 ° C for 5 hours with aeration. M13K0
7 helper phages were added to each well with moi 10 and 3 with gentle shaking
Incubated at 7 ° C for 45 minutes. The plate was centrifuged at 2000 rpm for 10 minutes and the supernatant was removed. Resuspend the cell pellet in 1 ml 2TYKA,
Incubate overnight at 25 ° C. The plates were centrifuged at 2000 rpm for 10 minutes and the phage supernatant collected from each well for use in ICC.

ICC Protocol Rat brain sections were established by immersion in acetone for 15 minutes at room temperature, P
Washed twice in BST for 3 minutes. Sections were encapsulated in 5 μg / ml streptavidin in PBST, washed 3 times in PBST for 3 minutes, PBST
Incubated in 10 μg / ml biotin for 15 minutes. The cross section was washed 5 times for 3 minutes in PBST, and then 10 minutes for washing containing 1% BSA was performed twice. 1% BSA
Was added to the phage supernatant and the phage were incubated on the cross section for 2 hours at room temperature.
The slides were washed 5 times in PBST for 3 minutes and 1 / in PBST containing BSA
Cultures were performed with anti-M13-HRP conjugate (Pharmacia) diluted to 500. The sections were washed 5 times in PBST for 3 minutes and then a biotintyramine amplification step was performed. Biotintyramine amplification is 0.0 at room temperature for 10 minutes.
Consists of culturing the sections with biotintyramine diluted 1/600 in 50 mM Tris-HCl, pH 7.4 containing 3% hydrogen peroxide, after which the slides were PBS.
Wash 3 times in T for 3 minutes. Next, a cross section of SAB diluted in PBST
Incubated in C-HRP complex (DAKO KO377) for 30 minutes, then washed 5 times in PBST for 3 minutes. 2.4 mg in dimethylformamide
The cross section was stained with 3-amino-9-ethyl-carbazole (AEC, Sigma) at a concentration of / ml. ACE incubation was carried out for 3 minutes and then washed in 0.1% Tween. This was repeated twice. The slides were further washed twice in 0.1% Tween for 5 minutes and then the slides were counterstained with hemotoxylin (DAKO) for 10 seconds.
Next, 7 changes of washing in water for 3 minutes were performed, and the cross section was covered with an aqueous slide base.

Summary of Results Eighteen of the 20 ELISA positive clones exhibited staining of the blood vessel wall in rat brain sections. The profiles differed in terms of staining intensity. No staining was found in tissues other than blood vessels. The control section, in which the phage antibody was deleted, showed no specific staining of rat brain.

Example 6 I of Rat Endothelial-Binding Phage Antibody Clones on a Panel of Human Tissue
CC Cross-Reactivity Testing Rat endothelial cell clones that were positive by ICC on rat brain were tested for cross reactivity in a range of normal and diseased human tissues.

Phage and ICC Preparation Phage were prepared for ICC as described in Example 5. The panel of human tissues prepared for the ICC consisted of: normal tissue disease tissue tonsils mammary adenocarcinoma kidney liver cirrhosis cerebellar lung cancer spinal cord Crohn's disease peripheral nerve ulcerative colitis cerebral rhabdomus testis skin spleen lung chest chest liver heart uterine muscle. layer

ICC Results ICC was performed exactly as described in Example 5. Six of the 18 clones tested (G65, G77, G102, G81, G101, G112) had no specific staining on the panel of human tissues.

Five clones showed staining in the cerebellum: G73 was cerebellar-specific; G11.
0 stained cerebellum plus adenocarcinoma and striated muscle; G95 stained cerebellum plus smooth muscle; G93 stained cerebellum plus spinal cord; G92 stained cerebellum plus spinal cord and cerebrum did.

[0136]   G76 stained cell bodies in peripheral nerves and spinal cord.

Of the remaining clones, many other staining patterns were observed. G88 exhibited a staining characteristic of lung cancer. G83 stained renal intraglomerular cells and testicular Leydig cells. G92 showed staining of Leydig cells of the testis, plus other punctate staining of the testis, and some staining of striated muscle. The ICC staining pattern is summarized in Table 6.

Example 7-Selection of SAP-Binding Antibodies Serum amyloid protein (SAP) is specifically transported through the blood brain barrier. A phage antibody that binds to human SAP (hSAP) was isolated as outlined below, and a phage antibody clone that gave a strong signal on hSAP by ELISA (
D5) was tested for its ability to be transported across the BBB in vitro.

Isolation of Phage That Bind to hSAP hSAP was coated on the surface of Maxisorp tubes with 1 ml of 10 μg / ml in PBS overnight at 4 ° C. After coating, the Maxisorp tube was rinsed 3 times with PBS to give 3% (w / v) in PBS (3MPBS).
) Overfilled with skim milk and capped at 37 ° C. for 1 hour. Library phage were blocked with 3M PBS for 1 hour at room temperature. The coated maxisorp tube was rinsed 3 times with PBS, and 1 ml of phage before sealing was added to the maxisorp tube. The phage were incubated at 37 ° C for 1 hour, then the tubes were PBST.
Rinse 20 times with PBS, then 20 times with PBS. Bound phages were eluted in Maxisorp tubes by addition of 1 ml of freshly made 100 mM triethylamine. The tubes were incubated at room temperature for 10 minutes (quiet) and the eluted phages were transferred to 1.5 ml microcentrifuge tubes. Phages were neutralized by the addition of 500 μl of 1 M Tris buffer pH 7.4. A single colony of E. coli TG1 taken from a minimal agar plate was used to inoculate 50 ml of 2TY culture and A. ₆₀₀ 1.
Incubated until 0 was achieved. 50 ml of 5 ml exponentially growing TG1
Infection was performed by placing in a Falcon tube, adding 750 μl (half) of the eluted phage, and incubating for 30 minutes at 37 ° C. gently and for 30 minutes with shaking (<200 rpm). The rest of the eluted phage was stored as a reserve at 4 ° C. The infected cells were placed at 3500 r.p. p. m. Centrifuge at 10 min, resuspend the cell pellet in 0.6 ml of 2TY medium, and use a single 243 x 243 mm 2TYA
A thin coat was applied on a G agar plate. The colonies were grown overnight at 30 ° C.

Scrape the colony into 10 ml of 2TY culture in a 50 ml Falcon tube and scrape 5
ml of sterile 50% v / v glycerol was added and mixed by placing the tube on an end-over-end rotator for 10 minutes at room temperature. 25 ml of 2TY
Inoculate G + 100 μg / ml ampicillin with about 50 μl of the above plate scraps,
Grow at 37 ° C. to an A ₆₀₀ of 0.5-1.0. M13K07 helper phage was added to a final concentration of 5x10 ⁸ pfu / ml and the cells were gently incubated at 37 ° C for 30
Infection for 30 minutes and for 30 minutes with shaking (<200 rpm). Transfer cells to 50 ml Falcon tubes and transfer to 3500 r. p. m. Centrifugation at 10 minutes, then the bacterial pellet was resuspended in 25 ml of preheated 2TY with kanamycin (50 μg / ml) and ampicillin (100 μg / ml). This was transferred to a new 250 ml flask and shaken rapidly at 30 ° C (300 r.p.m.).
p. m. ) Was grown overnight to produce phage particles. About 1 ml
Culture was transferred to a 1.5 ml microcentrifuge tube and placed in a microcentrifuge at 13000 r.p.m. p. m. It was centrifuged at. The phage-containing supernatant was transferred to a new tube and used as the input population for the second round selection performed exactly as described above.

Selection of Selected Clones by Phage ELISA on hSAP Individual colonies were picked in 100 μl of 2TYAG in 96-well microtiter plates and stained with 100 r. p. m. Grow overnight at 30 ° C with shaking. The culture in this plate was used to inoculate a new plate, 50 μl of 50%
v / v Glycerol was added to the original plate and it was stored frozen at -70 ° C.
Fresh plates were grown at 37 ° C for 5-6 hours until the culture became cloudy. In each well of the replica plate, 10μ in 2TYG + 100μg / ml ampicillin
1 of M13K07 (5 × ^{10 10} pfu / ml, moi ¹⁰ ) was added. The plate was shaken for 30 minutes at 37 ° C without shaking and then with shaking (100
r. p. m. ) Incubation at 37 ° C. for 30 minutes allowed superinfection of helper phage. Pellet the culture at 2,000 rpm for 10 minutes,
The supernatant was discarded. Resuspend the bacterial pellet in 100 μl 2TYAK,
Grow overnight at 30 ° C. with shaking at 100 rpm.

The culture was centrifuged at 2,000 rpm for 10 minutes and the supernatant transferred to a new plate. Phage were blocked by the addition of 20 μl of 6 × PBS / 18% Marvell, mixed by pipette and incubated for 1 hour at room temperature.

Antigen plates were prepared by adding 100 μl of 10 μg / ml hSAP in PBS to microtiter plate wells and incubating overnight at 4 ° C.
The plates were washed 3 times in PBS and blocked in 3% MPBS at 37 ° C for 1 hour. Phage ELISAs were performed as described in Example 3.

About 30% of the phage antibody clones selected by ELISA were found to be positive in terms of binding to hSAP compared to BSA or uncoated ELISA plates. One clone, D5, which gave the highest ELISA signal, was selected for further transport studies. Clone D5 was sequenced (as described in Example 3). Related sequences include (VH region SEQ
ID NO. SEQ ID NO. 73; VL area SEQ
ID NO. SEQ ID NO. 75).

Example 8: Characterization of SAP-binding phage antibody D5 based on its ability to cross the BBB in vitro in the presence or absence of SAP No serum to avoid competition between any endogenous SAP and added hSAP Experiments were carried out in the medium. Phage of either D5 or pCANTAB6 were added to the top reservoir of ice-cold BBB cultures in the presence or absence of 10 μg / ml hSAP at 10 ⁸ , 10 ⁶ , or 10 ⁴ phage per chamber. Culture 3 on ice
Incubate for 0 minutes, then remove liquid in lower reservoir for titration. The upper chamber was washed 3 times with 0.5 ml of ice-cold medium, then transferred to a new well and incubated at 37 ° C for 30
Incubate in CO ₂ incubator for minutes. The liquid from the lower reservoir was removed again and the phage titer present was determined by titration (Table 7).

Transport of D5 phage was increased approximately 2-fold in the presence of hSAP, while pCAN.
Transport of AB6 was slightly reduced in the presence of hSAP. Overall, D5 phage was 10-fold more effective than pCANTAB6 phage in the absence of SAP and
It was transported 100 times more effectively in the presence of AP. This indicates that D5 phages are actively transported and that higher levels of D5 transport in the absence of SAP may be a consequence of endogenous SAP prebinding to rat endothelial cells.

[0147]

[Table 1]

[0148]

[Table 2]

[0149]

[Table 3]

[0150]

[Table 4]

[0151]

[Table 5]

[0152]

[Table 6]

[0153]

[Table 7]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/00 A61P 25/00 4H045 25/00 25/16 25/16 25/18 25/18 25/20 25/20 25/28 25/28 35/00 35/00 C07K 14/47 C07K 14/47 16/00 16/00 19/00 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1 / 21 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 1/68 A C12Q 1/02 1/70 1/68 C12N 15/00 ZNAA 1/70 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA, ZW (72) Inventor Osborn, Jane Igiri Country, Cambridgeshire ESJ 8 6 Jay-Jay, Royston, Melborn, The Science Park, Cambridge Antibody Technology Limited (72) Inventor Ward, George United Kingdom, Oxfordshire Oex 4-5 Elwai, Oxford, Watling Road, British Biotech Pharmaceuticals Limited (72) Inventor Miller, Karen United Kingdom, Oxfordshire Ox 4 5 Elwai, Oxford, Watling Road, British Biotech Pharmaceuticals Limited F-Term (Reference) 4B024 AA01 BA61 CA04 CA07 CA09 DA02 DA06 EA02 EA03 EA04 GA11 GA18 GA19 HA03 HA12 4B063 QA01 QA05 QQ08 QQ10 QQ42 QR32 QR38 QR48 QR55 QR82 QS12 QS25 QS34 QS36 QS39 QX01 4B064 AG01 AG26 CA02 CA02 CA10 CA19 CC24 DA01 4B065 AA26X AA90X AA91X AA93Y AB01 AC14 BA01 CA24 CA44 4C085 AA13 AA14 AA33 BB11 CC22 CC23 DD62 DD63 EE01 4H045 AA10 AA20 AA30 BA10 BA41 CA40 DA75 EA21 FA74

Claims (38)

[Claims] 1. A mixture or panel of at least 5 various specific binding members each comprising an antibody VH variable region and / or an antibody VL variable region, said antibody VH variable region being shown in Table 4 (SEQ ID NO: .78, 80, 82, 8
4, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 1
06, 108, 110 and 112) VHCDR sequences and G65 (SEQ ID NO.2), G67 (SEQ ID NO.6) disclosed herein.
), G73 (SEQ ID NO.10), G76 (SEQ ID NO.14)
), G77 (SEQ ID NO.18), G78 (SEQ ID NO.22)
), G79 (SEQ ID NO.24), G81 (SEQ ID NO.30)
), G83 (SEQ ID NO.34), G85 (SEQ ID NO.38)
), G88 (SEQ ID NO.42), G92 (SEQ ID NO.46)
), G93 (SEQ ID NO.50), G95 (SEQ ID NO.54)
), G101 (SEQ ID NO.58), G102 (SEQ ID NO.
62), G110 (SEQ ID NO.66), G112 (SEQ ID N)
O. 70) and D5 (SEQ ID NO.74) VH region antibody V
The antibody VL variable region has an amino acid sequence selected from the group consisting of H variable regions and / or the antibody VL variable regions are shown in Table 4 (SEQ ID NO. 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 1
05, 107, 109 and 111) VLCDR sequences and G65 (SEQ ID NO.4), G67 (SEQ ID NO.8) disclosed herein.
), G73 (SEQ ID NO.12), G76 (SEQ ID NO.16)
), G77 (SEQ ID NO.20), G78 (SEQ ID NO.24)
), G79 (SEQ ID NO.28), G81 (SEQ ID NO.32)
), G83 (SEQ ID NO.36), G85 (SEQ ID NO.40)
), G88 (SEQ ID NO.44), G92 (SEQ ID NO.48)
), G93 (SEQ ID NO.52), G95 (SEQ ID NO.56)
), G101 (SEQ ID NO. 60), G102 (SEQ ID NO.
64), G110 (SEQ ID NO.68), G112 (SEQ ID N)
O. 72) and D5 (SEQ ID NO. 76) VL region sequence-containing antibody V
Has an amino acid sequence selected from the group consisting of L variable regions, wherein each specific binding member is displayed on the surface of a filamentous bacteriophage particle, or the specific binding member is a D5VH variable region and / or a human. The mixture or panel, which is capable of crossing the mammalian blood-brain barrier when it comprises the D5VL variable region when bound to serum amyloid protein. 2. The mixture or panel of claim 1 which comprises at least 10 said specific binding member. 3. The mixture or panel of claim 1, wherein the blood-brain barrier is the human or rat blood-brain barrier. 4. A mixture or panel according to claim 3 each consisting of 18 specific binding members each comprising a pair of antibody VH and VL variable region sequences as follows: VH region sequence VL region sequence 1) G65. (SEQ ID NO.2) G65 (SEQ ID NO.4); 2) G67 (SEQ ID NO.6) G67 (SEQ ID NO.8); 3) G73 (SEQ ID NO.10) G73 (SEQ ID NO.12); 4) G76 (SEQ ID NO.14) G76 (SEQ ID NO.16); 5) G77 (SEQ ID NO. 18) G 77 (SEQ ID NO. 20); 6) G 78 (SEQ ID NO. 22) G 78 (SEQ ID NO. 24); 7) G 79 (SEQ ID NO. 24) G 79 (SEQ ID NO. 28) ); 8) G81 (SEQ ID NO. 30) G81 (SEQIDNO.32); 9) G83 (SEQIDNO.34) G83 (SEQIDNO.36); 10) G85 (SEQIDNO.38) G85 (SEQIDNO.40); 11) G88 (SEQIDNO.42) G88 (SEQIDNO.42) .44); 12) G92 (SEQIDNO.46) G92 (SEQIDNO.48); 13) G93 (SEQIDNO.50) G93 (SEQIDNO.52); 14) G95 (SEQIDNO.54) G95 (SEQIDNO.56); ) G101 (SEQ ID NO. 58) G 101 (SEQ ID NO. 60); 16) G 102 (SEQ ID NO. 62) G 102 (SEQ ID NO. 64); 17) G 110 (SEQ ID NO. 66) G 110 (SEQ ID NO. 68); 18) G112 (SEQ ID NO.70) G112 (SEQ ID NO.72). 5. Contacting a mixture or panel of specific binding members according to any one of claims 1 to 4 and allowing them to cross the mammalian blood-brain barrier when displayed on filamentous bacteriophage particles. A method of obtaining one or more specific binding members comprising selecting one or more specific binding members of a possible mixture or panel. 6. A specific binding member in a mixture or panel is displayed on the surface of a filamentous bacteriophage particle, each containing a nucleic acid encoding the specific binding member displayed on its surface. Item 5. The method according to Item 5. 7. The method of claim 5 or claim, wherein the selection for the ability to cross the blood brain barrier is followed by one or more additional selections for the ability to bind cell types of the brain or central nervous system (CNS). The method according to 6. 8. The cell type is endothelial cells, meninges, parenchyma, choroid plexus, cerebrum,
The method according to claim 7, which is selected from cerebellum, spinal cord cells and microglial cells. 9. The method of claim 6, wherein the nucleic acid is derived from selected filamentous bacteriophage particles. 10. The nucleic acid having a nucleotide sequence within the nucleic acid derived from the selected filamentous bacteriophage particle is used for subsequent production of a specific binding member or antibody VH or VL variable region. Method. 11. The method of any of claims 5-10, comprising providing the selected specific binding member or the antibody VH or VL region of the selected specific binding member in isolated form. 12. The selected specific binding member or antibody VH or VL variable region of the selected specific binding member in isolated form is formulated in a composition comprising at least one additional component. Item 11. The method according to Item 11. 13. The selected specific binding member or the antibody VH or VL region of the selected specific binding member is provided in the form of a fusion protein with an additional amino acid according to any one of claims 5 to 12. the method of. 14. The method of claim 13, wherein the additional amino acid provides an antibody constant region. 15. A composition comprising a plurality of multispecific binding members or antibody VH or VL variable regions obtainable from the mixture or panel of any of claims 1-4. 16. A specific binding member or antibody VH or VL variable region obtainable from the mixture or panel of any of claims 1-3. 17. A specific binding member or antibody VH or VL variable region obtainable from the mixture or panel of claim 4. 18. The specific binding member according to claim 17, which comprises a G93 antibody VH region (SEQ ID NO.50) and a G93 antibody VL region (SEQ ID NO.52). 19. The specific binding member according to claim 17, which comprises a G73 antibody VH region (SEQ ID NO. 10) and a G73 antibody VL region (SEQ ID NO. 12). 20. D5 antibody VH region (SEQ ID NO. 74) and D
The specific binding member according to claim 16, which comprises a 5 antibody VL region (SEQ ID NO. 76). 21. The composition or specific binding member of any one of claims 15-20, wherein the specific binding member or antibody VH or VL variable region is in the form of a fusion with additional amino acids. 22. The composition or specific binding member of claim 21, wherein said additional amino acid provides an antibody constant region. 23. A method according to claim 15, which comprises a pharmaceutically acceptable excipient or carrier.
22. A composition comprising the specific binding member or antibody VH or VL variable region according to any one of claims 22 to 22 or a composition comprising the same. 24. An isolated nucleic acid comprising a nucleotide sequence encoding the specific binding member or antibody VH or VL region of any of claims 16-22. 25. A host cell transformed with the nucleic acid of claim 24. 26. A specific binding member or antibody VH or VL region comprising culturing the host cell of claim 25 under conditions to produce said specific binding member or antibody VH or VL region. How to produce. 27. The method of claim 26, further comprising isolating and / or purifying said specific binding member or antibody VH or VL variable region. 28. The method further comprising formulating the specific binding member or antibody VH or VL variable region into a composition comprising at least one additional component.
28. The method of claim 6 or claim 27. 29. A method of obtaining a specific binding member with desirable properties selected from the ability to cross the blood-brain barrier, specificity for brain antigens and specificity for endothelial cell antigens, as disclosed herein. G65 (SEQ ID NO.2), G67 (S
EQ ID NO. 6), G73 (SEQ ID NO.10), G76 (SE
Q ID NO. 14), G77 (SEQ ID NO.18), G78 (SE
Q ID NO. 22), G79 (SEQ ID NO. 24), G81 (SE
Q ID NO. 30), G83 (SEQ ID NO.34), G85 (SE
Q ID NO. 38), G88 (SEQ ID NO. 42), G92 (SE
Q ID NO. 46), G93 (SEQ ID NO.50), G95 (SE
Q ID NO. 54), G101 (SEQ ID NO.58), G102 (
SEQ ID NO. 62), G110 (SEQ ID NO.66), G11
2 (SEQ ID NO.70) and D5 (SEQ ID NO.74) VH
A VH region which is an amino acid sequence variant of the VH region is provided by means of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the antibody VH variable region selected from the region sequences, and the thus provided 1 The above VH region amino acid sequence variants are combined with one or more VL regions to provide one or more VH / VL combinations; and / or G65 (SEQ ID NO. 4), G67 (S) disclosed herein.
EQ ID NO. 8), G73 (SEQ ID NO.12), G76 (SE
Q ID NO. 16), G77 (SEQ ID NO.20), G78 (SE
Q ID NO. 24), G79 (SEQ ID NO. 28), G81 (SE
Q ID NO. 32), G83 (SEQ ID NO.36), G85 (SE
Q ID NO. 40), G88 (SEQ ID NO.44), G92 (SE
Q ID NO. 48), G93 (SEQ ID NO.52), G95 (SE
Q ID NO. 56), G101 (SEQ ID NO. 60), G102 (
SEQ ID NO. 64), G110 (SEQ ID NO.68), G11
2 (SEQ ID NO.72) and D5 (SEQ ID NO.76) VL
A VL region which is an amino acid sequence variant of the VL region is provided by means of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the antibody VL variable region selected from the region sequences, and thus provided Combining the above VL region amino acid sequence variants with one or more VH regions to provide one or more VH / VL region combinations;
And testing the VH / VL combination or combinations to identify a specific binding member by a desired property. 30. A method of obtaining a specific binding member with desirable properties, which encodes one or more VH regions, either comprising the CDR3 to be replaced or lacking the CDR3 coding region. And a starting amino acid sequence for VHCDR3 substantially as set forth in Table 4 (SEQ ID N
O. 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 1
00, 102, 104, 106, 108, 110 and 112) in a starting nucleic acid to provide a product nucleic acid that encodes a VH region, such that the donor nucleic acid encodes a VH region. There is provided a starting nucleic acid encoding one or more VL regions which is either inserted into the CDR3 region; or which contains the CDR3 to be replaced or lacks the CDR3 coding region, said starting nucleic acid comprising: To VL
The amino acid sequences substantially set forth in Table 4 for CDR3 (SEQ ID NO.
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109 and 111) in combination with a donor nucleic acid which results in the donor nucleic acid to provide a product nucleic acid which encodes a VL region. Inserted into; V expressing the nucleic acid of said product nucleic acid encoding the VH region, thus produced V
The H region is combined with one or more VL regions to provide a VH / VL combination and / or to express the nucleic acid of the product nucleic acid encoding the VL region, and the VL region thus produced is combined with one or more VH regions. Providing a VH / VL combination in combination with a region; selecting a specific binding member comprising a VH / VL combination having desired properties; and selecting said specific binding member having desired properties and / or a nucleic acid encoding it The method, comprising recovering. 31. Desirable properties are the ability to cross the blood-brain barrier, bind endothelial cells or other brain cell antigens, bind inflammatory moieties in brain or blood-brain barrier disruption, bind ICAM and transferrin. 31. The method of claim 30, selected from the ability to bind the receptor. 32. The method of any one of claims 29-31, further comprising formulating the specific binding member or antibody VH or VL variable region thereof in a composition comprising at least one additional component. . 33. The method of any one of claims 16-20, or any of claims 5-14, 26-32, wherein the specific binding member is an scFv antibody molecule. A specific binding member obtained by using. 34. The antibody according to claim 16, wherein the specific binding member is a whole antibody.
Specific binding member obtained by using the method according to any one of 0 to 33 or from any one of claims 5 to 14 and 26 to 32. 35. The specific binding member according to claim 34, wherein the whole antibody is an IgG4 antibody. 36. A mixture or panel of specific binding members according to any one of claims 1 to 4 against an antigen, a composition according to any one of claims 15 to 23, 33 to 35. An object or a specific binding member, or claims 5-14, 26
33. A method comprising causing or allowing binding of a specific binding member obtained using the method of any one of -32. 37. The method of claim 36, wherein the binding occurs in vitro. 38. The method according to claim 36 or 37, which comprises measuring the amount of binding of the specific binding member to the antigen.