CA2123307A1 - Process for the preparation of transferrin receptor specific antibody-neuro-pharmaceutical or diagnostic agent conjugates - Google Patents

Abstract

The present invention pertains to a method for delivering a neuropharmaceutical or diagnostic agent across the blood brain barrier to the brain of a host. The method comprises administering to the host a therapeutically effective amount of an antibody-neuropharmaceutical or diagnostic agent conjugate wherein the antibody is reactive with a transferrin receptor and the antibody is a chimera between the variable region from one animal source and the constant region from a different animal source. Other aspects of this invention include a delivery system comprising an antibody reactive with a transferrin receptor linked to a neuropharmaceutical or diganostic agent and methods for treating hosts afflicted with a disease associated with a neurological disorder. In embodiments of the present invention, the antibody that is reactive with a tranferrin receptor is a chimeric antibody. This antibody is composed of a variable region, immunologically reactive with the transferrin receptor, that is delivered from one animal source and a constant region that is derived from an animal source other than the one which provided the variable region. The chimeric antibodies of this invention can exist either as isolated entities or as conjugates with a neuropharmaceutical agent for transferal across the blood brain barrier. In the latter mode, the chimeric antibody-neuropharmaceutical agent conjugate forms a delivery system for delivering the neuropharmaceutical agent across the blood brain barrier.

Description

W O 93/10819 2 1 ~ 3 ~ 0 7 PC~/US92/10206 Process for the prepara~on of transferrin recep~or specific ant~body-neuro-pha~maceutical or d~agnost~c agent conjugates.

~escription Background : The capillaries that supply blood to the tissues of the brain constitute the ~lood bxain barrier ~Goldstein et al. tl986) Scientific American 255:7~-83; Pardridge, W~M. (1986) Endocrin. Rev.
7:314-330). The endothelial cells which form ~he ~ brain capillaries are different from those found in :~ ~ other tissues in the body. Brain capillary endo-thelial cells are joined together by tight inter-:~ cellular junctions which form a continuous wall against the passive movement of ~ubstances from the blood to the brain. These cells are also different ~: in that they have few pino~ytic vesicles which in other tissue:s allow somewhat unselective transport across the capillary wall. Also lacking are con-` tinuous gaps or channels running through the cells : which would allow unrestricted passage.

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The blood-brain barrier functions to ensure that the environment of the brain is constantly controlled. The levels of various substances in the blood, such as hormones, amino acids and ions, undergo f~equent small fluctuations which can be brought about by activities such as eating and exercise (Goldstein et al, cited supra). If the brain were not protected by the blood brain barrier from these ~ariations in serum composition, the result could be uncontrolled neural activity.
The isolation of the brain from the bloodstream is not complete. If this were the case, the brain would be unable to function properly due to a lack of nutrients and because of the need to exchange chemicals with the rest of the body. The presence of specific transport systems within the capillary endothelial cells assures that the brain receives, in a controlled manner, all of the compounds required for nor~m~al growth and function. In many instances, these transport systems consist of membrane-assoclated;receptors which, upon binding of their respective ligand~, are internalized by the cell Pardrid~e, W.~., cited supra). Vesicles containing the recept~r-ligand complex then migrate to the abluminal surface of the endothelial cell where the ligand~is rele~ased.
The problem posed by the blood-brain barrier is that, in the~process of protecting the brain, it excludes~many potentia71y useful therapeutic agents.
Presently, only substances which are sufficiently lipophilic can penetrate the blood-brain barrier ~ WO93/10819 PCT/US92/10206 ~123~07 (Goldstein et al, cited supra; Pardridge, W.M., cited supra~. Some drugs can be modified to make them more lipophilic and thereby increase their ability to cross the blood brain barrier. However, each modification has to be tested individually on each drug and the modification can alter the activity of the drug. The modification can also have a very general effect in that it will increase the ability of the compound to cross all cellular membranes, not only those of brain capillary endothelial cells.

Summarv of the Invention The present invention pertains to a method for delivering a neuropharmaceutical or diagnostic agent across the blood brain barrier to the brain of a host~ The method comprises administering to the host a therapeutically effective amount of an antibody-":
neuropharmaceu~ical or diagnostic agent conjugatewherein the antibody is reactive with a transferrin receptor and the antibody is a chimera between the ~ariable r gion from one animal source and the constant region ~rom:a different animal source. The con~u~ate is administered under conditions whereby binding of the ant~ibody to a transferrin receptor on a brain capillary~endothelial cell occurs and the neuropharmaceutical agent is transferred across the blood brain barrier in a pharmaceutically active form. Other aspects of this invention inciude a delivery system comprising an antibody reactive with a transferrin receptor linked to a neur:opharmaceutical agent and methods for treating hosts afflicted with a disease associated with a neurolog~cal disorder.

~4 Xn embodiments of the present invention, the antibody that is reactive with a transferrin receptor is a chimeric antibody. This antibody is composed of a variable region, immunologically reactive with the transferrin receptor, that is derived fro~ one animal source and a constant region that is derived from an animal source other than the one which provided the variable region. The chimeric antibodies of this invention can exist either as isolated entities or as conjugates with a neuropharmaceutical agent for transferal across the blood brain barrier. In the latter mode, the chimeric antibody-neuropharmaceut-ical agent conjugate forms a delivery system for ;delivering the neuropharmaceutical agent across the blood brain bar~ier.
Presently available means for delivering therapeutic agents~to the brain are llmited in that they a~e invasive~ The delivery system of the present invention~is non-invasive and can utilize readily available;antibodies reactive with a trans-ferrin receptor as carriers for neuropharmaceutical agénts. The delivery system is advantaqeous in that the~antibodies are capable of transporting neuropharmaceutical~agents across;~the blood brain barrier without being susceptible to premature release~of the~neuropharmaceutical agent prior to reaching the brain-side of the blood brain barrier.
Further, if the therapeutic activity of the agent to be~delivered t~o the brain is not altered by the addition of a linker~, a noncleavable linker can be :
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``~ WO93~10819 P~T/US92/102~6 2123~07 used to link the neuropharmaceutical agent to the antibody.

Description of the Drawings . Figure 1 is a graphic representation of rat brain uptake of 14C-labelled murine monoclonal antibody ~OX-26) to rat transferrin receptor in rats where the percent injected dose of radiolabelled antibody per brain and per 55 ~1 of blood is plotted versus time post-injection.
Figure 2 is a histogram illustrating time dependent changes in the disposition of radiolabelled OX~26 between brain parenchyma and vasculature.
Figure 3 is a histogram illustrating the enhanced delivery of methotrexate across the blood-brain barrier when administered as a conjugate with OX-26.
Fi-gure 4 illustrates in three histograms (A,B
and C) the distribution in the brain of both the :~
antibody and the AZT components of an OX-26-AZT
:~ conjugate.
Figuxe 5 is a histo~ram illustrating the experimental results of delivery of a protein~
horseradish peroxidase, across the blood-brain : barrier in rat brains in the form of a conjugate with OX-26. ~ ~
Figure 6 is a histogram illustrating the experimental results of delivering soluble CD4 to rat : brain parenchyma using CD4 in thP form of a conjuga~e with OX-26.

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WO93/10819 212 ~ 3 0 7 PCT/US92/102r Figure 7 is a histogram illustrating the biodistri~ution of antibody 128.l and control IgG in a cynomolgous monkey~
~ igure 8 is a flow diagram of the gener~l strateg~ for the expression of immunoglobulin variable region genes obtained by PCR.
Figure 9 illustrates the primers used for : variable region amplification, ~oth for first cloning and sequencing the V region and then for cloning into the final expression vector.
Figure lO iIlustrates the cloning of the 128.l heavy chain variable region.
Figure ll is the antibody coding sequence of - : h~avy chain expression vector pAH4602 containing the l isotype constant:region.
: Figure 12 illustrates the cloning of the 128.1 light ~hain variable region.
~;~ Figure 13 is the antibody coding sequence of light chain:expression vector pAG46ll.
: Figure 14 i11ustrates the plasmid map of the heavy chain expression vector pAH4625 containing the

2 i50type.
Flgure lS;illustrates the plasmid map of the heavy chain expression vector pAH4807 containing the ~ ~ ,

3 isotype. ~ ~
Figure 16~ lustrates the plasmid map of the heavy chain~ expression vector pAH4808 containing the

-4 isotype.
: Figure~17 is~the antibody coding sequence of heavy chain expression vector pAH4625 containing th~
~ 7-2 isotype constant region.

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, t W093/10819 PCT/US92/10206 Figure 18 is the antibody coding sequence of heavy chain expression vector pAH4807 containing the ~-3 isotype constant region.
Figure 1~ is the antibody coding sequence of heavy chain expression vector pAH4808 containing the ~-4 isotype constant region.

Detailed Descr ption ; The method for delivering a neuropharmaceutical agent across the blood brain barrier to the brain of a host comprises administering to the host a therapeutically effective amount of an antibody-neuropharmaceutical agent conjugate wherein the antibody is reactive with a transferrin receptor present on a brain capillary endothelial cell. The method is conducted under conditions whereby the antibody binds to~the transferrin receptor on the brain capillary endothelial cell and the neuropharm-aceutical agent is transferred across the blood brain barrier in a pharmaceutically active form.
The host can be an animal susceptible to a neurological disorder (i.e., an animal haviny a brain). Examples of hosts include mammals such as ; ~ humans, domestic animals (e.g., dog, cat, cow or horse),~mice and rats.
The neuroph~rmaceutical agent can be an agent ha~ing a therapeutic or prophylactic effect on a nPurological disorder or any condition which affects biological functioning of the central nervous system.
Examples of neurological disorders include cancer .

2123~7 (e.g. brain tumors), Autoimmune Deficiency Syndrome (AIDS), stroke, epilepsy, Parkinson's disease, multiple sclerosis, neurodegenerative disease, trauma, depression, Alzheimer's disease, migraine, pain, or a seizure disorder. Classes of neuropharmaceutical agents which can be used in this invention include proteins, antibiotics, adrenergic agents, anticonvulsants, small molecules, nucleotide analogs, chemotherapeutic agents, anti-trauma agents, peptides and other classes of agents used to treat or prevent a neurological disorder. Examples of proteins include CD4 (including soluble portions thereof), growth factors (e.g. nerve growth factor and interferon), dopamine decarboxylase and tricosanthin. Examples of antibiotics include amphotericin B, gentamycin sulfate, and pyrimethamine. Examples of adrenergic agents (including blockers) include dopamine and atenolol.
Examples of chemotherapeutic agents include adriam~cin, methotrexate, cyclophosphamide, etoposide, and carboplatin. An example of an anticonvulsant which can be used is valproate and an anti-trauma àgent which can be used is superoxide dismutase. Examples of peptides would be somatosta~in analogues and enkephalinase inhibitors. Nucleotide analogs which can~be used include azido thy~idine (hereinafter AZT), dideoxy Inosine (ddI) and dideoxy cytodine (ddc).
The antibody, which is reactive with a transferrin receptor present on a brain capillary ~ ` endothelial cell,~ may also be conjugated to a : : :

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diagnostic agent. In this method and delivery system, the neuropharmaceutical agent of the neuropharmaceutical agent - anti-transferrin receptor con3ugate has been replaced with a diagnostic agent.
The diagnostic agent is then delivered across the blood brain barrier to the brain of the host. The diagnostic agent is then detected as indicative of the presence of a physiological condition for which the diagnostic agent is intended. For example, the diagnostic agent may be an antibody to amyloid plaques. When conjug~ted`to an antibody reactive with a transferrin receptor present on a brain capillary endothelial cell, this diagnostic agent an~ibody can bé transferred across the blood brain barrier and can then subsequently immunoreact with amyloid plaques. Such an immunoreaction is indicative of Alæheimer's Disease.
Serum transferrin is a monomeric glycaprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports it to the various tis~sues~Aisen et al. ~1980) Ann. Rev.
Biochem. 49:357-39~;~ MacGillivray et al. (1981) J.
Bi~l~ Chem. 258:3543-3553). The uptake of iron by in~ividual:cells ~is~mediated by the transferrin re~ceptor, an lntegral membrane glycoprotein consisting of two identical 95,000 dalton subunits that are linked~by a: disulfide bond. The number of receptors on the surface of a cell appears to co~relate with cellular proliferation, with the : ~ :
highest number being:on actively growing cells and the lowest being~on resting and terminally i ~

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WO93/10819 212 3 3 0 7 PCT/US92/l02' differentiated cells. Jeffries et al (Nature Vol.
312 (November 1984) pp. 167-168) used monoclonal antibodies to show that brain capillary endothelial cells have a high density of transferrin receptors on their cell surface.
Antibodies which can be used within this invention are reacti~e with a transferrin receptor.
The term antibody is intended to encompass both polyclonal and monoclonal antibodies. The preferred antibody is a monoclonal antibody reactive with a transferrin receptor. The term antibody is also intended to encompass mixtures of more than one antibody reactive with a transferrin receptor (e.g., a cocktail of different types of monoclonal antibodies reactive with a transferrin receptor).
The term antibody is further intended to encompass whole antibodies, biologically functional fragments thereof, and chimeric antibodies comprising portions from more than one species, bifunctional antibodies, .
etc. Biologically functional antibody fragments which can be used are those ~ragments sufficient for binding of the antibody fragment to the transferrin receptor to occur.
The an~ibodies,~chimeric or otherwise, are not to be considered as being restricted to a specific isotype. Any of the antibody isotypes are within the present invention. For example, antibodies with identical light ;ch~ins but different heavy chains are intended. In addition, mutations of certain regions of the antibodies, e.g., in the ~ chains, are also intended. These mutations, particularly point ~^~ WO93/10~19 PCT/US92/1020~
21~3307 mutations, may occur anywhere provided functionality of the antibodies as reactive with a transferrin receptor is still maintained.
- The chimeric antibodies can comprise portions derived from two different species (e.g., human constan~ region and murine variable or binding region). The portions derived from two different species can be joined together chemically by conventional techniques or can be prepared as single csntiguous proteins using genetic engineering techniques. DNA encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody can be expressed as contiguous proteins.
One genetic engineering approach that can be used to produce or clone chimeric antibodies reactive with a transferrin receptor is to prime the DNAs encoding the variable region of functional antibodies for amplification by PCR using specific oligonucleotides~ The variable region of functional antibodles is~that portion of the antibody that immunologically reacts with the transferrin receptor antigen. Both the~heavy~chain and light chain of antibodies contribute to the variable region~ Thus, the DNA encoding the variable region has two , portions: a polynucleotide sequence encoding the variable region heavy chain and a polynucleotide sequence encoding the variable region light chain.
The primed variable regions can then be cloned into vectors which contain the DNA encoding the constant region of antibodies. A particularly useful vector is one which contalns DNA encoding the constant ; ~
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W0~3t1~819 2 12 3 3 0 7 PCT/US92/10~

region of human antibodies that has been designed to also express immunoglobulin variable regions from other sources. The DNA encoding the constant region is usually from a separate source than ~he one whose DNA encodes the variable region. Although different animals from the same spe~ies may be the sources of the DNA encoding the variable region and the constant region, the usual situation is where the animal species are dif~erent (e.g., human constant region and murine variable region). Following the cloning of the primed variable regions into vectors containing the constant region, chimeric antibodies can be expressed from such vectors.
A general strategy that can be used to amplify immunoglo~ulin variable regions has be~n previously described (~r1andi et al., Proc. Natl. Acad. Sci., 86: 3~33-3837 (1989); Larrick et al., Bio/kechnology, 7: 934-938 (19~9); Gavilondo et al., ~ybridoma, 9~5):
407-4l7 (l990)). Two approaches have been used in the general strategy. In one approach, 5' primers are designed to~pr1me the first framework region of the variable re~ion. The 3' primers are designed to prime either the J region or the constant region.
Priming in the frameworks (Orlandij takes advantage of the conserved nature of these sequences. This makes it feasible to use relatively few degenerate primers to clone the majority of the variable regions. The disadvantage of this approach is that it may introduce amino acid substitutions into the framework regions which affect ~antibody affinity.

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~, WO93/10819 PC~/US92/10206 2 1 ~33~7 In the second approach (Larrick, Gavilondo), 5' primers are designed to prime some portion of the leader sequence. The 3' primers are designed to prime either the J region or the constant region, as in the first approach. The second approach takes advantage of the relatively conserved nature of the leader sequences and uses a set of redundant oligonucleotides to prime this site. Priming in the leader sequences is generally the more powerful approach since this (leader) peptide is removed from ; the mature antibody molecule and variations in its sequence will have no effect on antibody affinity.
Many different leader peptide sequences are effective in targeting the immature antibody molecule to the endoplasmic reticulum~ This second approach is the preferred embodlment in this disclosure.
; ` The term transferrin receptor ls intended to encompass the entire~receptor or portions thereof.
Portions of the transferrin receptor include those portions sufficient for binding of the receptor to an `anti-trans~ferrin;receptor antibody to occur.
` Monoclonal antibodies reactive with at least a portion~of the transferrin receptor c~n be obtained (e.g., OX-26,~3/25 (Omary et al. (1980) Nature ; 286,888-891), T56/~14~ (Gatter et al. (19%3) J. Clin.
th.: 36 53~9: 545 j; Jefferles et al. Immunology ~1985) 54:333-341), OKT-9 ~(Sutherland et al. (1981) Proc.
' ~ Natl. ~cad. Sci`. USA 78:4515-4519), L5.1 (Rovera, CO
_. ~
(1982) Blood 59~ 671-678), 5E-9 ~Haynes et al.(1981) J. Immunol. 127:347-351), RI7 217 ~Trowbridge et al.
:
~ Proc. Natl. Acad.~Scl. USA 78:3039 (1981) and T58/30 WO93~10819 PCT/US92/10-~123307 (Omary et al. cited supra)or can be produced using conventional somatic cell hybridization techniques (Kohler and Milstein tl975) Nature 256, 495-497). A
crude or purified protein or peptide comprising at least a portion of the transferrin receptor can be used as the immunogen. An animal is vaccinated with the immunogen to obtain an anti-transferrin receptor antibody-producing spleen cells. The species of animal immunized will vary depending on the species of monoclonal antibody desired. The antibody produc-ing cell is fused with an immortalizing cell (e.g.
myeloma cell) to create a hybridoma capable of secreting anti-transferrin receptor antibodies. The ~nfused residual antibody-producing cells and iinmortalizing cells are eliminated. Hybridomas producing the anti-transferrin receptor antibodies are selected using conventional techniques and the selected anti-tranferrin receptor antibody producing hybridomas are cloned and cultured.
Polyclonal antibodies can be prepared by immunizing an animal with a crude or purified protein or peptide comprising at least a portion of a transferrîn receptor. The animal is maintained under conditions whereby antibodies reactive with a transferrin receptor are produced. Blood is collected from the animal upon reaching a desired titer of anti~odies. The serum containing the ~, ~
polyclonal antibodies (antisera) is separated from the other~blood components. The polyclonal antibody-containing serum can optionally be further , : ~ :

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~ WO93/10819 PCT/US92/10206 21~33~)7 separated into fractions of particular types of antibodies (e.g. IgG, IgM).
The neuropharmaceutical agent can be linked to the antibody using standard chemical conjugation techniques. Generally, the link is made via an amine or a sulfhydryl group. The link can be a cleavable link or non-cleavable link depending upon whether the neuropharmaceutical agent is more effective when released in its native form or whether the pharmaceutical activity of the agenk can be maintained while linked to the antibody. The determination of whether to use a cleavable or non-cleavable linker can be made without undue experimentation by measuring the activity of the drug in both native and linked forms or for some drugs can be determined based on known activities of the drug in both the native and linked form.
For some cases involving the delivery of proteins or peptides to the brain~ release of the free protein or peptide may not be necessary if the biologically active portion of the protein or peptide i5 uneffected by the link. As a result, antibody-protein or antibody peptide conjugates can be constructed using noncleavable linkers. Examples of such proteins or peptides include CD4, superoxide dismutase, inter~:eron, nerve growth factor, tricosanthin, dopamine decarboxylase, somatostatin analogues and enkephalinase inhibitorsu Terms such as "CD4l' are used herein to include modified versions of the natural molecule, such as soluble C~4, truncated CD4, etc. Examples of non-cleavable linker WO93/10819 212 3 3 0 7 PcTtus92/lo2~t systems which can be used in this invention include the carbodiimide ~EDC), the sulfhydryl-maleimide, the N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP;
Pharmacia), and the periodate systems. In the carbodiimide system, a water soluble carbodiimide reacts with carboxylic acid groups on proteins and activates the carboxyl group. The carboxyl group is coupled to an amino group of the second protein> The result of this reaction is a noncleavable amide bond hetween two proteins.
In the sulfhydryl-maleimide system, a sulfhydryl group is introduced onto an amine group of one of the proteins using a compound such as Traut's reagent.
The other protein ~is~reacted with an NHS ester (such as gamma-maleimidobutyric acid NHS ester (GMBS)~ to form a maleimide derivative that is reactive with :
sulfhydryl groups. The two modified proteins are then reacted to~form a covalent linkage that is noncleavable.
SPDP~is~a~h;eterobifunctional crosslinking reagent~that introduces thiol-reactive groups into either the~mono~lonal antibody or the neuropharma-ceutical agent. The~ thiol-reactive group reacts with a~free sulfhydry~l group forming a disulfide bondO
Periodate coupling requires the presence of ~ ~ . ~ : : : : :, :
oligosaccharide groups;on either the carrier or the protein to be delivered. If these groups are available on thé protein to be delivered (as in the ,:
ase of horseradish~peroxidase (HRP)), an active aldehyde is formed on the protein to be delivered which can react with an amino group on the carrier.

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3 wo ~3/108~9 PCT/US92/1~206 21 233~7 It is also possible to form active aldehyde groups from the carbohydrate groups present on antibody molecules. These groups can then be reacted with amino groups on the protein to be delivered generat-ing a stable conjugate. Alternatively, the periodate oxidized antibody can be reacted with a hydrazide derivative of a protein to be delivered which will also yield a stable conjugate.
Cleavable linkers can be used to link neuro-pharmaceutical agents which are to be deposited in the brain or when a non-cleavable linker alters the activity of a neuropharmaceutical agent. Examples of cleavable linkers include the acid labile linkers described in copending patent application Serial No.
07~308,960 filed February 6, 1989, the contents of which are hereby incorporated by reference. Acid labile linkers include cis-aconitic acid, cis-carboxylic alkadienes, cis-carboxylic alkatrienes, and poly-maleic anhydrides. Other cleavable linkers are linkers capable of attaching to primary alcohol groups. ~xamples of neuropharmaceutical agents which can be linked via a cleavable link include AZT, ddI, ddc, adriamycin, amphotericin B, pyrimethamine, valproate, methotrexate, cyclophosphamide, carboplatin and superoxide dimutase. The noncleavable linkers used generally to link proteins to the antibody can also be used to link other neuropharmaceutical agents to the antibody.
The antibody-neuropharmaceutical agent conjugates can be administered orally, by WO93~10819 212 3 3 0 7 PCT/US92/102 subcutaneous or other injection, intravenvusly, intramuscularly, parenternally, transdermally, nasally or rectally. The form in which the conjugate is administered (e.g., capsule, tablet, solution, emulsion) will depend at least in part on the route by which it is administered.
A therapeutically effective amount of an antibody-neuropharmaceutical agent conjugate is that amount necessary to significantly reduce or eliminate symptoms associated with a pa~ticular neurological disorder. The therapeutically effective amount will be determined on an individual basis and will be based, at least in part, on consideration of the individuals's size, the severity of symptoms to be treated, the result sought, the specific antibody, etc. Thus, the therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
Although the description above focuses on antibodies, any protein which interacts with the extracellular d~main of the transferrin receptor, including the 1igand binding site, could potentially ~erve as a vehicle for the delivery of drugs across the blood-brain barrier. In addition to anti-transferrin receptor antibodies, this would include transferrin, the ligand which binds to the receptor, and any transferrin derivatives which retain receptor-binding activity. In fact, any ligand which binds to the ~ransferrin receptors could potentially be employed~

' WO93/10819 PCT/US92/10206 2l2~)3a7 --19-- , A procedure for producing chimeric antibodies reactive with a transferrin receptor may be performed as follows: cDNA is synthesized from mRNA purified from a small number of cells producing the antibody of interest. A PCR reaction is performed .in order to obtain the antibody heavy and light chain variable regions which are then cloned and sequenced. After a second PCR reaction to modify the ends of these regions to make them compatible with the expression cassettes, they are cloned into novel expression ~ectors which contain human constant regions, immunoglobulin promoter and enhancers, and selection markers. In these vectors, a murine heavy chain promoter has been provided with restriction sites so that the l~ader sequences primed and expanded can be directly cloned into a functional promoter.
Restriction sites have also been provided for the direct cloning of the 3' end of the variable region into a constant region. In the heavy chain vector, a novel restriction site has been engineered into the CHl domain of the human ~l heavy chain gene. VH can ,.
then be joined at this site to provide a comple e heavy chain protein. For VL, a restriction site has ~een engineered just 3' of the splice site so that the cloned VL will then splice the kappa to produce a complete ~ light chain protein. The final constructs are then transfected into non-producer hybridoma cell lines as SP2/O or P3.X63.Ag~653 and the supernatants tested for antibody production (Figure 8).
Further procedures and materials r such as expression cassettes, for producing chimeric WO93/10819 212 3 3 0 7 PCT/US92/10~

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an~ibodies reactive with a transferrin receptor can be found in the patent application: Attorney Docket No. 38996, filed on the same date as the present application. Such teachings of this co-filed application are herein incorporated by reference.
The present invention will be illustrated by the following examples.

EXAMPLE 1- In Vitro .Binding of Murine Monoclonal Antibodies to Human Brain Endothelial Cells Two murine monoclonal antibodies, B3/25 and T58/30, described:~by Trowbr.idge (U.S. Patent :4,434,:156:issùed February 28, 1984, and Nature Vol.
294, pp.: 171-173 (19B1)), the contents of both are her~by incorporated by reference, which recognize the human transferrin receptor were tested for their ability;to bind to:human brain capillary endothelial :, ~
~ : cslls~ Hybridoma:cell lines which produce B3t25 and ~:
T58/30 antibodies were obtained from the American ; Type Culture~ollection (ATTC) in Rockville, aryl~nd, and g~own:in DMEM medium supplemented with 2.0~mM~glutamine,~ 10.0~mM`HEPES (pH 7,2), 100 ~M non-:essential amino acids and 10% heat-inactivated fetal calf~serum. The;hybridoma cultures were scaled-up in 225~ cm2 T-flasks~;for;~the production of milligram : quantities of IgG antibody. The hybridoma super-:~ natants were conce~ntrated 50x using vacuum dialysis and applied to ;a~protein-A sepharose column using the BioRad MAPS buffer~ s:ystem. Purified antib~dy was eluted from the~column, dialyzed against 0.1 M sodium ' W~93/1081g . 2 12 3 3 ~ 7 PCr/US92/1020$

phosphate (pH 8.0), concentrated and stored in aliquots at -20C.
Primary cultures of human brain endothelial cells were grown in flat-bottom 96-well plates until five days post-confluency. The cells were then fixed using 3.0% buffered formalin and the plate blocked with l.0% bovine serum albumin (BSA) in Dulbecco's phosphate buffered saline ~DPBS). Aliquots (lO0 ~l) of the B3/25 or T58/30 antibodies, either in the form of culture supernatants or purified protein, were then added to the wells (antibody concentrations were in the range of 1-50 ~gjml). Antibody which had specifically bound to the fixed cells was detected using a biotin~labeled polyclonal goat-anti-mouse IgG
antisera followed by a bio~inylated horseradish peroxidase (HRP)/avidin mixture (Avidin Biotin Complex technique). Positive wells were determined using a Titertek Multiscan Enzyme Linked Immunosorbent Assay (ELISA) plate reader. The results showed that both antibodies bind to human brain capillary endothelial cells with the T58/30 antibody::exhibiting a::~higher level of bindinqO
These same ~ntib~dies were also tested for binding to human brain capillaries using sections of human;brain tissue that were fresh frozen (without :
fixation), sectioned on a cryostat (section th.ickness was 5-20 ~m), pla~ed on gla~s slides and fixed in acetone (l~ minutes at room temperature). These :: sections were then stored a~ -20C prior to use.
The slides containing the human brain sections .
~ ~ were alIowed to come to room temperature prior to : :

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WO93/10819 PCT/US92/10~

use. The sections were then rehydrated in DPBS and incubated in methanol containing 0.3% H202 to block endogenous peroxidate activity. The sections were ocked for fifteen minutes in a solution containing 0.2% non-fat dry mllk and 0.2% methylmannopyrano-side.~ B3/25 and T58/30 antibodies, purified as '; ~ discussed above,~were~applied to the sections at a concentration of'5-50 ~g/ml and incubated at room temperature for~one to two hours. Antibody that specifically~bound to the tissue was detected using the Avidin-Biotin Complex (ABC) technique as described above~for the ELISA assay. Staining of capillaries in the human brain sections was observed with~both the~B3~/25~and~T58/30 antibo~ies. The T58~/~30 antibody~a~lso dlsplayed some binding to the wh'ite~mat~er~of~ the~brain cortex.

E ~ PLE~2- In-V~itro~Binding of Murine Monoclonal Antibody ~X-26~to~Rat~Transferrin Receptor The~;O -2 ine;antibody, which recognizes the rat tran`sferrin,~receptor, has be~en shown in vlvo to ;bi ~ to~bra''`~ c ~i;ll;ary~ endothel~ial cells (Jeffries et~al.~ cited~supra)~ The~murine hybridoma line whiCh~p~oduc" the~OX-26 murine antibody was obtained and~ e~'~hybri~d~ma~cel~l~line was grown in RPMI 1640 mediùm~supplemented~with 2.0 mM glutamine and 10%
heat-inà'cti~ated~Peta~l~ calf serum. The OX-26 antibody~;~was~purified~ using the affinity chroma-tography~technique~ descrlbed ln~Example l.

' ~ W093/lOX~9 ~ PCT/US92/10206 2123~U7 .
The purified antibody was tested in vitro as described for the anti-human transferrin receptor antibodies in Example l to determine whether it would ; bind to brain capillaries in fresh frozen, acetone-fixed rat brain sections. The results showed that the OX-26'anti-transferrin receptor antibody did 'bind to capillaries in rat~ brain sections in vitro.

EXAMPLE 3- ~n-Vivo Binding of OX-'26 Murine Monoclonal : ~, Antibody to Rat Transferrin Receptor Dose Ranae The rat an~tl-transferrin receptor antibody OX-26 was~tested in~-vivo~by inj~ecting purified antibody (pur~if~ication~as~described in Example l) into female Sprague-~Dawley~rat~s~ (lOO-l50 gm) via the tail vein.
Prior~to ~injection,~ the rats were anesthetized with halotha~ne.~ The~samples, ranging from 2.0 mg to 0.05 'mg~;of~àntibody~/rat~were~i~njected~into the tail ~ein in~400~ aliquots.~ All doses we~re tested in dup~ cate~;animals~ One hour;~post-lnjection, the animals~were~saarificed and;perfused through the héart~ wi-th DPBS~ o clear~the blood from the organs.
Imm~diatély ai~ter~the~perfusion was completed, the bra~in~'wàs~removéd~a~nd~quick frozen~in liquid nitrogen.~ Thè frozen~bra~ln was thén~sec~tioned (30-50 m)~on;~a~ cryostat~and;the sections placed on glass microscope slid~es.~The~brain sections were air dried ''at~ room~temperature~vne~to two~hours before fixation in a~etone~lO~minutes~at room~tempera~ure). After this tr~e;atment the sectlons could~be stored at -20C.

W093/10819 PCT/US92/10~
2~23307 The OX-26 antibody was localized in the brain sections using immunohistochemistry as described above for the _ vitro experiments in Example l. The addition of the primary antibody was unnecessary in that it is present in the brain sections. The results indicated that the OX-26 antibody binds to rat brain capillary endothelial cells and that doses of as little as 50 ~g result in detectable levels of antibody in the brain using the methods described herein. Doses above 0.5 mg did not appear to show significantly more antibody binding to the endothelial cells, suggesting that the sites for antibody binding may be saturated. No specific binding to capillary endothelium was detected in the liver, kidney, heart, spleen or lung.
A non-specific antibody of the same subclass as OX-26 (IgG~2aj was also tested in VlVO to show that the binding of OX-26 to rat brain endothelial cells that has been observed is specific to the OX-26 antibody. 0.5~mg of the control antibody was injected per rat~as described above. The results indicate that t~e~staining pattern observed with the OX-26 antibody lS specific to that antibody.

Time~Course A~ter establishlng that ~he OX-26 antibody is detectabIe in the rat brain capillaries after in vivo administration, the time frame in which this binding occurred was de~ermined. Using 0.5 mg of purified 0~-26 antibody~as the standard dose, brain sections taken from animals sacrificed 5 minutes, 15 minutes, , :

r ~ wO 93/10819 PCT/US92/10206 212~33~7 1 hour, 2 hours, 4 hours, 8 hours and 24 hours post-injection were examined for the presence of OX-26 antibody. All doses were administered in 400 ~1 aliquots and each time point was tested in duplicate animals. Samples were injected and the rats were processed at the various times post-injection as described above in the dose range section.
The results showed that the OX-26 antibody can be detected in or on the rat brain capillary endothelial cells as early as five minutes and as late as 24 hours post-injection. At 4 and 8 hours post-injection, the staining pattern of the antibody is very punctate suggesting that the antibody has a~cumulated in vesicular compartments either in endothelial or perivascular cells.

EXAMP~E 4- The Use of a Conjugate of OX-26 Murine Monoclonal Antibody for Tranferring Horseradish Peroxidase Across the Blood Brain Barrier Horseradish,Peroxidase (HRP; 40 kD) was chosen as a compound to be delivered to the brain because it is similar in size to several therapeutic agents and it~can be easily detected in the brain using an enzymatic assay.~ ~HRP was~conjugated to the OX-26 antibody using a non cleavable periodate linkage and the ability of the antibody to function as a carrier of compounds to the brain was examined. The antibody con~uga~e was tested in vivo to determine if the antibody could dellver HRP to the brain.

WO93/10819 212 3 3 0 7 PCT/US92/10l The antibody (10 mg) was first dialyzed overnight against 0.01 M sodium bicarbonate ~p~ 9.0).
The HRP ~10 mg) was dissolved in 2.5 ml deionized water, 0.1 M sodium periodate (160 ~1) was added and the mixture was incubated for five minutes at room temperature. Ethylene glycol (250 ~1) was added to the HRP solution followed by an additional five minute incubation. This solution was then dialyzed overnight against 1.0 mM sodium acetate buffer (pH
4.4). To the dialyzed OX-26 antibody (2.0 ml, 5.08 mg~ml) was added 200 ~1 of 1.0 M sodium bicarbonate buffer, pH 9.5 and 1.25 ml of the dialyzed HRP
solution. This mixture was incubated in the dark for two hours followed by the addition of 100 ~1 of 10 mglml sodium borohydride. The resulting mixture w~s incubated two additional hours in the dark at 4C.
The protein was precipitated from the solution by the addition of an e~ual volume of saturated ammonium sulfate and resuspended in a minimal volume of waterO
Free antibody was removed from the mixture by chromatography on a concanavalin A-sepharose column (a column which~binds HRP and the HRP-antibody conjugate and allows the free antibody to pass through). The free;HRP was removed by chromatography :
on a protein A-sepharose column which retains the antibody-HRP conjugate. The final product had an HRP/antibody ratio of 4/1.
A time course experiment identical to that described in Example 3 was performed using the antibody-HRP con~ugate. The antibody-HRP conjugate (0.5 mg) was injected in a 400 ~1 aliquot/rat. The ' WO93/10819 PCT/US92/10206 2i233 ;i7 animals were sacrificed at the various times post-injection and the brains processed as described above in Example 3~ The antibody HRP conjugate was localized in the brain either by staining for antibody immunohistochemically as described in Example l or by directly staining the brain sections for the presence of HRP. To detect HRP, the slides were first allowed to come to room temperature before incubating in methanol for thirty minutes. The brain sections were then washed in DPBS and reacted with 3,3'-diamino benzidine (DAB), the substrate for HRP.
The results showed that the OX-26 antibody HRP
conjugate binds to rat brain capillary endothelial cells in a manner identical to that of the unconjugated antlbody. The punctate staining 4-8 hours after injection which was seen with the antibody alone is~also seen with the a~tibody conjugate, suggesting that the conjugate can also be going into the pericy~es on the abluminal side of the blood brain barrier. Taken together, these results indicate that the OX-26 antibody can deliver a protein molecu1e~of at least 40 KD to the brain.

EXAMPLE 5~ The In-Vivo Delivery of Adriamycin to the -- : ~
Brain by Murine Monoclonal ~ntibody OX~26 A non-cleavable linker system similar to that used in Example 4, was used to couple the chemotherapeutic drug a:driamycin to the OX-26 anti~ody. The availability of antibodies that can detect adriamybin as well as the system previously ~ ' :~

WO93/10819 212 3 3 0 7 PCT/US92/10.

described in Example 1 for detecting the antibody carrier allowed the use of immunohistochemical tech-niques for monitoring the localization of the antibody carrier as well as the delivery of adriamycin to the brain.
To conjugate adriamycin to the antibody, the drug ~10 mg in 0~5 ml DPBS) was oxidized by the addition of 200 ~1 of 0.1 M sodiurn periodateO This mixture was incubated for one hour at room temperature in the dar~. The reaction was quenched by the addition of 200 ~1 of ethylene glycol followed by a five minute incubation. The OX-26 antibody (5.0 mg in 0.5 ml of carbonate buffer (pH 9.5)) was added to the oxidized adriamycin and incubated at ro~m temperature fo,~ one hour. Sodium borohydride (100 ~1 of 10 mg/ml) was added and the mixture was incubated for an additional two hours at room temperature. The free adriamycin was separated from ~he OX-26 antibody-adriamycin conjugate by chromatography on a PD-10 column. The adriamycin/OX-26 antibody ratio within the conjugate was 2/1. for this particular batch of conjugate.
The effectiveness of the OX-26 antibody as a carrier for delivering adriamycin to the brain was , determined by administering 0.5 mg of the antibody-adriamycin conjugate in a 400 ~1 aliquot per rat by injection via the tail vein. One hour post-injection, the rat was sacrificed and the brain processed as described in Example 1. All injections were performed in duplicate. As a control, 400 ~g of free adrlamycin in a 400 ~1 aliquot was also injected .
.
.

~ W0~3/tO819 PCT/US92/10206 21233~7 into a rat. Immunohistochemistry was used to detect both the carrier OX-26 antibody and the adriamycin in the rat brain sections. In the case of adriamycin, polyclonal rabbit anti adriamycin antisera was applied to the sections followed by a biotinylated goat anti-rabbit IgG antisera. This was then followed by the addition of a biotinylated HRP/avidin mixture and enzymatic detection of HRP.
The results indicate that both the OX-26 antibody and the conjugated adr:iamycin localized to the rat brain capillary endothelial cells after in vivo administration. There is no evidence that free adriamycin binds to brain capillary endothelial cells or enters the brain~
An adriamycin-OX-26 conjugate coupled via a carbodiimide linkage was also synthesized (drug/
antibody ratio of lO/l) and tested in vivo. The results of this experiment were essentially identical to that obtained with the periodate-linked antibody~drug conjugate. In both cases, staining for the antibody carrier was quite strong and was visualized in the capillaries in all areas of the brain. This staining was evenly distributed along the capillaries. Staining ~or adriamycin was less in*ense but again was seen in capillaries throughout the brain. Some punctate staining was observed which suggests accumulation in pericytes which lie on the brain side of the ~lood-brain ~arrier.

EXAMPLE 6- In Vivo Delivery of Methotrexate to the Brain by Murine Monoclonal Antibody OX-26.

:

WO93/10819 212 3 ~ 0 7 PCT/US92/107 A noncleavable carbodiimide linkage was used to couple methotrexate to the OX-26 murine monoclonal antibody. A system analogous to that described in Example 5 was used to monitor the delivery of both the methotrexate and the carrier antibody to the brain capillary endothelial cells.
Methotrexate was coupled to murine monoclonal antibody OX-26 via its active ester. Briefly, 81 mg (0.178 mM) of methotrexate (Aldrich) was stirred with 21 mg (0.182 mM) of N-hydroxysuccinimide (Aldrich) in 3 ml of dimethylformamide tDMF) at 4C.
Ethyl-3-dimethylaminopropyl-carbodiimide (180 mg;EDC;0.52mM) was added to this solution and the reaçtion mixture was stirred overnight. The crude ester was purified from the reaction by-products by flash chromatography over silica gel 60 (Merck) using a solution of 10% methanol in chloroform as an eluant~ The purified active ester fractions were pooled and concentrated to dryness. The ester was dissolved in 1 ml of DMF and stored at -20C until use. 50 mg (50%)`of active ester was recovered as determined by A372(~372 A solution of 0~-26 containing 2.1 mg (14 nmoles) of antibody in 0.9 ml of 0.1 M phosphate (pH
8 7 0) was thawed to 4 C. To this stirred antibody solution was added 1.4 ~L (140 nmoles) of the active ester prepared as described above. After 16 hours at 4C, the mixture was chromatographed over Sephadex PD-10 column (Pharmacia~ using phosphate buffered saline (PBS) to separate conjugate from free drug.
The fractions containing the antibody-methotrexate ;

. .

~`~ WO93/10819 PCT/US92/1~206 21233~7 conjugate were pooled. Antibody and drug concentration were determined spectrophotometrically as described by Endo et al. (Cancer Research (lg88) 48:3330-3335). The final conjugate contained 7 methotrexates/antibody.
The ability of the OX-26 monoclonal antibody to deliver methotrexate to the rat brain capillary endothelial cells was tested in vivo by injecting 0.2 mg of conjugate (in 400 ~1) into each of two rats via the tail vein. The animals were sacrificed one hour post-injection~and the brains processed for immunohistochemistry as descri~ed in Example 1. To detect methotrexate in the brain, a rabbit antisera :
raised against methotrexate was used as the primary antibody. A biotinylated goat-anti-rabbit antisera in conjunction with~;a mixture of biotinylated HRP and avidin was then used to visualize methotrexate in the rat brain. The carrier antibody was detected as described previ~ously.
The results of these experiments indicate that methotrexate ln~the form of a conjugate with OX-26 does~accumulate~along or in the capillary endothelial cells of the ~rain. The staining observed for m~thotrexate is comparable in intensity to that seen for~the carrier.~ ~The staining appears to be in all areas~of the brain~and is evenly~distributed along the capillaries.~

EXAMPLE 7- Antibody Derivatives ~: :

: ~ .

WO93t10B19 21~ 3 3 0 7 PCT/US92/102 The Fc portion of the OX-26 murine monoclonal antibody was removed to determine whether this would alter its localization to or uptake by the rat brain capillary endothelial cells. F~ab)2 fragments of OX-26 were produced from intact IgG's via digéstion with pepsin. A kit available from PiercP Chemical Co. contains the reagents and protocols for cleaving the antibody to obtain the fragments . The F(ab')2 frag~ent (0.2 mg doses) in 400 ~l aliquots were injected into rats via the tail vein~ A time course experiment identical to that done with the intact antibody (Example 2) was then performed. F(ab')2 fragment was detected immunohistochemically using a goat anti-mouse F(ab')2 antisera followed by a biotinylated rabbit anti-goat IgG antisera. A
biotinylated HRP/avidin mixture was added and the antibody complex was visualized using an HRP
enzymatic assay. The resul~s indicate that the F~ab)2 fragment of the OX 26 antibody ~inds to the capillary endothelial celIs of the rat brain.
, EXAMPLE 8 Measurement of OX-26 in Brain Tissue ~To quantitate,the amount of OX-26 which ;~accumulates in the brain, radioacti~ely-labelled ntibody was injected into rats via the tail vein~
Antib~dies were labelled with either 14C-acetic anhydride or 3H-succinimidyl proprionate essentially as des~ribed in Kummer, U., Methods in Enzymology, :`: ~
121: 670-678 (1986)~, Mondelaro, R.C., and Rueckert, J. of_Biological Chemistry, 250: 1413-1421 -~ :

: ~ :

:
:: :

~ ~ WO93/l~g19 PCT~US92/10206 21233~)7 tl975), hereby incorporated by reference. For all experiments, the radiolabelled compounds were injected as a 400 ~1 bolus into the tail vein of ~emale Sprague-Dawley rats (100-125 gms) under Halothane anesthesia and the animals were sacrificed at the appropriate time post-injection using a lethal dose of anesthetic. A 3H-labelled IgC2a control antibody was co-injected with the 14C-labelled OX-26 to serve as a control for non-specific radioactivity in the brain due to residual blood. At the appropriate time post-injection, animals were sacrificed and the brains were removed immedicately and homogenized in 5 ml of 0.5% sodium dodecysulfate using an Omni-mixer. An aliquot of the homogenate was incu~ated overnight with 2 ml of Soluene 350 tissue solubilizer prior to li~uid scintillation counting. All data were collected as disintegrations per minute (dpm). Blood samples were centrifuged to pellet red blood cells (which do not display significant binding of radiolabelled materials) and the radioactivity in an aliquot of serum determined using liquid scintillation counting.
The amount of antibody associated with the brain was determined at various~times post-injection to examine the pharmacokinetics of brain uptake. In addi~ion, the amount of labelled antibody in the blood was measur d so that the rate of clearance from the bloodstream~could be determined. This information was~also used to calculate the amount of radioactivity in the brain due to blood contamination, which was then subtracted from the WO 93/10819 21~ 3 3 0 7 PCT/US92/102 ~34 ~

total to give the amount of antibody that is sp~cifically associated with the brain.
A peak level of 14C-labelled OX-26 corresponding to approximately 0.9% of the injected dose was reached in the brain between 1 and 4 hours post-injection as illustrated in Figure 1 (with the values shown as means plus or minus standard error of measurement (SEM) and N-3 rats per time point). The amount of radioactivity associated with the brain decreased steadily from 4 to 48 hours post-injection, at which point it leveled off at approximately 0.3%
of the injected dose. The accumulation of 0X-26 in the brain was significantly reduced ~y the addition of unlabelled monoclonal antibody (0.5 or 2.0 mg in the bolus injection). As an additional control, a 3H-IgG2a control antibody was co-injected with the 4c-ox-26. The control antibody did not accumulate in the ~rain and represented the blood contamination of the brain.
In contrast to the levels in the brain, the blood level of OX-26 dropped quite dramatically immediately after injection such that by 1 hour post-înjection, the percent of injected dose in 55 ~l of blood (the volume of blood associated with the brain) was approximately 0.16% as illustrated in ~igure 1. This corresponds to a value of approximately 20% of the injected dose in the total blood volume of the rat. Extraction of total IgG
from serum followed by polyacrylamide gel electro- -~horesis (PAGE) and autoradiography did not reveal de~ectable levels of OX-26 degradation indicating ~ WO93/lOB1g PCT/US92/10206 ~1 233~7 .

that the antibody remains intact in the blood as long as 48 hours after injection.

EX~MPLE 9 - Distribution of OX-26 in Brain Parenchyma and Capillaries To demonstrate that anti-transferrin receptor antibody accumulates in the brain parenchyma, homogenates of brains taken from animals injected with labelled OX-26 were depleted of capil,laries by centrifugation through dextran to yield a brain tissue supernatant and a capillary pellet. Capillary depletion experiments followed the procedure of Triguero, et_al., J. of Neurochemîstry, 54: 18B2-1888 (1990~), hereby incorporated by reference. As for the brain uptake experiments of Example 8, the radiolabelled compounds were injected as a 400 ~l bolus in~o the tail vein of femals Sprague-Dawley rats (100 125 gm) under Halothane anesthesia and the a~imals were sacrificed at the appropriate time post-injection using a lethal dose of anesthetic. A
3H-labelled IgG 2a control antibody was co-injected with the 14C-labelled OX~26 to serve as a ~ontrol for non-specific radioactivity in the brain due to residual blood. After sacrifice, the brains were removed and kept on ice. After an initial mincing, the brains were homogenized by hand (8-10 strok~s) in 3.5 ml of ice cold physiologic buffer (100 mM NaCl, 4.7 mM KC1, 2.5 mM CaC121 1.2 mM KH2PO~, 1.2 mM
MgSO4, 14.5 mM HEPES, 10 mM D-glucose, pH 7.4). Four ml o~ 26% dextran svlution in buffer was added and ~: :

: :
~ ::

WO93J10819 212 3 3 ~ 7 PCT/US92/102f homogenization was continued (3 strokes). After removing an aliquot of the homogenate, the remainder was spun at 7200 rpm in a swinging bucket rotor. The resulting supernatant was carefully removed from the capillary pellet. The entire capillary pellet and aliquots of of the homogenate and supernatant were incubated overnight with 2 ml of Soluene 350 prior to liquid scintillation counting. This method removes greater than 90% of the vasculature from the brain homogenate (Triguero et al., cited supra).
A comparison of the relative amounts of radioactivity in the different brain fractions as a function of time indicates whether transcytosis of the la~elled antibody has occurred. The amount of OX-26 in total brain homogenate, the brain parenchyma fraction and the brain capillary fraction at an early time (30 minutes) and a later time (24 hours) pos~ injection is illustrated 1n Figure 2. The values in Figure 2 are shown as means+SEM with N=3 rats per time polnt. At the 30 minute time point, more of the radiolabelled antibody is associated with ~1'~
the capillary fraction than with the brain parenchyma fraction (0.36% of the injected dose (%ID) and 0.23%
ID, respectively). By 24 hours post-in~ection, the distribution is reversed and the majority of the radloactivity (0.36% IDj is in the parenchymal fraction as compared to the capillary fraction (0.12%
ID). The redistrlbution of the radiolabelled OX-26 from the capillary fraction to the parenchyma fraction is conslstent with the time dependent .

~ W~93/1~19 PCT/US92/10206 2 1 ~ 3 ~ 7 migration of the anti-transferrin receptor antibody across the blood-brain barrier.

EXAMPLE lO - Distribution of an OX-26-methotrexate Conjugate in Brain Parenchyma and Capillaries Capillary depletion studies following the procedures described in Example 9 were performed with an OX-26-methotrexate (MTX) conjugate linked via a gamma-hydrazid as described in Kralovec, et al., J.
of Medicinal Chem., 32: 24~6-2~31 (1989), hereby incorporated by reference, in which the MTX moiety was labelled with 3H. As with unconjwgated antibody, the amount of label in the capillary fraction at 30 minutes post-injection is greater than the parenchyma fraction (approximately 2-fold as illustrated in Figure 3, with the data expressed as means+SEM and N=3 rats per time point). This distribution changes over time such that by 24 hours post-in~ection9 approximately 4.5-fold more of the labelled MTX is in the ~rain parenchyma than in the capillaries. These ;~
results are consistent to those obtained with unconjugated antibody and, again, suggest that these compounds cross the blood-brain barrier.
To ensure that these results were not due to contaminating amounts of free H-MTX or ~-MTX that had been cleaved from the conjugate after injection, a co-mix of labéIled drug and antibody was injected into rats and a capillary depletion experiment performed. The amount of ~-MTX in the different brain fraction is significantly lower for the co mix WO93/10819 2 1 2 3 3 0 7 PCT/US9~/102f as compared to the conjugate (as much as 47 fold in the case of the capillary fraction at 30 minutes post-injection as illustrated in Figure 3). The 3H-MTX and the co-mix also does not show the change in distribution of the label between the different brain fractions over time as was seen with the antibody~MTX conjugate or antibody alone. These results demonstrate that delivery of 3H-MTX across the blood-brain barrier to khe brain parenchyma .is greatly enhanced by the conjugation of the drug to the anti-transferrin receptor antibody OX-26.

EXAMPLE 11 - Distribution of OX-26-AZT in Brain Parenchyma and Capillaries Capillary depletion studies following the procedures of Example 9 were performed with an OX-26-AZT conjugate using a pH-sensitive succinate linker. These studies employed a dual-labelled conjugate in which the AZT was 14C-labelled and the antibody carrier was 3H-labelled. The use of such a conjugate allowed independent monitoring of the disposition of both the antibody and AZT within the brain.
The linker was synthesized as follows. Succinic anhydride was ~sed to acylate the AZT by reacting equimolar amounts of these two compounds for 3 hours at room temperature under argon in the presence of dimethylaminopyridine and sodium bisulfate in freshly distilled pyridine. The product was isolated by chromatography on a DEAE sephadex A50 column run with !, ~ , W~93/10819 PCT/US92~t~206 21~3~7 a triethylammonium bicarbonate buffer~ The succinate derivative of ~ZT was activated at the carboxyl group as the NHS ester by reaction with equimolar amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) in freshly distilled THF at 4C for 2 hours.
The product was purified by flash charomatograph~ on silica gel. The resulting NHS-ester of AZT-succinate was used to acylate amine groups on OX-26, resulting in an AZT-OX-26 conjugate. A 15-fold molar excess of AZT-NHS ester was reacted with OX-26 in HEPES buffer overnight at 4C. The antibody-drug conjugate was isolated from free drug on a PD-lO column. The molar ratio of drug to antibody was 7:l. These studies employed a duaI-labelled conjugate in which the AZT
was L4C-labelled and the antibody carrier was H-labelled.
Similar levels of VX-26 and AZT are seen in the capillary fraction of the brain and ~hese levels decrease with time~, suggesting that the materials are not being retained by the capillary endothelial cells as il~lus~trated in Figure 4c. As the levels of OX-26 ,~-~in khe capillary fraction decrease, the levels in the parenchyma fract~ion increase, indicating that the antibody is migrating from the capillaries to the parenchyma in a~time-dependent manner as illustrated in Figure 4b. In contrast, the levels of AZT in the brain parenchyma do not rise significantly, suggesting that the majority of the drug is released in the endothelial~cells and is not transported across the blood-brain barrier. The levels of OX-26 .
~ and AZT remai~ned similar in unfractionated : ~

~ .

W093/lOX19 212 ~ 3 0 7 PCT/US92/102~

homogenates over time as illustrated in Figure 4a.
The data in Figure 4 are expressed as means~SEM with N=3 rats per time point. These results indicate that the linker is cleaved within the endothelial cells and may represent a method for delivering compounds to those cells.

EXAMPLE 12 - Distribution of OX-26-Horseradish Peroxidase (HRP) in Brain Perenchyma and Capillaries .~ ,.. .
~: Capillary depletion st~dies following the procedures described for OX-26 in Example 9 were performed with a 3H-labelled OX-26-HRP conjugate that : was prepared uslng a~non-cleavable periodate linkage as des~rlbed in Example 4. The tritium label was distributed between the.antibody and the HRP portion of the:con}ugate. At 1 hour post-injection, the majority of the radioactivity associated with the brain is in the caplllary fraction as illustrated in Figure 5. The data~in Figure 5 are expressed as :means+SEM with N=3~rat~s~per time;point. By 4 hours post-injection, the distribution of radioactivity associated~with the~:brain changed such that the majority is in the~ fraction which represents the brain parenchyma.~At~24 hours post-in~ection, ` essenti~ally all of~the 3~-labelled OX 26-HRP
conjugate is in the:parenchyma fraction of the brain indicating that th~e material has crossed the :blood-brain:barrier.:: Similar results were obtained -in~experiments in~which only the HRP portion of the conjugate was radlolabelled. "

~ ~ WO93/1081~ PCT/US92/10206 21233~7 The percent of injected dose of the OX-26-HRP
conjugate that reaches the brain is somewhat lower than that for antibody alone or the OX-26 HRP
conjugate. This is most likely due to the presence of 2 to 3 40 kD HRP molecules attached to each carrier and that these "passenger" molecules are randomly attached to the carrier. Due to this, many of the HRP passengers may be attached to the antibody in such a way as to interfere with antigen recognition. This problem can be alleviated by directing the attachment of the passenger to regions of the carrier removed from critical functional domains.

E~AMPLE 13 - Distrlbution of OX-26-CD4 in Brain :Parenchyma and:Capillaries ~ , .
': :
:::: :
A soluble form of:CD4, consisting of amino acids 368, was conjugated~to OX-26 using a linkage that directed the:at~tachment of the C~4 to the carbohydrate groups~located in the Fc portion of the antibody~. : By~dire~ing the site of attachment in this~way, the~chance~that the passenger molecules will interfere~with an~ibody-antigen recongition is lessened.: The linkage ~etween the proteins was achieved by first introducing a~ sulfhydryl group onto CD4 using SATA (N-Succinimidyl S-acetylthioacetate), a commerically available compound. A hydrizid derivat:ive ~of SDPD, ~ another: commercial cross-linking agent, was attached to OX-26 vi~ carbohydrate groups : :

W093/10~19 2 1 2 3 ~ ~ 7 PC~/US9~/102f on the antibody. Reaction of the two modified proteins gives rise to a disulfide-linked conjugate.
More specificallyk the linkage between the proteins was achieved by first introducing a sulfhydryl group onto CD4 using N-succinimidyl S-acetylthioacetate (SATA), a commercially available compound. A 4-fold molar excess of SATA was added to

5 mg of CD4 in 0.1 M sodium phosphate buffer containing 3 mM EDTA (pH 7.5). This mixture was reacted at room temperature in the dark for 30 minutes. Unreacted starting materials were removed by passage over a PD-10 column. A hydrizid derivative of SPDP, another commercially available cross-linking agent, was attached to OX 26 via carbohydrate groups on the antibody. T~n milligrams of OX-26 in 2.0 ml of 0~1 M sodium acetate, 0.15 M
sodium chloride (pH 5.0) was reacted with a 1000-fold molar excess of sodium periodate for 1 hour at 4C in the dark. Unreacted starting materials were removed by passage over a PD-10 column. The oxidized antib~dy was reacted with a 30-fold molar excess of hydra3ido-SPDP overnight at 4C with stirring.
Reaction of the two modified proteins gives rise ~o a disulfide-linked conjugate. Vne tenth volume of 0.5 M hydroxylamine was added to the thioacetylated CD4 (CD4-DATA) and derivatized antibody was then added such that ~he ratio of CD4 to antibody was 7.5:1.
This mixture was reacted at room temperature in the dark for 2 hours. Conjugate was purified ~y running the reaction mixture over a protein A column followed by a CD4 affinity column.

` ~ WO 93/10~19 P~T/US92/10206 2l233a7 Capillary depletion experiments following the prosedures described in Example 9 with OX-26 were performed with an OX-26-CD4 conjugate in which only the CD4 portion was 3H-labelled. Time dependent changes in the distribution of the labelled conjugate between the capillary and parenchyma fractions of the brain which are consistent with transcytosis across the blood-brain barrier were observed as illustrated in Figure 6. The da~a in Figure 6 are expressed as means~SEM with N=3 rats per time point.

EXAMPLE 14 ~ Biodistribution and Brain Uptake of Anti-Human Transferrin Receptor Antibodies in Cynomolgous Monkeys A collection of 32 murine monoclonal antibodies which recognize various epitopes on the human transferrin receptor were examined for reactivity with brain capillary endothelial cells in sections from human, monkey (cynomolgous), rat and rabbit brain samples by the immunohistochemical methods ;~
described in Examp~e 1. These antibodies were ~btained from Dr. Ian Trowbridge of the Salk Institute, LaJolla, CA. All 32 antibodies displayed some reactivity with human brain endothelial cells.
Two antibodies reacted very weakly with rabbit brain capillaries and nonP reacted with rat. While 21 of the antibodies reacted with monkey brain capillaries, only 2 displayed strong reactivity comparable to that seen with human brain oapillaries. These 2 WO93/1081~ 21~ 3 ~ ~ 7 PCT/US92~102( antibodies are herewithin referred to as 128.l and Z35.2.
These antibodies were used to determine the tissue distribution and blood clearance of the 14C-labelled anti-human transferrin receptor antibodies 128.l and Z35.2 in 2 male cynomolgous monkeys. 128.l or Z35.2 was administered concurrently with a 3H-labelled control IgG to one of the monkeys with an intravenous catheter. During the course of the study, blood samples were collected to determine the clearance of the an~ibodies from the circulation. At 24 hours post-injection, the animals were euthanized and selected organs and representa-tive tissues were collected for the determination of isotQpe distribution and clearance by combustion. In addition, samples from different regions of the brain were processed as described for the capillary deple-tion experiments in Example 9 to determine whether the antibodies had crossed the blood-brain barrier.
The results of the capillary depletion experiments were performed on samples from the cortex, frontal cortex, cerebellum and striatum. All samples had greater than 90% of the 128.l or Z35.2 in the brain parenchyma, suggesting that the antibodies crossed the blood-brain barrier. The levels of the control antibody in the same samples were from 5 to lO-fold lower. Using the average brain homogenate value for dpm/G tissue, the percent injected dose of 128.l in the whole brain is approximately 0.2-0.3%. This compares to a value of 0.3-0.5% for OX-26 in the rat at 24 hours post-injection. A comparison of the 21~3307 ratios of 128.1 to the control antibody for variousorgans is illustrated in Figure 7. Similar results were obtained for Z35.2. These results suggest that 128.1 is preferentially taken up by the brain as compared to control antibody. For the majority of organs:and tissues tested, the ratio of 128.1 to control is less:than 2.

EXAMPLE~15 - Cloning and Expressing of ALK 128.1: An Anti~-Human Transfe~rrln:Receptor Chimeric Antibody RNA EXTRACTION~
~:

RNA was extracte~ foIlowing the single step guan~idinlumtphen~ol method~(P.Chomczynski and S.
;:Sacchi.;1~987,~Ana~ Bioch. 162:156-259). All the instruments~and~;conta~i:ners~u;sed were~previously autocl~aved~and~r,i:nsed~-with~diethyl~pyrocarbonate (depc):~treated~water~to avoid degradat:ion due to 'RNAases~ Se~vé`ral~samples each containlng~5xlO~ cells ^f~rom~the~:128.~l~hybr1doma~:which secretes a murine anti ,~
human`~transferr~in~`recéptor monoclonal anti~ody, were washéd~tw:ice~wlth~PBS. The pellets~were quick frozen in~ iguid~;n~itrogen~and~either kept~at -70C for later use~,or extracted~i'mme~d~iately.~
For~ the~extractlon, in~a::R~Ias~e:free~microfuge ;tube,; 1/2 ml of: soluti~on D (Solution D-3~ ~l 2~mercaptoethànol~per 5Iml of lX GITC [lx GITC: ~o g guanidinium-~thiocya~nate, 17.6 ml 0.75 M Na citrate pH7;~ 26~.~4~:ml 10%~sarcosyl,~ 293~ ml ~H20]):,:~50 ~l of 2M
Na~acetate~:pH~4~ :0~ 5~ml~:phenol (dH20 equllibrated) ~'~93/10819 212 ~ ~ 0 7 PCT/US92/102Q
-and 100 ~1 of chloroform:isoamylalcohol (49:1) were added to the cell pellet mixing by inversion after each addition. The extraction was left on ice for 15 minutes and centrifuged at ~13000g for 20 min at 4C, The upper aqueous phase containing the RNA was removed to a new tube and precipitated with 2 volumes of cold absolute ethanol for 2 hr. at -70C. After two 70% depc-ethanol washes the RNA pellet was dried briefly and resuspended in dH20 0.5% SDS.
FIRST STRAND cDNA SY~NTHESIS

, ~ ~ S
~ Total RNA~from~5xlO cells was resuspended in , 18 ~1 of 0.5% SDS. 9~1 of RNA were annealed with 2~1 of ~3'~primer (~lmg/ml~) at 60C;for 10 minutes.
For ~light chain~V;~region amplifications,; an oligo dT
;primer;~was used,~whereas~for the amplification of he`avy~chain V;reglons~a~ CH1 antisense primer, containi~ng~an XbaI~ site;~(underlined in Table 13, with `d`egeneracies~introduced so that~;lt will prime all isotypes~of murlne heavy~cha~lns~except 73 was used Table~ ?~

~ } WO93/10X19 PCT/US92/10206 212~3~7 -~7-After annealing, the samples were cooled on ice, 4 ~l of first strand cDNA buffer (50 mM Tris pH 8.3, 50 ~ KCl, l0 mM MgCl2, l mM DTT, l mM EDTA, 0.5 mM
spermidine), l ~l of RNAse inhibitor (Promega), 2 ~l of l0 mM dNTP:'s and~2 ~l of prediluted l.l0 Promega AMV Reverse Transcriptase were added and the reaction incubated for l hour at 42C. The cDNA was kept at .
~ 20C until used for PR.
~ : ~

: : Table l: PRINERS~FOR c~NA S~NTHESIS

PRIMER FOR SYNT~ES15 OF IIG~T C~AIN ~ REGION cDNA

OLIGO~ dT.Rl.XBA.H3 GccGGAAT~cTAGAAGc(T)~l7: ~; :

;PF~ R~FOR SYNT~ESIS~OF HEAVY: ~ IN V REG~ON cDNA

N~C:.C~I AS:~ egeneracies at a single position are shown in parenthesis~

5~'::AGG:~ CTAGA`A(CT)~:TC~ ACA CAC AGG ~AG) (AG)C CAG TGG ~TA GAC

. ~, , :

WO93/10819 2 1 2 3 ~ ~ 7 PCT/US92~102 PRIMERS AND PCR REACTION:

A first PCR reaction was performed in order to amplify the variable regions and determine their sequence. To achieve this the PCR primers were designed to hybridize to the leader sequence (5' primer) and to the constant region immediately downstream of the V~J region (3' primer).
The oligonucleotides were synthesized in an Applied Biosystem 391 DNA Synthesizer, eluted without purification, diluted to 20 ~M and kept at 4C.
All primers were designed with a restriction site with three additional bases upstream to protect the site and facilitate en~yme digestion. The sites were chosen to make possible the cloning of the PCR
product into a subcloning vector and into the final expression cassett vectors.
For the leader region, the primers contain a ribosome recognition site (Kozak's sequence CACC;
Kozak M. 1981, Nucl. Acid. Res., 9:20, 5233-5252) 5' of the start codon, and an EcoR V site (underlined in Tables 2 and 3) protected by three 5' G's. A set of 4 universal 5' sense primers was used simultaneously in ~he light variable region amplification, and a set of 3 universal 5' sense primers in the case of heavy variable regions (Coloma et al. l99l, Biot~chniques ll,2,152-156~. An equimolar amount of each primer was used in the PCR reaction. These primers contain degeneracies in order to hybridize with all the families of murine leader sequences reported in Kabat's database. (Kabat E. l987, Sequences of ~ '. WO93/108~9 . PCT/US9~/10206 21 233~7 Proteins of Immunological Interest, NIH). The 3' primers were designed in the constant region 20 bases downstream of the V-J region and contain an XbaI site (underlined in Tables 2 and 3) for subcloning purposes (Tables 2 and 3).

: ~ :

::;

:~.: ~ :
, ~ ~ . :j : ~, . ~ ~

, : ~ :

W093/10819 2 1 2 3 ~ ~ 7 P~T/U~92/102' Table 2: PRI~ERS FOR MURINE ~E~Y CHAIN VARIABLE REGION
AMPLIFICA~IO~. (Degeneracies at a single position are shown in parenthesis.) LBADER REGION PR ~ RS (5'SENSE) Tl.RV #085 Leader ~urine Heavy IgV
':
5' GGG GAT~TC CACC ATG G~AG)A TG(CG) AGC TG(TG) GT(CA) AT(CG) CTC
TT
, MHAL~2~RV #086 Leader Murine Heavy IgV

5' GGG 9~Ta~_ QCC ATG (AG)AC TTC GGG (TC3TG AGC T(TG)G GTT TT

~ X~AlT~.RV ~ #087 `; Leader:Murine Heavy IgV

~5'~GGG GATATC CACC ATG GCT GTC TTG GGG CTG CTC TTC T

ONSTANT REGION:PRIN~R~`(3'ANTIS~NSE) ,~
~ Primer designed to hybridize:at aminoacids 130-120 în ~l of :~ Ig~ :mis: primer is identical to the primer used for heavy chain first~stra~d cDNA synthesis.

MC~ ~ElAS.XBA ~ #097 ~:: CHl antisense primer for murine Ig~, except Igy3 S' AGG ~5~a~_ A(CT)C TCC~ACA CAC AGG (AG)(AG)C CAG ~GG ATA GAC

:` :: ~:

:

, .
: ' 1~ WO93/10819 251~ 3 ~ 0 7 PCT/US92/10206 Table 3: PRIME~S FOR MnRINE LIGHT CHAIN VARIABLE REGION
AMPLIFIC~TION. (Degeneracies at a single position are shown in p~renthesis.) LE~ER REGION P~IMERS (5'SENSE3 NLALT1.~ #088 Leader ~urine Light IgV

5' GGG GATATC QCC ATG GAG ACA GAC ACA CTC CTG CTA T

ML~LT2~R~ #089 Ieader Murine Light IgV
, S' GGG GAT~TC CACC ATG GAT TTT CAA GTG CAG ATT TTC AG

ML~LT30RV ~o90 Leader Murine Light IgV

5' GGG GATATC C~CC ~TG GAG ~TA)GA CA(GT) (TA)CT CAG GTC TTT
~; (GA)TA

: ~rALT4.~V ~O91 ~Leader~ Mu~ine Light Ig~
,: ~
,; ~.
~: :`
~ ~5~' ~GGG; ~TATC: ~acc ATG (GT)CC CC(~T) ~GA)CT CAG (CT)TtCT~ CT(TG) : : :
GT : ~ ~ ~

.
: CONSTA~T REGION PRIN~R (~3'ANTISENSE) - ~ Primer designed to hybridize to amino acids 122-116 of kappa constant region.

: MCK ~S.XBA ~ #096 Constant Murine Light 5' GCG ~s~a_~ ACT GGA TGG TGG GAA GAT GGA

:

wo 93/1081g 2 1 2 3 ~ Q 7 PCT/US92/102~

The primers for the second PC~ reaction (Table 4) have the actual sequence of the V-J regions, determined by sequencing of the subcloned products (Figure 9). These primers have a Nhe I site in the case of the VH primer and Sal I for the VL primer, -~
which permits the cloning into the expression vectors. (The restriction enzyme sites are underlined in Table 4). The Nhe I site in the 3' prirner for the VH allows the direct ligation of the VH-J region ko the first two amino acids of the CH1 of the ~1 constant region. The VL 3' primer has a donor splice sequence before its Sal I site which is necessary to splice the VL to C K in the expression vector.
-~ WO93/10819 2 1 2 3 ~ ~ 7 PCT/US92/10206 Table 4: PRI~æRS FOR 128.1 VJ REGION MODI~IC~ION BY
S~COMD PCR PRIOR TO THE CLONING INTO EXPRESSION VECTORS

~EAVY CHaIN PRIMER ~3'ANTISENSE):
Primer designed to hybridize to amino acids 111-113 in J4 region of 128.1 heavy chain V region. It includes a Nhe I site for cloning into the expression vector (links J4 to C~I) and Sal I for subcloning (upstream Nhe I).

4 AS~NHE~SAL1 #098 Antisense of VHJ4 ~ ~1 CH1 5 ' TGG ~a~ AGA TGG GGG TGT TGT GCTAGC TGA GGA GAC

..
: LIG~T ~NN PRIMER (3'ANTISENSE):
Prime~ designed to hybridize to amino acids 101-107 in J4 region~of 128~.1 light chain V region. It includes a donor splicing sequence which is highlighted.

J4AS~SAL1 : #1~1 Antisense of VL J4 ~ splicing donor ~ 5'~AGC 3~5B_ T~ACG TCT GAT TTC CAG CCT GGT CCCT

: ~
: ::
.
:::: : : :

: : -'~.

WO93/10819 21~ ~ 3 0 7 PCT/US92/102 -54~

PCR reactions were performed in a volume of l00 ~l with the following final conditions: 2~1 of cDNA, 0.5 ~l Taq polymerase (Cetus Corporation), lX
buffer (l0 mM Tris pH8, 1.5 mM MgC12, 50 mM KCl, l00 ~g BSA), 200~M each dNTP, l~M of each primer and 50 ~l of mineral oil. PCR was carried out for 30 cycles in a PTC l00 Thermal Controller (M~J. Research Inc.) with l min. denaturing (94C), l min. annealing ~55C), 1.5 min. extension (72C), and a final extension of l0 min.
The size of the PCR products was verified by agarose gel electrophoresis in a 2% TAE gel stained with ethidium bromide. The correct products were approximately 380 base pairs for the light chain and 420 base pairs for the heavy chain variable region.

5UBCLONING AND -SEQUE~CING:

After the PCR reaction the oil was removed by chloroform extraction and the samples kept at 4C.
For subcloning, ~he products were either directly cloned into Bluescript KS T-A (blunt ended by digestion at EcoR V site and tailed with dideoxythymidine triphosphate using terminal transferase) prepared ~ollowing the procedure by Holton (T.A. Holton and M.W. Graham. l990 Nucl.
Acid. Res., l9:5, lI56), or gel isolated, cut with the appropriate restr1ction enzymes (EcoR V and Sal I~ and cloned into Bluescript KS previously cut with the same enzymes.

/ ~I WO93/10~19 PCT/US92/10206 212~7 For TA cloning 3 ~1 of the PCR product was directly ligated with 50 ng of T-A vector in a 15 ~l reaction for 4-12 hours at 16C. For sticky end ligations 200 ng of cut Bluescript was ligated with 200~400 ng of cut product in 20 ~l ligation reactions. 5 ~l of the ligation was used for transformation of E. Coli. XL1-blue (Stratagene) competent cells prepared by calcium chloride treatment. White colonies, containing inserts were picked above a blue colony background. Miniprep DNA
was restriction digested, analyzed and the apparently correct clones sequenced.
Dideoxynucleotide chain termination sequencing was carried out using T7 DNA polymerase (Pharmacia, Uppsala, Sweden or Sequenase, US Biochemical Corp., Cleveland, Ohio) according to the manufacturer's protocol. Four independent clones from different PCR
reactions were sequenced in both directions, to obtain the concensus sequence.
The obtained sequences were compared against other murine sequences in Genbank and aligned with reported V regians in Kabat's database to identify ~ ~their family and conserved amino acids. (See Tables - 5 and 6.) :~ ' '~

WO 93/10819 2 1~ 3 ~ 0 7 P~/US92/102~ -Table 5 : COMPIETE SEQUENCE OF CHIMERIC 128 .1 (Anti--Human Transferrin Receptor) LIG~ CHAIN VARIABLE REGIOM, MOUSE
KAPPA SUBGROU~
--22 I,EADE~
ATG GAT m CA~ GTG CAG Arr Met Asp Phe Gln Val Gln Ile TTC AGC T~TC CTG CTA ATC AGT GCC TCA GTC ATA CTG TCC AGA
Phe Se~ Phe leu ~u Ile Ser Ala Ser Val Ile Leu Ser Arg -1 1 F~l GGA - -- CAA ATT GTT CTC ACC ~G TCT CCA GCA ATC ATG TC r Gly --- Gln Ile ~ I~ Thr GL~I 8~a PRO Al~ I~E Met Ser FRl 2 4 CDR1 GCA TCT CCP, GGG GP~G AAG GTC ACC ATG ACC I GC AGT GCC AGC
A~ ~ER Pro t;LY Gl u I,Y~ 'V~ TER Met T~ CY~ Ser A~A ~r~R
27-29:: * CD~l 35 FR2 ~CA ~GT A~ GAT TAC ATT CAC TGG TAC CAG ~G AAG TCA GGC
8ER 8~ Ile Asp TYR Ile His T~L~ Tyr Gl~ GLpa I.Y~ Ser Gly F}~2 ~ 50 C:DR2 ACC TCC CCC AAA AGA TGG ATT TAT GAC ACA l~CC ~ C~rG GCT
Thr 8~la PR0 LY8 Ar~ Trp I~ Asp Thr ~L Lys I~lJ Ala s7 GGA GTC CCT GCT CGC TTC AG~ GGC AGT GS;G ~rCT GGG ACC
~BR ~LY Y~ PR0 Ala ~Ra P~s 8~ G~Y 8X~ GLY ~E~ GI,Y Thr T~T T~T TCT CTC ACA ATC ~C AGC ATG G~Ç; CC~ GAA S:;~T GCT
Ser Tyr Ser ~E~ :q~r II-E: Ser Ser Met GLlJ Pro G~ A8P Ala GCC A&T TAT q~C TGC~ ~T CAG_CG~; ~AT ~GT ~AC CCA TGG_CG
TY~CY8~His G~ Arg Lys Ser q~yr Pro Trp TlIR
98 : F~- ~ * 107 CONST.
TTC ~GT GG~ GGG ACC ~ ~AS;G CTG GAA ~TC AGA ----> C :;G GCT
P~E: G~ GI-Y S:I.Y q!~R~Arg L~n GI~ I:le ARG --> ~G
~ J~_ Conserved amino acids are capitaliz~d and bold.
~ NOTE: Amino acid # 30:is a c~nserved Val and amir~o acid ~ 103 and #107 a conser-~ed Lys in 98% o~ the sequences ~eported in Kabat's database for this ~ ~ family.

:~

' .r~ ~A WO 9~/10819 _57_ 2 1 2 3 ~ 0 7 PCI'/US92/10206 Table 6: COMPLETE SEQ~JENCE OF CHIMERIC 12$.1 (Anti-Human l~ransferrin ~eceptor) H13AVY CHAIN VA~IABIE REGION. MOUSE
GAMMP~ S~BGROUP IIBo ATG GAA TGG AGC: TGG GTA
Met Glu Trp Ser Trp Val ~DEa - 1 ATG CTC TTC CTC CTG TCA GGA ACT GCA GGT GTC CGC TCr ---Met Leu Phe I~:~ L.eu Ser Gly Thr Ala Gly Val Arg Ser - -FRl GAG GTC ~G CTG CAA CAG TCT G~;A CCT GAA CrG GTG AAG GCT
Glu ~ Gl~a I~l~ Gln GL~7 Ser GI.Y Pro Glu I-E~ YAI~ I.ys PRO
*18 FRl GGA GC'r TCA ATG AAG ATT TCC TGC AAG GGT TCT GGT TAC TCA

6$Y Ala ~ER Met LYS Ile 8~R CY8 LY8 AL~ 8ER GLY TYR Ser 31 CDRl 3 6 FR2 TTC: ACT GGC TAC ACC ATG AAC TGG GTG AA(; CP.G AGC CAT GGA
Phe Thr Gly Tyr Thr Met Asn T~P Vi~I. Lys S;I,N Ser His ~ly 52~-a~ 53 CDR2 GAG AAC CTT GA~ TGG Al~ GGA CGT A$~ C~ CAC AAT GGT
Glu Asn Leu Glu Trp Ile Gly Arg Ile Asn PRO His Asn Gly C~2 66 *68 ~ AAG GCC CCT TTA
Gly Thr P.sp TYR Asn Gln I.Y8 ~ Lys Asp LY8 Ala Pre~
FR3 82--a-A~T G~A ~;AC AAG TCA TCC AAC ACA GCC TAC A~G GAG CTC CTC
Val AE;P Lys 8ER Ser ~sn l~R Ala ~YR Met Glu L~E~ Leu 82b-c- 83 FR3 ,;~ :
AGT CTG ACA TCI GAG GAC TCT GCA t ;TC TAT TAC TGT GCA AGA
Ser Leu T~ 8E~II Gl.~ ? Ser AI~a Val ~YR Tyr C:YI~ Ala Arg 9S CDR3 100-a- 103 FR4 ~ A D~ ~GG GGT C~A GGA ACC ::
îy Tyr Tyr Tyr Tyx Ser Leu Asp qyr ~P ~ Y Gln ~I.~ T~
:
ER4 ~13 CHl GTC. Aec GT~ TCC T~ ----> GCC AAA
Ser V~ lR ~ 8~:~ Ser --~ Ala Lys :-a-,~ :
~:' ~onserved an~ino acids are capitalized and bald. Amino acid # 18 is a conselved Val and amino acid ~ 68 a conselved Thr in 9~% of the s~quences reported in K~bat's dalabas~ ~or this ~amily~

W093/l0819 21 ? 3 ~ o 7 PCT/US92/10^

The final clones were named pBKS4600 for the VH
region and pBKS4601 for the VL region.

CLONING INTO EXPRESSION VECTORS:

Plasmid pAH4274 is the vector for expression of heavy chain variable regions obtained by PCR with leader/J region priming. V region cloning into this cassette is performed by a complete digestion of vector and product with EcoR V and Nhe I. This vector has a human yl constant region whose CH1 is directly linked with the 3/ end of the VH-J region by means of a Nhe I site. This 11 kb vector contains an ampicillin resistance gene for procaryotic selection, a heavy chain immunoglobulin enhancer and a histidine (histidinol) selection marker for selection of transfectants (Hartman, S~, R. Mulligan, Proc. Natl.
Acad. Sci. 85, 8047-B051); transcription is from the VH promoter of the murine 27.44 gene.
The 400 bp. EcoR V-Nhe I fragment (VH of 128.1) from pBKS4600 was used to replace the EcoR V-Nhe I
fragmen in plasmid pAH 4274. HB101 competent cells `
were transformed and plated on LB plates with 50 ~g/ml of ampicillin~ ~Colonies were screened by colony hybridizat1on with a 32p end labelled leader region o1igonucleotide. Positive clones were restriction mapped and maxi plasmid preps prepared using the QIAGEN maxi prep kit (QIA~EN In~., Studio City, California;). The final expression vector wi~h the VH of 128.1 joined to human ~1 constant region ;:

'~ WO93/10819 21~ 3 3 ~ 7 PCT/USg2/10206 was named pAH4602 (Figure lO). The coding sequence for this expression vector is given in Figure ll.
Plasmid pAG4270 is the expression vector for light chain variable regions obtained by PCR with leader/J region priming. The 14 kb vector has an ampicillin resistance gene, a ~ (mycophenolic acid resistance) selected marker, an immunoglobulin H
enhan~er and an introl for V-Constant region ~plicing; transcription is from the murine VH
promoter from the 27.44 gene.
Due to the presence of an EcoR V within the ~E~
gene in the vector, the cloning of the anti-transferrin receptor V~ was performed in two steps to avoid inef~icient partial digestions. The 380 bp ~coR V - Sal I fragment (VL) from pBKS4601 was cloned into pBR460x (6.9 kb), a subcloning vector with the VH promoter, previously cut with the same enzymes. The resulting construct (pBK4608) was then cut with Pvu I - Sal I and the 4 kb fragment containing the promoter, the V region and part of the ;ampicillin resistance gene was ligated to the 9.7 kb Pvu I - Sal I fragment of pSV427l an intermediate vector which lacks thé promoter. HBlOl competent cells were transformed and positives screened by colony hybridi~ation and restriction digestion.
Maxipreps were prepar~ed as described above. The final expression vector was named pAG4611 (Figure 12). The coding sequence of this expression vector is shown in Figure 13.

..

W093/10819 2 ~ 2 3 ~ Q 7 PCT/US92/1~2~

TRANSFECTION AND SELECTION:

Ten ~g of maxiprep DNA from each final expression vector was linearized by BSPCl (Stratagene, Pvu I isochizomer) digestion and lxl0 SP2/0 cells were cotransfected by electroporation.
Prior to transfection the cells were washed with cold PBS, then resuspended in 0.9 ml of the same cold buffer and placed in a 0.4 cm electrode gap electroporation cuvet*e tBio-Rad) with the DNA. For the:electrical pulse, the Gene Pulser from Bio-Rad (Blo-Rad, Richmond, California) was set at a capacitance of 960 ~F and 200 V. After the pulse the cells were incubated on ice for l0 minutes then washed once in IMDM with 10% calf serum and resuspended in ~MDM wlth 10% calf serum at a concentratio~ of 105 cells/ml.
:
The transfected cells:were:plated into five 96 well plates at a concentration of 104 cells/well~
Selection:was started~after 48: hours. Two plates were selected with 5~mM histidinol ~heavy chain s~election), 2 plat~es~were selected with l ~l/ml mycophenolic àcid (light chain s lection) and l plate was~selected~with histidinol and mycophenolic acid (heavy:~and~ light chain selection).
Twelve d:ays~post selection supernatants were screened ~y~ELISA o test for the secretion of both chains. Immulon:II 96 well plates were coated with 5 yglml of goat anti~human ~l in carbonate buffer at pH9.6,~ and blocked wlth 3~ BSA. Supernatants from the~transfectants were added and the plates were ~ W093~10819 PCT/US92/10206 21233~7 incubated overnight at 4C. ~fter washing, plates were developed with goat anti-human k conjugated with alkaline phosphatase and wells secreting H and L
chains identified (Table 7).
Table 7: R~SU~TS OF TRaNsFEcTIoNs Results of cotransfe~tion with vectors p~H4602 and pAG4611 in SP2/0 cells. 2 plates were selected with 5mM histidinol (HIS), 2 plates with l~g/ml mycophenolic acid (HXM~ ~nd 1 plate selected with both (HIS+HXM). Wells containing clones were analyzed by ELISA to determine those containing secreted antibody (# positive wells)~
, SEIECTION

: :HIS HXM ~IS ~ ~XM

#WE~IS:~WITH 78/96 76/96 13/96 :
~LONES: 83/96 64/96 :~ :: ~:: : ~ :
~ ~POSITIVE 20/78 28/76 lO/13 ~
.
WELLS: ~ ~ ~ 25/83 :~ 20/64 High:producers~were expanded for further analysis; ~:
selected~ transfectants were subcloned.
, A~TIBODY ANALYSIS~

To dstermine the nature of the protein being :~ produced, transfeetan~s~were biosynthetically lab:el1ed:with~35S mèthionine, cytoplasmic and secreted antibodies~immunoprecipitated with rabbit . .

: ' .:

WO 93J1081g 2 1 ? 3 ~ ~ 7 PCT/US92/102' anti-human Ig and protein-A and the immunoprecipi-tates fractioned on SDS polyacrylamide gels.
Clones with the highest production identified by EL~SA were expanded to 5 ml petri dishes and removed from selection. lxlO cells were pelleted at 220xg for 5 minutes a~ 4~C and washed twice with labelling medium (high glucose DME deficient in methionine:
GIBC0). Cells were finally resuspended in l ml labeling medium containing 25 ~Ci35S-Methionine (Amersham Corp.) and allowed to incorporate label for 3 hours at 37C under tissue culture atmospheric conditions.
Cells were pelleted and supernatants drawn off for immunoprecipitation of secreted IgG. Cell pellets ~ere lysed in NDET (1% NP-40, 0.4%
deoxycholate, 66 mM EDTA, lO mM Tris, pH 7.4), centrifuged, and the supernatants removed and ~-incubated l hour a~ 4C with rabbit anti-human IgG Fc polyclonal antiserum (5 ~l/ml). To the labelled ~: ~s~pernatants, lO0 ~l/ml of protein A (10% in NDET, IgG Sorb) was added and mixed by rotation at 4C for 15 minutes. Protein-A bound IgG was washed by centrifuging through l ml 30% sucrosP in ioo ~1 NDET
~ 0.3% SDS. The protein A pellet was then : resuspended in lO0 ~1 NDETt3% SDS, transferred to a l.5 ml polypropylene tube with lO0 ~l of the same buffer, and the previous tube rinsed wi~h lO0 ~l.
The 300 ~l suspension was centrifuged and washed with deionized water. Finally, the protein A pellet was resuspended in 50 ~l of loading buffer (25 mMTris pH
6.7, 0.2% SDS, 10% glycerol, 8% ~g/lOO ml bromophenol 21~.3307 blue) and boiled for two minutes prior to gel loading. Antibodies were analyzed by SDS-PAGE ~5%
acrylamide gels, 0.1% sodium phosphate buffered) to confirm proper assembly of H and L chains. In addition, a portion of the labelled sample was reduced by treatment with 0.15 M 2-mercaptoethanol, :
37~C for l hour and analyzed on 12% acrylamide gels to confirm the size of the unassembled H and L
chains. The gels were stained, dried and exposed for autoradiograms.
The resultant autoradiograms revealed the expected patterns for fully functional antibodies.
The secreted antibodies that were in the cell supernatant exhibited the expected molecular wei~ht ~pattern of free light chain, light chain dimer and the tetramer formed ~rom two light chains and two ~heavy ch~ins for fully expressed and assem~led functional antibodies~ The pattern for antibody parts in the cell cytoplasm was also as expected for fully expressed ant;.body constitutents.

EXAMPLE 16 - Further~MnuselHuman Chimeras of the :.
:
Anti-Human Transferrin Receptor Antibody 128.l.
:.
: As described~in Example 15, the initial cloning ~ ~ : of:the gene encoding the h:eavy chain of the murine : monoclonal antibody 128.l, which binds the hu~an transferrin receptor, involved placing the se~uences :~ ~: encoding the variable region of the heavy chain into : ; ~ an expression vector c~ntaining the human ~l constant : region framework. This:created a mouse/human chimera ' .
: ~

WO~3/10819 ~CT/US~2/102r 2123~07 in which the sequences encoding the variable region of the antibody heavy chain ~VH) were derived from a murine source and the sequences encoding CH1, CH2 and CH3 were derived from a human source. Because the different human gamma isotypes (y~ 2, -3 and -4) have different biological properties, it was necessary to create chimeric antibodies with constant region sequences from each isotype in order to obtain mousethuman chimeras for each of these isotypes. The production of these chimeras was accomplished by cloning the 400 bp Eco RV-Nhe 1 fragment containing the VH region of antibody 128.1 from plasmid pBSK4600 into expression vectors containing the ~-2, ~-3 and ~-4 constant regions in a fashion similar to that previously described ln Example 15 for the cloning of the ~H region of antibody 128~1 into the expression vector containing the ~-1 constant region. These cloni~ngs wlth the y-2, ~-3 and ~-4 constant regions resulted~in respective plasmids pAH4625, pAH4807 and pAH4808 whose plasmid maps are shown in Figure 14, -~
Figure 15 and Figure 16, respectively. The antibody coding~sequences of the heavy chain expression vector~pAH4625, pAH4807 and pAHA808 are shown in Fig~ure~17, Figure 18~and Figure` 19, respectively.
These vectors, in~combination with the chimeric ;light~chain vector pAG4611, were transfected into SP2/~ cells ~and clones selected as described in Example 15. Initial antibody analysis using biosynthetically labeled~proteins,~immunoprecipi-tation and SDS-PAGE as previously described gave rise to~the appropriate bands for the heavy and light ~: : : : :
:: : :

:

`~ WO93/10819 PCT/US92/10206 21~3~97 chains as well as the assembled antibody for the ~ 3 and ~-4 chimeras. No detectable protein was made by the ~-Z transfectants.

EXAMPLE 17 - Antibody Production by Transfectants Antibody production by selected transfectanks was assessed by ELISA. Cells were diluted in fresh medium to a density of 1o6 cells/ml and 1 ml was aliquoted into each of 3 wells on a 24-well culture plate. The plates ~ere then incubated for 24 hours at 37C with 5% CO2. The media was then collected from the wells and the cells and debris were spun down to give a clarified supernatant. For the ELISA, a 96-well microtiter dish was coated with a goat antisera against human IgG. After blocking with 3%
BSA, the plate was washed and a series of dilutions of both the cell supernatants and human IgG standard of known concentration were applied to the plate and incubated for 1 hour at room temperature. The plate was then washed and biotinylated goat antisera against human IgG was added, followed by a mixture of avidin and biotinylated horseradish peroxidase (HRP).
The amount of antibody present in the samples was then determined, based on the amount of substrate converted by the HRP.
Three clones resulting from the ~-1 chimera transfection were tested for antibody production.
The average values from three experiments were 39, 21 and 24 ~g/ml IgG/10 cells/24 hours, respectively, for the dlfferent clones. One ~-3 clone has been .

W~ 93/10819 2 1 2 ~ 3 ~ 7 PCT/VS~2~102' tested and it was found to produce approximately 1 ~g/ml IgG/106 cells/24 hours. Two different clones of the ~-4 chimera have been tested and were found to produce 2.8 and 0.2 ng/ml IgG/106 cells/24 hours, respectively.

Equivalents Those skilled in the art will know, or be able to ascertain using no more than routine experimenta-tion, many equivalents to the specific embodiments expressly described herein. These are intended to be within the scope of the invention as described by the cl.aims herein.

Claims (24)

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1. An antibody-neuropharmaceutical or diagnostic agent conjugate for the manufacture of a medicament for delivering the neuropharmaceutical or diagnostic agent across the blood-brain barrier to the brain of a host whereby the antibody binds to a transferrin receptor present on brain capillary endothelial cells and the neuropharmaceutical or diagnostic agent is transferred across the blood-brain barrier in a pharmaceutically active form, wherein the antibody is a chimeric antibody that is reactive with said transferrin receptor.

2. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 1 wherein the chimeric antibody is a chimera between the variable region of a murine antibody and the constant region of a separate antibody.

3. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 2 wherein the constant region is of an animal source other than murine.

4. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 3 wherein the animal source is human.

5. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 2 wherein the variable region is from a monoclonal antibody produced by the 128.1 hybridoma.

6. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 4 wherein the variable region is from a monoclonal antibody produced by the 128.1 hybridoma.

7. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 6 wherein the constant region is from plasmids pAH4274 and pAG4270.

8. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 6 wherein the constant region is from plasmids pAH4625 and pAG4270.

9. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 6 wherein the constant region is from plasmids pAH4807 and pAG4270.

10. An antibody-neuropharmaceutical or diagnostic agent conjugate according to Claim 6 wherein the constant region is from plasmids pAH4808 and pAG4270.

11. A delivery system for delivering a neuropharma-ceutical or diagnostic agent across the blood brain barrier comprising a chimeric antibody, reactive with a transferrin receptor present on brain capillary endothelial cells, linked to a neuropharmaceutical or diagnostic agent, whereby the delivery system transports the neuropharma-ceutical or diagnostic agent across the blood brain barrier when administered in vivo.

12. A delivery system according to Claim 11 wherein the chimeric antibody is a chimera between the variable region of a murine antibody and the constant region of a separate antibody.

13. A delivery system according to Claim 12 wherein the constant region is of an animal source other than murine.

14. A delivery system according to Claim 13 wherein the animal source is human.

15. A delivery system according to Claim 12 wherein the variable region is from a monoclonal antibody produced by the 128.1 hybridoma.

16. A delivery system according to Claim 14 wherein the variable region is from a monoclonal antibody produced by the 128.1 hybridoma.

17. A delivery system according to Claim 16 wherein the constant region is from plasmids pAH4274 and pAG4270.

18. A delivery system according to Claim 16 wherein the constant region is from plasmids pAH4625 and pAG4270.

19. A delivery system according to Claim 16 wherein the constant region is from plasmids pAH4807 and pAG4270.

20. A delivery system according to Claim 16 wherein the constant region is from plasmids pAH4808 and pAG4270.

21. A chimeric antibody comprising a variable region reactive with a transferrin receptor present on brain capillary endothelial cells and a constant region of a separate antibody.

22. A chimeric antibody of Claim 21 wherein the variable region is of murine origin.

23. A chimeric antibody of Claim 22 wherein the constant region is of an animal source other than murine.

24. A chimeric antibody of Claim 23 wherein the animal source is human.