CA2146854A1 - Recombinant specific binding protein - Google Patents
Recombinant specific binding proteinInfo
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- CA2146854A1 CA2146854A1 CA 2146854 CA2146854A CA2146854A1 CA 2146854 A1 CA2146854 A1 CA 2146854A1 CA 2146854 CA2146854 CA 2146854 CA 2146854 A CA2146854 A CA 2146854A CA 2146854 A1 CA2146854 A1 CA 2146854A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
A specific binding protein having first and second binding regions, e.g. antibody Fv frag-ments, which specifically recognise and bind to target entities, said binding regions being con-tained at least in part on respectively first and second polypeptide chains, said chains addition-ally incorporating respectively first and second associating domains, e.g. antibody VH and VL
domains, which are capable of binding to each other, causing the first and second polypep-tide chains to combine, thereby providing a single protein incorporating the binding specif-icities of said first and second binding regions. The first and second binding regions may re-cognise different target entities, giving a bispecific binding protein. Preferably the associat-ing domains are derived from a human protein (i.e. one which has been exposed to the human immune system), so that the protein is less likely to provoke the human immune sys-tem when administered therapeutically. The binding protein is suitably produced by recom-binant DNA expression.
domains, which are capable of binding to each other, causing the first and second polypep-tide chains to combine, thereby providing a single protein incorporating the binding specif-icities of said first and second binding regions. The first and second binding regions may re-cognise different target entities, giving a bispecific binding protein. Preferably the associat-ing domains are derived from a human protein (i.e. one which has been exposed to the human immune system), so that the protein is less likely to provoke the human immune sys-tem when administered therapeutically. The binding protein is suitably produced by recom-binant DNA expression.
Description
21~6854 ~ '0 94/09131 PC~r/G B93/02133 RECOMBINANT SPBCIFIC BINDING PROTBIN
This invention relates to recombinant bispecific (heterodimeric) and/or monodimeric bivalent specific bin~;ng proteins, for example antibodies.
Since the development, in 1975, of monoclonal antibody hybridoma technology (ref 1) it has been possible to produce a large number of specific antibodies which have been used widely as research, therapeutic and industrial lQ tools. However, general experience indicates that murine monoclonal antibodies display reduced therapeutic efficacy which may be attributed to elicited immllnoglobulin responses by the hllmAn ;mmllne system, or alternatively poor initiation of defence mech~n;sms such as complement lS activation or the recruitment of human effector cells or have inappropriate pharmcokinetics in vivo.
Approaches to circumvent these limitations have been achieved by the use of hllm~n;sed monoclonal antibodies (ref 2), antibody fragments (refs 3 and 4) and ~Ibispecific"
antibodies (monovalent bispecific reagents)(refs 5 and 6).
Bispecific antibodies may be described as reco-m-binant antibodies capable of binding two different antigenic sites and thus contain antigen binding domains derived from two different sources, and which are brought into association by complementary interactive domains within the antibody molecule.
Bivalent antibodies of a monospecific nature may be derived from hybridomas and similarly, bispecifics by the WO94/09131 21~ ~ ~ 5 ~ PCT/GB93/0213 ~
fusion of two hybridoma lines expressing antibodies with different specificities. However, using this strategy, the application of bispecifics has been limited by the difficulty in efficiently producing and purifying such molecules and additionally, the effector functions intrinsic to complete antibody molecules (such Fc receptor and complement b;n~;ng) have led to undesirable interactions.
Recombinant DNA technology coupled with the recent advances in the fields of monoclonal antibody and protein engineering has enabled access to large selection antibodies and antibody fragments which have monospecific recognition and b;n~;ng properties for a wide variety of different antigens. The lack of effector functions and the a~ility of immllnoglobulin Fab, Fv and single chain Fv (sCFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates for drug delivery systems and imaging tools (ref 7). However, unlike complete antibodies, these fragments are monovalent, each carrying a single antigen binding site Dimerisation techniques which promote the formation of bivalent complexes (or even the formation of multivalent complexes) may therefore be of great importance when one considers constructs, for example Fv or Fab fragments, in which the monovalency of the product precludes polyvalent b; n~i ng to the antigen, providing an avidity factor.
In addition, most bacterially produced Fv, Fab and even scFv fragments have shown lower avidities than their 2l~c8~i ~'094/09131 PCT/GB93/02133 progenitor monoclonal antibodies (refs ~ and 9). Since there is no extra stabilization provided by the association of other ~om~; n~, as in the case of a complete antibody, the stability of Fv's is substantially dependant on heavy and light chain variable domain association. Since the heavy and light chain ~ i n association stability is dependant on the residues displayed at the domain interface (which is partly formed by residues belonging to the CDR
regions) the dissociation constants for Fv and similiar constructs may vary greatly from antibody to antibody. Thus antibodies with poorly associating variable regions are likely to show lower binding affinities, relative to their parent molecules, when in a small.fragment configuration.
Therefore there are good reasons to develop methodologies for the production of dimeric antibodies which may allow one to increase the binding affinity of a particular antibody via bivalent binding and/or additional stability due to further ~om~; n association.
In addition to the production of bivalent antibodies, a major application of mol~cnl~r dimerisation lies in the production of "bispecific~ or ~bifunctional~ antibodies in which the association is by definition ~heterodimeric"
(each subunit cont~in;ng different V ~om~;n~)~
In vitro experiments have shown that bispecific antibodies can be effective in cross-linking cytotoxic effector cells to target cells and stimulating the cytotoxic destruction mechanism. Similarly, bispecific antibodies may be utilized to associate and bring into WO~4/09131 PCT/GB93/0213 close proximity certain molecules eg. a drug or a toxin, a radiolabelled hapten or inactivating protein to other entities, such as a particular protein or cell type, via the specific recognition and binding of the individual binding ~om~;n~. In addition, such ~IbifurLctional~
antibodies may also be used to develop novel ;m~llnoassays and/or diagnostic assays. Since the bispecific antibodies are capable of binding to two distinct antigens, they may be designed to bind enzymes, therapeutic agents, radiolabels or cells to a target without the need for chemical modification. Hence bispecific entities are likely to demonstrate more efficient and specific cell killing than antibodies directly conjugated to cytotoxic agents and furtherm~re avoid any of the adverse ;mmllne lS responses which may be elicited by chemically modified antibodies.
Bispecific antibodies are likely also to prove useful for accelerating and promoting the association/dimerization of other molecules which may have themselves signalling, effector or reporter functions when in a associated/dimeric state. Therefore, bifunctional antibodies may also have an important role as ~Imolecular switches~ as well as ~cross-linkersll together with a multitude of other uses (both diagnostic or therapeutic) to which a dual binding specificity may be applicable.
Although the possible roles for which bispecific antibodies may be advantageous are numerous, the clinical applications and other uses for bispecific antibodies have ~ 094/09131 ~8~-~ PCT/GBg3/02133 been limited by the difficulty in efficiently producing and purifying such molecules.
Methods used to assemble bispecific or other dimeric antibodies include chemical cross-linking, disulphide exch~nge or the utilization of hybrid-hybridomas or heterotransfectomas. These methods have proved as yet unsatisfactory due to the purity of the products required for successful construction, the production of heterogeneous and ill defined products and especially in the case of Fab fragments the lo~ affinity of the partners (Fd,VLCL) for each other.
To address these problems a recent development for the production of heterodimeric F(ab)2 molecules has employed the C-t~rm;n~l fusion of peptide sequences corresponding to 1~ the leucine zipper regions of the Fos and Jun transcription factors (Kostelny et al., J. Tmmllnol, 148, 1547-1553, 1992)(reflO). These se~uences preferentially form heterodimers and therefore are effective in promoting bispecific formation when fused to two different Fab fragments.
To achieve bivalency (i.e. two bin~ing sites with the same specific within one antibody based entity), Pack and Pluckthun (1992)(ref 11) designed mini-antibodies based upon single chain Fv fra~ments with a flexible hinge region and an amphiphilic helix fused to the C-t~rminlls of the antibody fragments. They have shown that such molecules have antigen binding characteristics nearly identical to whole antibodies. A different approach has been described WO94/09131 ''` S ~ 6 PCT/GB93/0213 by Carter et al., (1992)(refl2) based upon the expression of Fab in E. coli and subsequent chemical linking of free thiols to generate (Fab') 2 .
According to one aspect of the present invention there is provided a specific binding protein having first and second binding regions which specifically recognise and bind to target entities, said binding regions being contained at least in part on respectively first and second polypeptide ~h~ i n~, said ch~; n~ additionally incorporating respectively first and second associating domains which are capable of binding to each other, causing the first and second polypeptide chA; n~ to combine, thereby providing a single protein incorporating the binding specificities of said first and second b;n~;nq regions.
Preferably, said first and second binding regions recognise different target entities.
Suitably said associating ~om~;n~ are derived from an antibody (eg IgA, IgD, IgE, IgG, IgM), and much of the description which follows uses antibody ~om~;n~ by way of illustration. However, they could be derived from other molecules which are capable of associating, such as natural ligand/receptor combinations or binding regions of natural dimeric proteins. Preferably the associating ~om~;n~ are derived from a human protein (ie one which has been exposed to the human immune system).
Said associating domains may be two identical domains which are capable of association, even if they do not nonmally associate in nature. Conveniently said 094/09131 ~6~S~ PCT/GB93/02133 associating domains are selected from:
antibody VH and VL regions, antibody CH1 and CL regions, antibody VH.CH1 and VL.C~ regions.
S Preferably the first and second binding regions are antibody antigen-binding ~mA; ns ~ eg comprising VH and VL
regions contained in a Fab fragment or in a single-chain Fv fragment.
However, either or both of the binding domains could be derived from just one of the VH or VL regions of an antibody, most suitably from the VH region of an antibody (ref 13).
The invention also includes DNA encoding the polypeptides of such a specific binding protein; optionally contained in one or more expression vectors; host cells cont~i n i ng such recombinant DNA and capable of expression of the DNA to produce the polypeptides of the specific binding protein.
The invention also provides a process which comprises expressing the recombinant DNA in such a host cell to produce the polypeptides encoded thereby, and where necessary performing post-translation manipulation or processing of the polypeptides, to produce the specific binding protein.
The invention further provides a process for producing a specific binding protein as set forth above, which comprises:
(i) cloning or synthesising DNA encoding said first 2 1~ 6g51 8 and second bindi~g regions, (ii) joining in reading frame to the DNA of said first and second binding regions DNA encoding respectively said first and second associating domains, (iii) providing one or more expression vectors con~;n;ng said fused DNA constructs of (i) and (ii), (iv) inserting the expression vectors into a host organism, (v) culturing the host organism to express the encoded polypeptides, and (vi) causing or allowing said first and second associating domains to combine to form the specific binding protein.
Thus, in general terms the invention provides recombinant bispecific (heterodimeric) and/or monodimeric ~ivalent specific binding proteins, for example antibodies, in which the specific association of the component modules is accomplished by using the recognition and natural homo-or hetero-dimerization of additionally fused associating ~l~ m;~; ng, Methods for the production of the aforementioned bispecifics and/or dimerics (and variants thereof) are also disclosed.
Unless the context requires otherwise, the tenms ~antibody" and "antibody fragments~ are hereinafter used synonymously. Similarly the terms ~antibodyl' and 'l;mmllnoglobulinll are to be treated in a likewise m~nner.
The accompanying drawings show diagrammatically:
Fig 1 a general scheme for bivalent and bispecific ~ 094/09131 PCT/GB93/02133 9 ~ 8S~
antibody formation, Fig 2 an experimental example for bivalent recombinant antibody formation, Fig 3 an example of m; n;m~l configuration bispecific recombinant antibody formation, and Figs 4 to 9 examples of bispecific recombinant antibody formation.
Fig 10 shows the arrangement of genes in the various vectors used for antibody fragment expression in E.coli.
All coding regions were cloned downstream from a lac promotor in pUC 19. VH and VK and CH and CK are the antibody fragment variable (V) and constant (C) domains respectively, Pb denotes a Pel B leader sequence and the Tag gene encodes a peptide for antibody fragment detection with a secondary antibody. The Tag gene product was not used in these studies. The linker and termination signal were engineered into the vectors as described in the materials and methods. pSW1-Fab/Pa is identical to the pSW1-~HD1.3-VKD1.3-TAG1 vector descri~ed by Ward et al.
(1989) with the exception that the VH and HK ~om~;n~ encode - anti-P. aeruginosa rather than anti-lysozyme binding m~in~. pDMl iS a derivative of pSW1-Fab/Pa.
Fig 11 shows denaturing SDS-PAGE of affinity purified anti-P. aeruginosa Fab (lane 2) and ScFv* (lane 3). 2-5 mg of each sample were electrophoresed through a st~n~rd denaturing 10% SDS-PAGE gel.
Fig 12 show the relative antigen binding capacities of affinity purified anti-P. aeruginosa Fab (-) ScFv*(-) and WO94/09131 21~ 6 8 5 ~ PCT/GB93/0213 ~
the original ~h i meriC antibody (O).
Fi~ 13 shows a Western blot showing the cross-reactivity of the anti-P. aeruginosa with a number of Gram-ve bacteria.
s Fig 14 shows the different confonnational forms of anti-P. aeruginosa ScFv* and Fab. (a) A 7.5% non-denatruring polyacrylamide gel showing multimeric forms (I-III) of ScFv* (lanes 1-3) and a single monomeric form of Fab (lane 4). HPLC purification of monomeric (III), dimeric (II) and trimeric (I) forms of ScFv*.
Fig 15 shows a comparison of the ability of HPLC
purified monomeric ScFv* (-), dimeric ScFv* (~) and the original ch;meric antibody (o) to bind P. aeruginosa.
The advantage of the present invention, for the association of the bispecific heterodimer or simple homodimeric bivalent formation, over existing techniques lies in the given nature of the fused domains utilized for this purpose. One of the major disadvantages of chemical modification or the Jun/Fos type approaches concerns their 2~ potential to elicit an immune response against llforeign'' chemical constituents and peptide sequences when used in human therapy. The technique of usin~ antibody domains to achieve dimerization of antibody constituents avoids this problem by using natural antibody domains which should not induce an immune response in hl]m~n~ especially where the antibody ~om~;n~ are reshaped (htlm~n;sed) or derived from natural hllm~n antibodies. Such dimeric antibodies ought to closely approach the ;mmllnogenic characteristics of a ~ 094/09131 2 i ~ 6 ~ tl~ ~ PCT/GBg3/02133 natural human antibody and thus should have an extended half-life in circulation compared to bispecifics prepared by other means as discussed previously.
In addition to promoting dimerisation through specific intermolecular recognition, the use of fused C-terminal ; n~ (with known dimerisation properties) is likely to lead to stabilization of the dimeric constructs produced due at least to the increase in area of hydrophobic interface interaction. For example the association of heavy and light ch~;n~ (KA >11 M-l) is derived from the com~ination of relatively weak VH_VL (KA-106 M-l) and CH1 CL
(KA-107 M-l) interactions, indicating that the association of the two ~om~; n pairs is at least additive (ref 14).
The present invention relates to the development of lS bivalent and other dimeric antibodies especially for use in therapy or for in vivo diagnosis. The successful targetting of antibody molecules to sites of disease upon repeated administrations is dependant on these antibodies provoking little or no immune reaction. M~;mi sation of the natural human antibody sequence content of the bispecific antibodies will prospectively enh~nce the perspective for long-term repeat administration of these antibodies, for example in the treatment of chronic disease.
Notwithst~n~i ng this, other applications as yet 2~ undetermined or not described herein for which mono-, bi-, or multi-specific antibodies/antibody fragments (produced via the means of association described in this invention) may be applied will be also encompassed by this patent.
W O 94/09131 2~ 8 5 ~ 12 PC~r/GB93/021 The general concept of association of specific b;nfling ch~ i n~ which is applicable to the present invention may be illustrated by reference to figure 1. In this diagram, the entity has been divided into four components A, B, C and D
in order to simplify discussion of the inventive process.
As previously discussed, a wide variety of antibodies (and fragments thereof) are readily available. Thus, there are numerous combinations of constructs which may be produced within the parameters of this invention. One may consider the specific specific binding regions or the associating ~mAin~ as derived variously from such entities as Fab~ FV~ SCFV' Fd~ VL/CL~ Fc or F(ab)~ fragments, or individual ~lom~; n~ such as CH3~ C~2, CH1, CL~ VH or VL (or even various combinations of these domains).
The specific binding regions and the associating ~om~; n~ are fused to each other either directly or via peptide linkers of different lengths (the linker sequence composition and length, being tailored to the particular antibody configuration and foreseen application). The components B and D may be n~m;n~lly seen as additionally fused ~om~;n~ although in certain configurations they may themselves be part(s) of the same antibody from which A and C are derived. The fragments or domains may be derived from one or more sources depending again on the chosen format and foreseen application of the particular construction. A further augmentation, also foreseen in this invention, is that for particular molecular configurations and destined applications it may be ~ 094/09131 2~ ~ 685~cT/GBg3/o2l33 advantageous to introduce or use existing cysteine residues to create inter-domain disulphide bridges in order to further stabilize the constructs. The reduction to produce these covalent bonds may be performed in vivo or in vitro depending on the construct and relative merits of this additional process.
It will be apparent to those skilled in the art that the choice of any particular type of antibody or antibody fragment is not flln~mPntal to the inventive concept described here. Similarly any immllnogobulin which provides the desired specificity may be employed. An extension of the invention also foresees the use of any natural molecules (or fragments thereof) which form specific associations (such as cell surface receptors and their ligands) which have been exposed to the immune system and show little or no ;mml~nogenicity within the said system.
A valuable embodiment of this invention foresees the dimerization of the constituent partners via the homodimeric association of additionally fused antibody ~oma;n~ (or other ;mml~nQ-silent molecules). Such a strategy may be used to produce dimeric antibodies. This embodiment is expected to be more suited towards the dimerisation of single antibody species to form homodimeric, bivalent antibodies, since is recognised that a mix of product assemblies may arise when this technique is used for the co-expression and association of two different antibody species to form a heterodimeric, bispecific antibody.
WO94/09131 2 1~ PCT/GB93/0213 The following specific description illustrates how the invention may be carried out. The ~ReferenCe example"
provides an illustration of the basic technology by which specific binding and associating fragments can be fused and caused to associate, while Examples 1 and 2 which follow provide specific embodiments of the invention to whose manner of execution can draw on the technology of the Reference example.
Reference Example The concept of using dimeric association of fused domains to enable formation of a bivalent species may be simply illustrated by the following example designed for production of a bivalent homodimeric (scFv) 2 antibody. With reference to figure 1, in this embodiment, the components A and D are V~ and VL ~m~; n~ respectively fused via a linker to a cognate VL and VH ~om~; n (components B and C
respectively). This format for the production homodimeric bivalent antibodies has been experimentaly tested using a simple model system (see Fig. 2) in which VH and VL ~nm~in~
of an anti-Pseudomonas aeruginosa antibody were fused via a linker peptide. This construct, when expressed in Escherichia coli and subsequently purified, was found to exist in monomeric, dimeric and multimeric forms as analysed by HPLC and non-denaturing PAGE. The dimeric fonm was pre~om;n~nt, and was found to have an antigen binding profile similar to the parent antibody, furthermore, it was found that the bivalent form was produced spontaneously.
~ 094/09131 2:~ 4 6 8 ~ ~ PCT/GB93/02133 ExPerimental For this study the pUC based pSW1-VHD1.3-VKD1.3-TAG1 expression vector (Ward et al., 1989)(refl3), encoding anti-P. aeruginosa (PSV) antibody heavy and light chain variable region genes was employed. A novel single chain antibody construct was generated a fourteen amino acid linker described by Chaudhary et al., (l990)(ref 15) by modification of the original Fab encoding vector. This novel scFv* construct possesses a hllm~n kappa chain domain fused to the 3~ end of the VK domain for detection purposes. Results indicated that when expressed, this scFv*
construct formed multivalent products spontaneously.
Bacterial strain All vectors were transformed and expressed in the E.
coli strain XL-1-Blue (supE44 hsdR17 recA1 endA1 gyrA46 the relA1 lacF' [proAB+ lacl9 lacZ M15 TnlO (tetr)]. The tetacycline selectable F' pilus allows strict control of expression of pUC based vectors.
Vectors construction The PSV scFv* expression vector was constructed form a pSW1-VHD1.3-VKD1.3-TAG1 vector derivative (pSW1-Fab/Pa) using the polymerase chain reaction (PCR) mutagenesis method of Higuchi (1989)(refl6). The linker was incorporated using a two stage procedure. Two separate amplifications were carried with the primers VHBAK:5l-AGGT(C/G)(C/A)A(G/A)CTGCAG(G/C)AGTC(T/A)GG
WO94/09131 21 ~ 6 8 5 ~ PCT/GB93/0213 with LINKFOR:5'-CGATGTCATCCACTTTAGATTCAGAGCCAGAGCCAGAAGATT
TGCCTTCTGAGGAGACGG
and VKFOR:5'-GTTTGATCTCGAGCTTGGTGCC
with T-T~RR~: 5'-GTCACCGTCTCCTCAGAAGGCAAATCTTCTGGCTCTGGCTC
TGAATCTAAAGTGGATGACATCGAGCTG.
10The vector pSWl-VHDl.3-VKDl.3-TAGl, expressing an anti-P.aeruginosa (pSWl-Fab/Pa), was used as template DNA.
The PCR con~ine~ 2 units of Taq polymerase (BCL, Lewes, East Sussex, England), 1.25mM dNTPs, lmM of each either VHBAK and LINKFOR or VHFOR and TT~RRAK, 50 ng pSWl-15Fab/Pa and 5~1 l0x reaction buffer (BCL) made up to 50~1 with de-ionised water. All reactions were W treated for ten minutes prior to the addition of polymerase and template, and then overlaid with mineral oil. PCR was performed using a Techne PHC-2 thermal cycler (Scotlab, Aberdeen, Scotland) using the following parameters: 94 C, l minute, 60'C, l minute, 72 C, l minute for 30 cycles. The purified products of these reactions were mixed and seven cycles of PCR were perfonmed without primers (94 C, minute; 72 C, l minute). This reaction was then maintained at 94 C while VHBACK and VKFOR primers were added. A
further 30 cycles of PCR were then performed (94 C, minute; 65'C, l minute; 72 C, l minute). The reaction products were electrophoresed through a 2% (w/v) agarose-094~09131 ~6 PCT/GB93/0213317 S~
TAW gel and the 65~ bp product purified into 10~1 of de-ionised water by the Prep-a-gene procedure (Bio-Rad Ltd, Hemel Hempsted, Herts., England). The product was digested with PstI and XhoI then ligasted into PstI/XhoI digested pSW1-Fab/Pa under st~n~Ard conditions to construct the pDM1 vector (fig 10).
Antibody fraqment exPression The expression vectors pDM1 encoding the anti-P.
aeruginosa scFv* antibody fragment and pSW1-Fab/Pa encoding the equivalent Fab fragment (each with the fused light chain constant region ~m~; n for detection purposes) were assessed. Expression conditions were modified from Ward et al, (1989)(ref 13): XL1-blue transformed with one of the above vectors was grown in 5ml 2xTY cultures contA;n;ng 1%
glucose, lOO~g/ml ampicillin and 12.s~g/ml tetracycline at 37 C, overnight. Ten microlitres of overnight culture was then used to inoculate fresh medium for expression of antibody fragments. Expression was carried out at 26 C for six to eighteen hours in Terrific broth or 2xTY contA;n;ng O.lmM IPTG, lOO~g/ml ampicillin and 12.5~g/ml tetracycline.
Antibody fragments were detected in culture supernatants by ELISA or could be purified directly from cell pellets as described below.
Affinity purification of antibody fraqments Anti-P. aeruginosa scFv* and Fab fragments were 2~468~
WO94/09131 ~ PCT/GB93/0213 purified by affinity chromatography. Five hundred millilitre cultures of bacteria producing either anti-P.
aeruginosa Fab or scFv* were centrifuged at 4000 rpm for 20 minutes. The supernatant was discarded and the cell pellet sonicated for l minute in lOml of PBS to release periplasmic antibody fragments. This solution was concentrated to a lml volume in a Centricon-lO column (Amicon, Stonehouse, Glos, England) and loaded onto an anti-human FAb affinity column (Pierce, Warriner, Cheshire England) prepared according to the manufactures instructions. Seven lml fractions were eluted in O.lM
glycine buffer pH2.8. The fractions were dialysed overnight against PBS and analyzed by polyacrylamide gel electrophoresis.
Detections of antibody fraqments for sandwich ELISA
Bacterial supernatants and antibody fragments tFAb and scFv*) purified from cell pellets were assayed in 96 well flat bottomed polystyrene plates (Nunc, Denmark). The plates were coated overnight at 4'C with l~g affinity purified goat anti-human IgG Fab antibody (Sigma Chemical Co., Poole, Dorset, England) in 50~1 phosphate buffered saline (PBS). The plates were blocked for l hour at 37 C
with 200~1 of 2% (w/v) BSA (fraction V, Sigma) in PBS and then washed twice with 200~1 PBS. Fifty microlitres of culture supernatant or purified antibody fragments were added to each well and the plates incubated at 37 C for l hour. After this time the plates were washed four times ~ 094/09131 ~6~ PCT/GBg3/02l33 withe 200~1 0.05% (v/v) Tween 20 in PBS (Tw20/PBS). Fifty microlitres of horse radish peroxidase coniugated goat anti-human IgG Fab antibody (Sigma), diluted 1: 2000 in TW20/PBS was added to each well and incubated at 37 C for 1 hour. The plates were then washed six times in 200~1 TW20/PBS and the assay developed with 50~1 lmg/ml 0-phenylendiamine dilydrochloride (Sigma) and 1~1/ml 30%
(v/v) hydrogen peroxide (Sigma) in O.lM citrate-phosphate buffer pH 6Ø The reaction was stopped with 100~1 0.5M
citric acid and the optical density at 450nm was read using a Bio -Rad M450 plate reader. Positive control anti-P
psell~omon~s mouse-hllm~n ~h;m~ric IgG1 antibody was provided by Scotgen Ltd, Aberdeen. This antibody was derived from the original murine patent ;mmllnoglobulin from which the Fab and scFv* variable ~om~; n~ were obtained.
Detection of antiqen bindinq of ELISA
This assay was performed essentially as the ELISA
described above but with the following modifications. P.
aeruginosa were grown overnight in Luria broth, pelleted an resuspended in PBS as a volume representing half of the volume of the original culture. The cells were then heat treated at 65 C for 10 minutes. 96-well polystyrene plates were coated overnight with 50~1 of heat killed P.
aeruginosa. After blocking the plates with TW20/PBS and washing with PBS, 50~1 of bacterial supernatant, purified antibody fragments or the mouse/hllm~n ch;mPric anti-P.
aeruginosa antibody supernatant were added to each well and WO94/09131 214 ~ ~ S ~ PCT/GB93/0213~
incubated at 37 C for one hour. After the plates were washed four times with PBS the assay proceeded as described in the previous section.
Western blottinq The specificity of the scFv* expressed was investigated by Western blotting. One million P. aeruginosa, P.
fluorescens, Al. xylosidans, Ac. calcoaticus and O.
anthropi were electorphoresed through a st~n~rd 10% SDS
polyacrylamide gel with Rainbow size markers (Amersham Int., Aylesbury, Bucks, England). The gel was electorblotted at 0.8 mA/cm2 in a Hoeffer T70 semi-dry blotter onto Immobilion-P membrane (Millipore Corp., Bedford, Mass, USA). The membranes were blocked overnight in 5% (w/v) milk powder in PBS. scFv* (l~g/ml) was incubated for one hour in 0.1% (v/v) Tween 20 in PBS followed by three l0 minute washed in 1% (v/v) Tween 20 in PBS. The membrane was then incubated with a l:5000 dilution of an anti-hllm~n kappa chain or anti-human Fab antibody conjugated to horse radish peroxidase in 5% (w/v) milk powder, 1% (v/v) Tween 20 in PBS for 30 minutes at 37 C.
scFv* binding was detected using the ECL chemiluminescent system (Amersham Int.).
Non-denaturinq polyacrylamide qel electrophoresis Affinity purified Fab and s~Fv* were separated on a 7.5% non-denaturinq polyacrylamide gel using the method of Ornstein (1964)(ref 17) to determine the conformation of 094/09131 ~ 8 S~ PCT/GB93/02133 the molecules.
HPLC purification of scFv* fractions Forty microgram samples of scFv* were analyzed using a Zorbax GF250 size exclusion column on a Gilson HPLC
comprising a model 302 pump, an 802L mAnom~tric module, a 811 dynamic mixer, a 116 W detector and a 201 fraction collector controlled by Gilson 714 software. The analysis was done using both PBS and 0.2M sodium phosphate buffer at a flow rate lml/minute. Eluate was monitored at 280nm, OlAUFS and 0.5 ml fractions were collected and analyzed by ELISA and non-denaturing polyacrylamide gel electrophoresis.
scFv* and Fab Purification and antiqen b;n~;nq The presence of the light chain constant region allowed purification of both Fab and scFv* directed against P. aeruginosa. Similar amounts (2-5mg) of both proteins were obtained when periplasmic cell extracts derived from 500 ml of IPTG induced overnight cultures were loaded onto affinity columns. The purity of the eluted proteins was assessed by denaturing SDS-PAGE with Coomassie Blue st~;ning (fig ll).
The relative antigen binding capacities of affinity purified Fab (pSWl-FAb/Pa), scFv* and chimeric antibody were compared. The c~;mPric antibody and the scFv* gave similar binding profiles, indicating that the scFv* had an affinity close to the original monoclonal antibody. Fab binding was WO94/09131 2-~ 4 ~ 8 ~ ~ 22 PCT/GB93/0213 ~
2-3 times weaker than either that of c~;m~ric or scFv* (Fig 12). Western blGtting was employed to test the specificity of the scFv* protein. One band of approximately 21 kd was observed when scFv* was tested against P. aeruginosa antigens (Fig 13). Cross ~eactivity with Al. xylosidans and Ac. calcoaticus but not O. anthropi, F. mulitivoran or P.
fluorescens was noted (Fig 13). These are all Gram negative bacteria.
Analysis of scFv*
Differences in conformation of bacterially produced Fab and scFv* molecules was thought to be responsible for the changed affinities observed in ELISA. Non-denaturing gel electrophoresis revealed that Fab (pSWl-Fab/Pa) was identifiable as a single unit while scFv* was present as a series of multimeric forms (Fig 14a). ~onomeric and dimeric forms of scFv* appeared to pre~om;n~te. HPLC purification by size exclusion allowed separation of the monomeric and dimeric scFv* forms. Comparison of the elution times with those of molecular weight marker proteins confirmed the presence of both monomeric and dimeric forms of scFv* (Fig 14b). An ELISA was employed to determine the ability of the monomeric and dimeric sc~v* fragments to bind P.
aeruginosa and revealed that the dimeric form had affinity similar to the ~;meric antibody while the monomeric form had a lower affinity (Fig 15).
Discussion 094/09131 ~1 ~ 6~ PCT/GB93/02133 The bivalent component thus shows similar binding characteristics to the intact antibody while the monomeric fragment shows greatly reduced binding. Dimeric and m~om~ric forms can be easily separated by gel filtration, offering a simple method for preparing pure dimeric antibody.
The scFv* dimers dissociate into monomers in non-reducing SDS gels and appear, therefore, not linked by disulphide bonds (data not shown). Our interpretation of the data is that the dimer formation of the (scFv*)2 is due to non covalent intermolecular VH/VL domain association; the association occurring between ~omAin~ of different scFv*
polypeptides as indicated in figure 2. The resulting dimer is bivalent and shows a binding affinity to the ch;m~ric antibody for the P. aeruginosa antigen.
Further Methods For Bispecific Recombinant AntibodY
Formation The present invention discloses novel bispecific antibodies in which dimerization of the constituent partners is achieved via the heterodimeric association of additionally fused natural antibody domains. For example, antibodies may consist of two different Fab or scFv antibody fragments (or any other antibody/antibody fragment combination) linked to either a VH or VL polypeptide by gene fusion. The desired bispecific antibody is therefore specifically associated by the recognition and natural heterodimerization of the fused VH or VL domains. The =
W O 94/09131 PC~r/GB93/0213 ~
2~;~68~4 24 present invention also discloses methods for the production of novel bispecific antibodies.
The simplest format envisaged for the production of a bispecific antibody would be the association of two single chain antibodies scFvl and scF~2. If one relates to the general scheme shown in figure 1, the ~o~;n~ A and C would be VH1 and VL2 respectively, and domains B and D; VL1 and VH2 respectively (see Fig.3). In this m;m;nAl configuration it is clear that both the two domains of each individual peptide chA; n~ may associate to form normal scFvs and additionally, that homo- and heterodimers (and possibly larger multimeric associations) of the two peptide c~;n~
may be produced by intermol~clllAr ~om~;n association. A
preferred embodiment of this invention is a bispecific antibody, ~ormed by a C-term; n;l 1 fusion of either a VH or VL antibody domain (component B) to one scFv antibody fragment (component A) and its corresponding partner (component D) to the second scFv (component C). Such a species is illustrated in Figure 4.
Similarly for bispecific F(ab)2 heterodimers, either the VH or VL ( components B and D) could be added by C-term;nAl fusion to the VH/CH1 polypeptide ~A;n~ of the Fab fragments (components A and C). Using this approach, Fab dimerization, to form the bispecific antibody, should be 2S promoted by the spontaneous and specific association of the attached VH and VL ~lomA i n~; . See Figure 5.
Examples of other alternative embodiments of this invention may involve the use of other naturally occuring 094/09131 ~ ~683~ PCT/GB93/02133 antibody domains which form heterogeneous (dimers such as VH.CH1/VL.CL or CH1/CL) as C-terminal fusions (components B
and D) which are illustrated in figure 6-9. This and other possible confonmations have been alluded to previously in the text.
It will be apparent to those skilled in the art that the choice of any particular type of antibody or antibody fragment is not fl~n~Am~ntal to the inventive co~cPpt but it is clear that the specificity and affinity of the C-terminal ~om~;n~ chosen will greatly influence the endproduct(s). Any domain(s) which provides the desired dimerization and recognition specificity required may be employed. However, it is recognised that, for example, specific VH ~m;~;n~ will associate more efficiently with certain VL ~ ; nC than others (KD values for Fv~s varying from 10-4 M - 10-7 M)(ref 18) and thus the VH/VL binding pair of a specific bifunctional should possibly be optimised to promote heterodimer fonmation and also to m; n; m; se homodimer formation (especially when producing bispecifics). Similiarly it is also foreseen that molecules may be modified in order to improve the specificity and/or binding affinity of the molecules for each other and thus improve their dimerisation properties (ref l9). This, for example, may be achieved via engineering, eg mutation or chemical modification, of the inter-domain hydrophobic contacts, introduction of a metal (or other molecule binding site) between the ~om~;ns to stabilize association, introduction of strategic cysteine residues to form WO94/09131 ~ 26 PCT/GB93/0213 disulphide bridges, etc.
If supplementary Fv ~om~;n~ are created when producing a bivalent or bispecific antibody (as may be the case when using the VH/VL ~om~;n combination as fusions), the antibody may, in fact, be trivalent. This extra antigen binding site may be useful itself. If the binding site recognizes a different type of antigen or ligand than the other sites, this property may be used for purification (ligand bound to matrix), stability (ligand added and bound to site thereby ~cross-linking~l the two ~m~; n~ of the Fv) or as a label (added ligand-reporter complex). This idea would also be valid for bispecifics, if one type of binding site is used for ligand binding it may be used for the same purposes as described above for the trimer.
The present invention provides an antibody with a m;n;mllm of two Fv ~omA;nC each comprising of structural framework with the relevant CDRs which provide the antibody with the capability to bind specific molecules such as antigens. The invention may be exemplified ,but not limited, in a configuration in which the antigen recognition and binding ~nm~;n~ (and any other affiliated sequence such as constant regions etc) are fused at the C-t~rm;n~l end to "association~ domains such as either a VH
or VL antibody domain respectively. Association of the constituent partners is achieved via the natural heterodimerisation of the fused VH and VL antibody domains (see figs 4 and 5).
Processes for the production of such antibodies and 094/09131 27 ~ ~
genes encoding such antibodies are also provided by the present invention.
Corresponding to a preferred process of the present invention, there is provided a method to produce bispecific (scFv) 2.VH/VL or F(ab) 2.VH/VL entities (see figs, 4 and 5) or derivatives of these with either CH1/CL or VH.CH1/VL.CI, fused ~nm~;n~ (see figs.6-9). This method comprises the following basic scheme:
(i) Cloning or synthesis of the antibody Fv and~or Fab genes from the antibodies which exhibit the desired binding properties. The genes should be inserted into plasmid or phage vectors suitable for proliferation and /or gene expression.
(ii) The addition of the VH or VL t30m;l; n genes by C-term;n~l gene fusion to the respective antibody partners such that on correct heterodimerization of these added ~o~;n~ the bispecific construct should be created.
(iii) Transfer of the complete individal gene constructs to an appropriate expression vector or vectors (if not performed previously) and subsequent introduction to the host organism.
(iv) Growth of the host organism and expression of the installed recombinant genes to achieve antibody production.
(v) Purification of the antibody/antibodies and any ensuing biochemical manipulation to obtain the bispecific construct.
Naturally, the details of the individual steps will 2 ~ S ~ 28 be dependant on the form of the bispecific antibody to be produced e.g (FV)2~V~/VL or F(ab) 2.VH/VL, (see figs 4 and 5) whether they are to be co- or individually expressed and the host organism (e.g prokaryote or eukaryote) which is to be used.
The invention is illustrated but not limited by the following examples.
Example 1 This example illustrates the production of a bispecific antibody from two different scFv fragments expressed in Escherichia coli.
(a) Starting with the mouse hybridoma cell lines producing the monoclonal antibodies of interest, a suitable method for determ; n; ng the corresponding heavy and light chain variable sequences from hybridoma in RNA
is described by Orlandi et al., Proc. Natl. Acad. Sci.
USA, 86, 3833-3837, 1989.
(b) Genes encoding reshaped human antibodies (comprising the necessary mouse CDR and framework residues) which correspond to the original mouse monoclonals can be produced by site directed mutagenesis (see Riechm~n et al.). Alternatively genes encoding the reshaped human antibodies can be asse-m~bled by gene synthesis (Jones et al.)(ref 20).
(c) These genes can be manipulated using various molecular biology techniques to form scFv fra~ments as described by Bird et al., Science 242, 423-426, 1988 (ref 21) or Huston et al., Proc. Natl. Acad. Sci. USA 85, ~CYO 94/09131 2 PC~r/G B93/02133 _ 29 ~ ~ 6 ~ S
5879-5883, 1988 (ref 22).
Complete human VH or VL domains (naturally paired but preferably showing no specific binding relative to the antibodies utilized) can be added by C-terminal gene fusion, one to either of the two constituent scFv constructs.
(d) The completed genes can then be cloned into an appropriate expression vector(s) depending on whether they are to be expressed as in a two cistron system on one vector under a single promotor, expressed singularly from each carried on its own vector yet together in a co-transformed host or each individual grown in different cultures. Examples of such vectors may be obtained from the literature of Skerra et al., Science, 240 1038-1041, 1988 (ref 3), Ward et al., Nature, 341, 544-546, 1989 (ref 13), Bird et al., Science 242, 423-426, 1988 (ref 21~, Hoogenboom et al., Nuc. Acid Res. 19, 4133-4137, 1991 (ref 23)., etc. Further changes to obtain vectors suited to the needs of bispecific production may be necessary.
(e) Depending on whether the bispecific formation is to be performed in vitro or in vivo and the state of the products formed it may be pertinent to incorporate an additional reduction and reoxidation of the purified antibody fragments in order to obtain the desired bispecific (Bird et al., Science 242, 423-426, 1988 (ref 21), Skerra et al., Science, 240 1038-1041, 1988 (ref3), Kostelny et al., J. Tmml~nQl. 148, 1547-1553, 1992 (ref WO94/09131 ~ ~ ~ PCT/GB93/0213 10 ) . ) Similarly F(ab) 2.VH.VL fragments can be produced in E.coli by similar means and the VH/VL association technique may utilized for for other antibody fragments and other dimerization purposes (see fig 5) Example 2 This example illustrates the production of a bispecific antibody from two different Fab fragments expressed mAmm~ 1; an cell lines. The initial procedures are quintessencally the same as in example 1 (steps a and b) The construction of the bispecific shown in fig.5, could be made by the same technique as was used for the F(ab) 2 Jun.Fos. construction as described by Kostelny et al., J. Tmmllnol~ 148, 1547-1553, 1992 (ref 10). in which the one the additional dimerization ~m~ inS were fused to the first few residues of the CH2 intron such that it would be spliced to the CH1 of the VH gene thus replacing the nonmal CH2 and CH3. The VL/CL genes are expressed from a separate vector which is co-transfected with the VH carrying plasmid.
Again various strategies for expression and bispecific production may be attempted as have ~een detailed previously for E. coli expression.
094/09l3l 1~6 ~ PCT/GB93/02133 REF~RENCES
The following references are all here~y incorporated by reference.
1. Kohler G., & Milstein C., (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 265, 495-497.
2. Verhoeyen M., Milstein C., & Winter G., (1988).
Reshaping human antibodies: grafting an anti-lysozyme activity. Science, 239, 1098-1104.
This invention relates to recombinant bispecific (heterodimeric) and/or monodimeric bivalent specific bin~;ng proteins, for example antibodies.
Since the development, in 1975, of monoclonal antibody hybridoma technology (ref 1) it has been possible to produce a large number of specific antibodies which have been used widely as research, therapeutic and industrial lQ tools. However, general experience indicates that murine monoclonal antibodies display reduced therapeutic efficacy which may be attributed to elicited immllnoglobulin responses by the hllmAn ;mmllne system, or alternatively poor initiation of defence mech~n;sms such as complement lS activation or the recruitment of human effector cells or have inappropriate pharmcokinetics in vivo.
Approaches to circumvent these limitations have been achieved by the use of hllm~n;sed monoclonal antibodies (ref 2), antibody fragments (refs 3 and 4) and ~Ibispecific"
antibodies (monovalent bispecific reagents)(refs 5 and 6).
Bispecific antibodies may be described as reco-m-binant antibodies capable of binding two different antigenic sites and thus contain antigen binding domains derived from two different sources, and which are brought into association by complementary interactive domains within the antibody molecule.
Bivalent antibodies of a monospecific nature may be derived from hybridomas and similarly, bispecifics by the WO94/09131 21~ ~ ~ 5 ~ PCT/GB93/0213 ~
fusion of two hybridoma lines expressing antibodies with different specificities. However, using this strategy, the application of bispecifics has been limited by the difficulty in efficiently producing and purifying such molecules and additionally, the effector functions intrinsic to complete antibody molecules (such Fc receptor and complement b;n~;ng) have led to undesirable interactions.
Recombinant DNA technology coupled with the recent advances in the fields of monoclonal antibody and protein engineering has enabled access to large selection antibodies and antibody fragments which have monospecific recognition and b;n~;ng properties for a wide variety of different antigens. The lack of effector functions and the a~ility of immllnoglobulin Fab, Fv and single chain Fv (sCFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates for drug delivery systems and imaging tools (ref 7). However, unlike complete antibodies, these fragments are monovalent, each carrying a single antigen binding site Dimerisation techniques which promote the formation of bivalent complexes (or even the formation of multivalent complexes) may therefore be of great importance when one considers constructs, for example Fv or Fab fragments, in which the monovalency of the product precludes polyvalent b; n~i ng to the antigen, providing an avidity factor.
In addition, most bacterially produced Fv, Fab and even scFv fragments have shown lower avidities than their 2l~c8~i ~'094/09131 PCT/GB93/02133 progenitor monoclonal antibodies (refs ~ and 9). Since there is no extra stabilization provided by the association of other ~om~; n~, as in the case of a complete antibody, the stability of Fv's is substantially dependant on heavy and light chain variable domain association. Since the heavy and light chain ~ i n association stability is dependant on the residues displayed at the domain interface (which is partly formed by residues belonging to the CDR
regions) the dissociation constants for Fv and similiar constructs may vary greatly from antibody to antibody. Thus antibodies with poorly associating variable regions are likely to show lower binding affinities, relative to their parent molecules, when in a small.fragment configuration.
Therefore there are good reasons to develop methodologies for the production of dimeric antibodies which may allow one to increase the binding affinity of a particular antibody via bivalent binding and/or additional stability due to further ~om~; n association.
In addition to the production of bivalent antibodies, a major application of mol~cnl~r dimerisation lies in the production of "bispecific~ or ~bifunctional~ antibodies in which the association is by definition ~heterodimeric"
(each subunit cont~in;ng different V ~om~;n~)~
In vitro experiments have shown that bispecific antibodies can be effective in cross-linking cytotoxic effector cells to target cells and stimulating the cytotoxic destruction mechanism. Similarly, bispecific antibodies may be utilized to associate and bring into WO~4/09131 PCT/GB93/0213 close proximity certain molecules eg. a drug or a toxin, a radiolabelled hapten or inactivating protein to other entities, such as a particular protein or cell type, via the specific recognition and binding of the individual binding ~om~;n~. In addition, such ~IbifurLctional~
antibodies may also be used to develop novel ;m~llnoassays and/or diagnostic assays. Since the bispecific antibodies are capable of binding to two distinct antigens, they may be designed to bind enzymes, therapeutic agents, radiolabels or cells to a target without the need for chemical modification. Hence bispecific entities are likely to demonstrate more efficient and specific cell killing than antibodies directly conjugated to cytotoxic agents and furtherm~re avoid any of the adverse ;mmllne lS responses which may be elicited by chemically modified antibodies.
Bispecific antibodies are likely also to prove useful for accelerating and promoting the association/dimerization of other molecules which may have themselves signalling, effector or reporter functions when in a associated/dimeric state. Therefore, bifunctional antibodies may also have an important role as ~Imolecular switches~ as well as ~cross-linkersll together with a multitude of other uses (both diagnostic or therapeutic) to which a dual binding specificity may be applicable.
Although the possible roles for which bispecific antibodies may be advantageous are numerous, the clinical applications and other uses for bispecific antibodies have ~ 094/09131 ~8~-~ PCT/GBg3/02133 been limited by the difficulty in efficiently producing and purifying such molecules.
Methods used to assemble bispecific or other dimeric antibodies include chemical cross-linking, disulphide exch~nge or the utilization of hybrid-hybridomas or heterotransfectomas. These methods have proved as yet unsatisfactory due to the purity of the products required for successful construction, the production of heterogeneous and ill defined products and especially in the case of Fab fragments the lo~ affinity of the partners (Fd,VLCL) for each other.
To address these problems a recent development for the production of heterodimeric F(ab)2 molecules has employed the C-t~rm;n~l fusion of peptide sequences corresponding to 1~ the leucine zipper regions of the Fos and Jun transcription factors (Kostelny et al., J. Tmmllnol, 148, 1547-1553, 1992)(reflO). These se~uences preferentially form heterodimers and therefore are effective in promoting bispecific formation when fused to two different Fab fragments.
To achieve bivalency (i.e. two bin~ing sites with the same specific within one antibody based entity), Pack and Pluckthun (1992)(ref 11) designed mini-antibodies based upon single chain Fv fra~ments with a flexible hinge region and an amphiphilic helix fused to the C-t~rminlls of the antibody fragments. They have shown that such molecules have antigen binding characteristics nearly identical to whole antibodies. A different approach has been described WO94/09131 ''` S ~ 6 PCT/GB93/0213 by Carter et al., (1992)(refl2) based upon the expression of Fab in E. coli and subsequent chemical linking of free thiols to generate (Fab') 2 .
According to one aspect of the present invention there is provided a specific binding protein having first and second binding regions which specifically recognise and bind to target entities, said binding regions being contained at least in part on respectively first and second polypeptide ~h~ i n~, said ch~; n~ additionally incorporating respectively first and second associating domains which are capable of binding to each other, causing the first and second polypeptide chA; n~ to combine, thereby providing a single protein incorporating the binding specificities of said first and second b;n~;nq regions.
Preferably, said first and second binding regions recognise different target entities.
Suitably said associating ~om~;n~ are derived from an antibody (eg IgA, IgD, IgE, IgG, IgM), and much of the description which follows uses antibody ~om~;n~ by way of illustration. However, they could be derived from other molecules which are capable of associating, such as natural ligand/receptor combinations or binding regions of natural dimeric proteins. Preferably the associating ~om~;n~ are derived from a human protein (ie one which has been exposed to the human immune system).
Said associating domains may be two identical domains which are capable of association, even if they do not nonmally associate in nature. Conveniently said 094/09131 ~6~S~ PCT/GB93/02133 associating domains are selected from:
antibody VH and VL regions, antibody CH1 and CL regions, antibody VH.CH1 and VL.C~ regions.
S Preferably the first and second binding regions are antibody antigen-binding ~mA; ns ~ eg comprising VH and VL
regions contained in a Fab fragment or in a single-chain Fv fragment.
However, either or both of the binding domains could be derived from just one of the VH or VL regions of an antibody, most suitably from the VH region of an antibody (ref 13).
The invention also includes DNA encoding the polypeptides of such a specific binding protein; optionally contained in one or more expression vectors; host cells cont~i n i ng such recombinant DNA and capable of expression of the DNA to produce the polypeptides of the specific binding protein.
The invention also provides a process which comprises expressing the recombinant DNA in such a host cell to produce the polypeptides encoded thereby, and where necessary performing post-translation manipulation or processing of the polypeptides, to produce the specific binding protein.
The invention further provides a process for producing a specific binding protein as set forth above, which comprises:
(i) cloning or synthesising DNA encoding said first 2 1~ 6g51 8 and second bindi~g regions, (ii) joining in reading frame to the DNA of said first and second binding regions DNA encoding respectively said first and second associating domains, (iii) providing one or more expression vectors con~;n;ng said fused DNA constructs of (i) and (ii), (iv) inserting the expression vectors into a host organism, (v) culturing the host organism to express the encoded polypeptides, and (vi) causing or allowing said first and second associating domains to combine to form the specific binding protein.
Thus, in general terms the invention provides recombinant bispecific (heterodimeric) and/or monodimeric ~ivalent specific binding proteins, for example antibodies, in which the specific association of the component modules is accomplished by using the recognition and natural homo-or hetero-dimerization of additionally fused associating ~l~ m;~; ng, Methods for the production of the aforementioned bispecifics and/or dimerics (and variants thereof) are also disclosed.
Unless the context requires otherwise, the tenms ~antibody" and "antibody fragments~ are hereinafter used synonymously. Similarly the terms ~antibodyl' and 'l;mmllnoglobulinll are to be treated in a likewise m~nner.
The accompanying drawings show diagrammatically:
Fig 1 a general scheme for bivalent and bispecific ~ 094/09131 PCT/GB93/02133 9 ~ 8S~
antibody formation, Fig 2 an experimental example for bivalent recombinant antibody formation, Fig 3 an example of m; n;m~l configuration bispecific recombinant antibody formation, and Figs 4 to 9 examples of bispecific recombinant antibody formation.
Fig 10 shows the arrangement of genes in the various vectors used for antibody fragment expression in E.coli.
All coding regions were cloned downstream from a lac promotor in pUC 19. VH and VK and CH and CK are the antibody fragment variable (V) and constant (C) domains respectively, Pb denotes a Pel B leader sequence and the Tag gene encodes a peptide for antibody fragment detection with a secondary antibody. The Tag gene product was not used in these studies. The linker and termination signal were engineered into the vectors as described in the materials and methods. pSW1-Fab/Pa is identical to the pSW1-~HD1.3-VKD1.3-TAG1 vector descri~ed by Ward et al.
(1989) with the exception that the VH and HK ~om~;n~ encode - anti-P. aeruginosa rather than anti-lysozyme binding m~in~. pDMl iS a derivative of pSW1-Fab/Pa.
Fig 11 shows denaturing SDS-PAGE of affinity purified anti-P. aeruginosa Fab (lane 2) and ScFv* (lane 3). 2-5 mg of each sample were electrophoresed through a st~n~rd denaturing 10% SDS-PAGE gel.
Fig 12 show the relative antigen binding capacities of affinity purified anti-P. aeruginosa Fab (-) ScFv*(-) and WO94/09131 21~ 6 8 5 ~ PCT/GB93/0213 ~
the original ~h i meriC antibody (O).
Fi~ 13 shows a Western blot showing the cross-reactivity of the anti-P. aeruginosa with a number of Gram-ve bacteria.
s Fig 14 shows the different confonnational forms of anti-P. aeruginosa ScFv* and Fab. (a) A 7.5% non-denatruring polyacrylamide gel showing multimeric forms (I-III) of ScFv* (lanes 1-3) and a single monomeric form of Fab (lane 4). HPLC purification of monomeric (III), dimeric (II) and trimeric (I) forms of ScFv*.
Fig 15 shows a comparison of the ability of HPLC
purified monomeric ScFv* (-), dimeric ScFv* (~) and the original ch;meric antibody (o) to bind P. aeruginosa.
The advantage of the present invention, for the association of the bispecific heterodimer or simple homodimeric bivalent formation, over existing techniques lies in the given nature of the fused domains utilized for this purpose. One of the major disadvantages of chemical modification or the Jun/Fos type approaches concerns their 2~ potential to elicit an immune response against llforeign'' chemical constituents and peptide sequences when used in human therapy. The technique of usin~ antibody domains to achieve dimerization of antibody constituents avoids this problem by using natural antibody domains which should not induce an immune response in hl]m~n~ especially where the antibody ~om~;n~ are reshaped (htlm~n;sed) or derived from natural hllm~n antibodies. Such dimeric antibodies ought to closely approach the ;mmllnogenic characteristics of a ~ 094/09131 2 i ~ 6 ~ tl~ ~ PCT/GBg3/02133 natural human antibody and thus should have an extended half-life in circulation compared to bispecifics prepared by other means as discussed previously.
In addition to promoting dimerisation through specific intermolecular recognition, the use of fused C-terminal ; n~ (with known dimerisation properties) is likely to lead to stabilization of the dimeric constructs produced due at least to the increase in area of hydrophobic interface interaction. For example the association of heavy and light ch~;n~ (KA >11 M-l) is derived from the com~ination of relatively weak VH_VL (KA-106 M-l) and CH1 CL
(KA-107 M-l) interactions, indicating that the association of the two ~om~; n pairs is at least additive (ref 14).
The present invention relates to the development of lS bivalent and other dimeric antibodies especially for use in therapy or for in vivo diagnosis. The successful targetting of antibody molecules to sites of disease upon repeated administrations is dependant on these antibodies provoking little or no immune reaction. M~;mi sation of the natural human antibody sequence content of the bispecific antibodies will prospectively enh~nce the perspective for long-term repeat administration of these antibodies, for example in the treatment of chronic disease.
Notwithst~n~i ng this, other applications as yet 2~ undetermined or not described herein for which mono-, bi-, or multi-specific antibodies/antibody fragments (produced via the means of association described in this invention) may be applied will be also encompassed by this patent.
W O 94/09131 2~ 8 5 ~ 12 PC~r/GB93/021 The general concept of association of specific b;nfling ch~ i n~ which is applicable to the present invention may be illustrated by reference to figure 1. In this diagram, the entity has been divided into four components A, B, C and D
in order to simplify discussion of the inventive process.
As previously discussed, a wide variety of antibodies (and fragments thereof) are readily available. Thus, there are numerous combinations of constructs which may be produced within the parameters of this invention. One may consider the specific specific binding regions or the associating ~mAin~ as derived variously from such entities as Fab~ FV~ SCFV' Fd~ VL/CL~ Fc or F(ab)~ fragments, or individual ~lom~; n~ such as CH3~ C~2, CH1, CL~ VH or VL (or even various combinations of these domains).
The specific binding regions and the associating ~om~; n~ are fused to each other either directly or via peptide linkers of different lengths (the linker sequence composition and length, being tailored to the particular antibody configuration and foreseen application). The components B and D may be n~m;n~lly seen as additionally fused ~om~;n~ although in certain configurations they may themselves be part(s) of the same antibody from which A and C are derived. The fragments or domains may be derived from one or more sources depending again on the chosen format and foreseen application of the particular construction. A further augmentation, also foreseen in this invention, is that for particular molecular configurations and destined applications it may be ~ 094/09131 2~ ~ 685~cT/GBg3/o2l33 advantageous to introduce or use existing cysteine residues to create inter-domain disulphide bridges in order to further stabilize the constructs. The reduction to produce these covalent bonds may be performed in vivo or in vitro depending on the construct and relative merits of this additional process.
It will be apparent to those skilled in the art that the choice of any particular type of antibody or antibody fragment is not flln~mPntal to the inventive concept described here. Similarly any immllnogobulin which provides the desired specificity may be employed. An extension of the invention also foresees the use of any natural molecules (or fragments thereof) which form specific associations (such as cell surface receptors and their ligands) which have been exposed to the immune system and show little or no ;mml~nogenicity within the said system.
A valuable embodiment of this invention foresees the dimerization of the constituent partners via the homodimeric association of additionally fused antibody ~oma;n~ (or other ;mml~nQ-silent molecules). Such a strategy may be used to produce dimeric antibodies. This embodiment is expected to be more suited towards the dimerisation of single antibody species to form homodimeric, bivalent antibodies, since is recognised that a mix of product assemblies may arise when this technique is used for the co-expression and association of two different antibody species to form a heterodimeric, bispecific antibody.
WO94/09131 2 1~ PCT/GB93/0213 The following specific description illustrates how the invention may be carried out. The ~ReferenCe example"
provides an illustration of the basic technology by which specific binding and associating fragments can be fused and caused to associate, while Examples 1 and 2 which follow provide specific embodiments of the invention to whose manner of execution can draw on the technology of the Reference example.
Reference Example The concept of using dimeric association of fused domains to enable formation of a bivalent species may be simply illustrated by the following example designed for production of a bivalent homodimeric (scFv) 2 antibody. With reference to figure 1, in this embodiment, the components A and D are V~ and VL ~m~; n~ respectively fused via a linker to a cognate VL and VH ~om~; n (components B and C
respectively). This format for the production homodimeric bivalent antibodies has been experimentaly tested using a simple model system (see Fig. 2) in which VH and VL ~nm~in~
of an anti-Pseudomonas aeruginosa antibody were fused via a linker peptide. This construct, when expressed in Escherichia coli and subsequently purified, was found to exist in monomeric, dimeric and multimeric forms as analysed by HPLC and non-denaturing PAGE. The dimeric fonm was pre~om;n~nt, and was found to have an antigen binding profile similar to the parent antibody, furthermore, it was found that the bivalent form was produced spontaneously.
~ 094/09131 2:~ 4 6 8 ~ ~ PCT/GB93/02133 ExPerimental For this study the pUC based pSW1-VHD1.3-VKD1.3-TAG1 expression vector (Ward et al., 1989)(refl3), encoding anti-P. aeruginosa (PSV) antibody heavy and light chain variable region genes was employed. A novel single chain antibody construct was generated a fourteen amino acid linker described by Chaudhary et al., (l990)(ref 15) by modification of the original Fab encoding vector. This novel scFv* construct possesses a hllm~n kappa chain domain fused to the 3~ end of the VK domain for detection purposes. Results indicated that when expressed, this scFv*
construct formed multivalent products spontaneously.
Bacterial strain All vectors were transformed and expressed in the E.
coli strain XL-1-Blue (supE44 hsdR17 recA1 endA1 gyrA46 the relA1 lacF' [proAB+ lacl9 lacZ M15 TnlO (tetr)]. The tetacycline selectable F' pilus allows strict control of expression of pUC based vectors.
Vectors construction The PSV scFv* expression vector was constructed form a pSW1-VHD1.3-VKD1.3-TAG1 vector derivative (pSW1-Fab/Pa) using the polymerase chain reaction (PCR) mutagenesis method of Higuchi (1989)(refl6). The linker was incorporated using a two stage procedure. Two separate amplifications were carried with the primers VHBAK:5l-AGGT(C/G)(C/A)A(G/A)CTGCAG(G/C)AGTC(T/A)GG
WO94/09131 21 ~ 6 8 5 ~ PCT/GB93/0213 with LINKFOR:5'-CGATGTCATCCACTTTAGATTCAGAGCCAGAGCCAGAAGATT
TGCCTTCTGAGGAGACGG
and VKFOR:5'-GTTTGATCTCGAGCTTGGTGCC
with T-T~RR~: 5'-GTCACCGTCTCCTCAGAAGGCAAATCTTCTGGCTCTGGCTC
TGAATCTAAAGTGGATGACATCGAGCTG.
10The vector pSWl-VHDl.3-VKDl.3-TAGl, expressing an anti-P.aeruginosa (pSWl-Fab/Pa), was used as template DNA.
The PCR con~ine~ 2 units of Taq polymerase (BCL, Lewes, East Sussex, England), 1.25mM dNTPs, lmM of each either VHBAK and LINKFOR or VHFOR and TT~RRAK, 50 ng pSWl-15Fab/Pa and 5~1 l0x reaction buffer (BCL) made up to 50~1 with de-ionised water. All reactions were W treated for ten minutes prior to the addition of polymerase and template, and then overlaid with mineral oil. PCR was performed using a Techne PHC-2 thermal cycler (Scotlab, Aberdeen, Scotland) using the following parameters: 94 C, l minute, 60'C, l minute, 72 C, l minute for 30 cycles. The purified products of these reactions were mixed and seven cycles of PCR were perfonmed without primers (94 C, minute; 72 C, l minute). This reaction was then maintained at 94 C while VHBACK and VKFOR primers were added. A
further 30 cycles of PCR were then performed (94 C, minute; 65'C, l minute; 72 C, l minute). The reaction products were electrophoresed through a 2% (w/v) agarose-094~09131 ~6 PCT/GB93/0213317 S~
TAW gel and the 65~ bp product purified into 10~1 of de-ionised water by the Prep-a-gene procedure (Bio-Rad Ltd, Hemel Hempsted, Herts., England). The product was digested with PstI and XhoI then ligasted into PstI/XhoI digested pSW1-Fab/Pa under st~n~Ard conditions to construct the pDM1 vector (fig 10).
Antibody fraqment exPression The expression vectors pDM1 encoding the anti-P.
aeruginosa scFv* antibody fragment and pSW1-Fab/Pa encoding the equivalent Fab fragment (each with the fused light chain constant region ~m~; n for detection purposes) were assessed. Expression conditions were modified from Ward et al, (1989)(ref 13): XL1-blue transformed with one of the above vectors was grown in 5ml 2xTY cultures contA;n;ng 1%
glucose, lOO~g/ml ampicillin and 12.s~g/ml tetracycline at 37 C, overnight. Ten microlitres of overnight culture was then used to inoculate fresh medium for expression of antibody fragments. Expression was carried out at 26 C for six to eighteen hours in Terrific broth or 2xTY contA;n;ng O.lmM IPTG, lOO~g/ml ampicillin and 12.5~g/ml tetracycline.
Antibody fragments were detected in culture supernatants by ELISA or could be purified directly from cell pellets as described below.
Affinity purification of antibody fraqments Anti-P. aeruginosa scFv* and Fab fragments were 2~468~
WO94/09131 ~ PCT/GB93/0213 purified by affinity chromatography. Five hundred millilitre cultures of bacteria producing either anti-P.
aeruginosa Fab or scFv* were centrifuged at 4000 rpm for 20 minutes. The supernatant was discarded and the cell pellet sonicated for l minute in lOml of PBS to release periplasmic antibody fragments. This solution was concentrated to a lml volume in a Centricon-lO column (Amicon, Stonehouse, Glos, England) and loaded onto an anti-human FAb affinity column (Pierce, Warriner, Cheshire England) prepared according to the manufactures instructions. Seven lml fractions were eluted in O.lM
glycine buffer pH2.8. The fractions were dialysed overnight against PBS and analyzed by polyacrylamide gel electrophoresis.
Detections of antibody fraqments for sandwich ELISA
Bacterial supernatants and antibody fragments tFAb and scFv*) purified from cell pellets were assayed in 96 well flat bottomed polystyrene plates (Nunc, Denmark). The plates were coated overnight at 4'C with l~g affinity purified goat anti-human IgG Fab antibody (Sigma Chemical Co., Poole, Dorset, England) in 50~1 phosphate buffered saline (PBS). The plates were blocked for l hour at 37 C
with 200~1 of 2% (w/v) BSA (fraction V, Sigma) in PBS and then washed twice with 200~1 PBS. Fifty microlitres of culture supernatant or purified antibody fragments were added to each well and the plates incubated at 37 C for l hour. After this time the plates were washed four times ~ 094/09131 ~6~ PCT/GBg3/02l33 withe 200~1 0.05% (v/v) Tween 20 in PBS (Tw20/PBS). Fifty microlitres of horse radish peroxidase coniugated goat anti-human IgG Fab antibody (Sigma), diluted 1: 2000 in TW20/PBS was added to each well and incubated at 37 C for 1 hour. The plates were then washed six times in 200~1 TW20/PBS and the assay developed with 50~1 lmg/ml 0-phenylendiamine dilydrochloride (Sigma) and 1~1/ml 30%
(v/v) hydrogen peroxide (Sigma) in O.lM citrate-phosphate buffer pH 6Ø The reaction was stopped with 100~1 0.5M
citric acid and the optical density at 450nm was read using a Bio -Rad M450 plate reader. Positive control anti-P
psell~omon~s mouse-hllm~n ~h;m~ric IgG1 antibody was provided by Scotgen Ltd, Aberdeen. This antibody was derived from the original murine patent ;mmllnoglobulin from which the Fab and scFv* variable ~om~; n~ were obtained.
Detection of antiqen bindinq of ELISA
This assay was performed essentially as the ELISA
described above but with the following modifications. P.
aeruginosa were grown overnight in Luria broth, pelleted an resuspended in PBS as a volume representing half of the volume of the original culture. The cells were then heat treated at 65 C for 10 minutes. 96-well polystyrene plates were coated overnight with 50~1 of heat killed P.
aeruginosa. After blocking the plates with TW20/PBS and washing with PBS, 50~1 of bacterial supernatant, purified antibody fragments or the mouse/hllm~n ch;mPric anti-P.
aeruginosa antibody supernatant were added to each well and WO94/09131 214 ~ ~ S ~ PCT/GB93/0213~
incubated at 37 C for one hour. After the plates were washed four times with PBS the assay proceeded as described in the previous section.
Western blottinq The specificity of the scFv* expressed was investigated by Western blotting. One million P. aeruginosa, P.
fluorescens, Al. xylosidans, Ac. calcoaticus and O.
anthropi were electorphoresed through a st~n~rd 10% SDS
polyacrylamide gel with Rainbow size markers (Amersham Int., Aylesbury, Bucks, England). The gel was electorblotted at 0.8 mA/cm2 in a Hoeffer T70 semi-dry blotter onto Immobilion-P membrane (Millipore Corp., Bedford, Mass, USA). The membranes were blocked overnight in 5% (w/v) milk powder in PBS. scFv* (l~g/ml) was incubated for one hour in 0.1% (v/v) Tween 20 in PBS followed by three l0 minute washed in 1% (v/v) Tween 20 in PBS. The membrane was then incubated with a l:5000 dilution of an anti-hllm~n kappa chain or anti-human Fab antibody conjugated to horse radish peroxidase in 5% (w/v) milk powder, 1% (v/v) Tween 20 in PBS for 30 minutes at 37 C.
scFv* binding was detected using the ECL chemiluminescent system (Amersham Int.).
Non-denaturinq polyacrylamide qel electrophoresis Affinity purified Fab and s~Fv* were separated on a 7.5% non-denaturinq polyacrylamide gel using the method of Ornstein (1964)(ref 17) to determine the conformation of 094/09131 ~ 8 S~ PCT/GB93/02133 the molecules.
HPLC purification of scFv* fractions Forty microgram samples of scFv* were analyzed using a Zorbax GF250 size exclusion column on a Gilson HPLC
comprising a model 302 pump, an 802L mAnom~tric module, a 811 dynamic mixer, a 116 W detector and a 201 fraction collector controlled by Gilson 714 software. The analysis was done using both PBS and 0.2M sodium phosphate buffer at a flow rate lml/minute. Eluate was monitored at 280nm, OlAUFS and 0.5 ml fractions were collected and analyzed by ELISA and non-denaturing polyacrylamide gel electrophoresis.
scFv* and Fab Purification and antiqen b;n~;nq The presence of the light chain constant region allowed purification of both Fab and scFv* directed against P. aeruginosa. Similar amounts (2-5mg) of both proteins were obtained when periplasmic cell extracts derived from 500 ml of IPTG induced overnight cultures were loaded onto affinity columns. The purity of the eluted proteins was assessed by denaturing SDS-PAGE with Coomassie Blue st~;ning (fig ll).
The relative antigen binding capacities of affinity purified Fab (pSWl-FAb/Pa), scFv* and chimeric antibody were compared. The c~;mPric antibody and the scFv* gave similar binding profiles, indicating that the scFv* had an affinity close to the original monoclonal antibody. Fab binding was WO94/09131 2-~ 4 ~ 8 ~ ~ 22 PCT/GB93/0213 ~
2-3 times weaker than either that of c~;m~ric or scFv* (Fig 12). Western blGtting was employed to test the specificity of the scFv* protein. One band of approximately 21 kd was observed when scFv* was tested against P. aeruginosa antigens (Fig 13). Cross ~eactivity with Al. xylosidans and Ac. calcoaticus but not O. anthropi, F. mulitivoran or P.
fluorescens was noted (Fig 13). These are all Gram negative bacteria.
Analysis of scFv*
Differences in conformation of bacterially produced Fab and scFv* molecules was thought to be responsible for the changed affinities observed in ELISA. Non-denaturing gel electrophoresis revealed that Fab (pSWl-Fab/Pa) was identifiable as a single unit while scFv* was present as a series of multimeric forms (Fig 14a). ~onomeric and dimeric forms of scFv* appeared to pre~om;n~te. HPLC purification by size exclusion allowed separation of the monomeric and dimeric scFv* forms. Comparison of the elution times with those of molecular weight marker proteins confirmed the presence of both monomeric and dimeric forms of scFv* (Fig 14b). An ELISA was employed to determine the ability of the monomeric and dimeric sc~v* fragments to bind P.
aeruginosa and revealed that the dimeric form had affinity similar to the ~;meric antibody while the monomeric form had a lower affinity (Fig 15).
Discussion 094/09131 ~1 ~ 6~ PCT/GB93/02133 The bivalent component thus shows similar binding characteristics to the intact antibody while the monomeric fragment shows greatly reduced binding. Dimeric and m~om~ric forms can be easily separated by gel filtration, offering a simple method for preparing pure dimeric antibody.
The scFv* dimers dissociate into monomers in non-reducing SDS gels and appear, therefore, not linked by disulphide bonds (data not shown). Our interpretation of the data is that the dimer formation of the (scFv*)2 is due to non covalent intermolecular VH/VL domain association; the association occurring between ~omAin~ of different scFv*
polypeptides as indicated in figure 2. The resulting dimer is bivalent and shows a binding affinity to the ch;m~ric antibody for the P. aeruginosa antigen.
Further Methods For Bispecific Recombinant AntibodY
Formation The present invention discloses novel bispecific antibodies in which dimerization of the constituent partners is achieved via the heterodimeric association of additionally fused natural antibody domains. For example, antibodies may consist of two different Fab or scFv antibody fragments (or any other antibody/antibody fragment combination) linked to either a VH or VL polypeptide by gene fusion. The desired bispecific antibody is therefore specifically associated by the recognition and natural heterodimerization of the fused VH or VL domains. The =
W O 94/09131 PC~r/GB93/0213 ~
2~;~68~4 24 present invention also discloses methods for the production of novel bispecific antibodies.
The simplest format envisaged for the production of a bispecific antibody would be the association of two single chain antibodies scFvl and scF~2. If one relates to the general scheme shown in figure 1, the ~o~;n~ A and C would be VH1 and VL2 respectively, and domains B and D; VL1 and VH2 respectively (see Fig.3). In this m;m;nAl configuration it is clear that both the two domains of each individual peptide chA; n~ may associate to form normal scFvs and additionally, that homo- and heterodimers (and possibly larger multimeric associations) of the two peptide c~;n~
may be produced by intermol~clllAr ~om~;n association. A
preferred embodiment of this invention is a bispecific antibody, ~ormed by a C-term; n;l 1 fusion of either a VH or VL antibody domain (component B) to one scFv antibody fragment (component A) and its corresponding partner (component D) to the second scFv (component C). Such a species is illustrated in Figure 4.
Similarly for bispecific F(ab)2 heterodimers, either the VH or VL ( components B and D) could be added by C-term;nAl fusion to the VH/CH1 polypeptide ~A;n~ of the Fab fragments (components A and C). Using this approach, Fab dimerization, to form the bispecific antibody, should be 2S promoted by the spontaneous and specific association of the attached VH and VL ~lomA i n~; . See Figure 5.
Examples of other alternative embodiments of this invention may involve the use of other naturally occuring 094/09131 ~ ~683~ PCT/GB93/02133 antibody domains which form heterogeneous (dimers such as VH.CH1/VL.CL or CH1/CL) as C-terminal fusions (components B
and D) which are illustrated in figure 6-9. This and other possible confonmations have been alluded to previously in the text.
It will be apparent to those skilled in the art that the choice of any particular type of antibody or antibody fragment is not fl~n~Am~ntal to the inventive co~cPpt but it is clear that the specificity and affinity of the C-terminal ~om~;n~ chosen will greatly influence the endproduct(s). Any domain(s) which provides the desired dimerization and recognition specificity required may be employed. However, it is recognised that, for example, specific VH ~m;~;n~ will associate more efficiently with certain VL ~ ; nC than others (KD values for Fv~s varying from 10-4 M - 10-7 M)(ref 18) and thus the VH/VL binding pair of a specific bifunctional should possibly be optimised to promote heterodimer fonmation and also to m; n; m; se homodimer formation (especially when producing bispecifics). Similiarly it is also foreseen that molecules may be modified in order to improve the specificity and/or binding affinity of the molecules for each other and thus improve their dimerisation properties (ref l9). This, for example, may be achieved via engineering, eg mutation or chemical modification, of the inter-domain hydrophobic contacts, introduction of a metal (or other molecule binding site) between the ~om~;ns to stabilize association, introduction of strategic cysteine residues to form WO94/09131 ~ 26 PCT/GB93/0213 disulphide bridges, etc.
If supplementary Fv ~om~;n~ are created when producing a bivalent or bispecific antibody (as may be the case when using the VH/VL ~om~;n combination as fusions), the antibody may, in fact, be trivalent. This extra antigen binding site may be useful itself. If the binding site recognizes a different type of antigen or ligand than the other sites, this property may be used for purification (ligand bound to matrix), stability (ligand added and bound to site thereby ~cross-linking~l the two ~m~; n~ of the Fv) or as a label (added ligand-reporter complex). This idea would also be valid for bispecifics, if one type of binding site is used for ligand binding it may be used for the same purposes as described above for the trimer.
The present invention provides an antibody with a m;n;mllm of two Fv ~omA;nC each comprising of structural framework with the relevant CDRs which provide the antibody with the capability to bind specific molecules such as antigens. The invention may be exemplified ,but not limited, in a configuration in which the antigen recognition and binding ~nm~;n~ (and any other affiliated sequence such as constant regions etc) are fused at the C-t~rm;n~l end to "association~ domains such as either a VH
or VL antibody domain respectively. Association of the constituent partners is achieved via the natural heterodimerisation of the fused VH and VL antibody domains (see figs 4 and 5).
Processes for the production of such antibodies and 094/09131 27 ~ ~
genes encoding such antibodies are also provided by the present invention.
Corresponding to a preferred process of the present invention, there is provided a method to produce bispecific (scFv) 2.VH/VL or F(ab) 2.VH/VL entities (see figs, 4 and 5) or derivatives of these with either CH1/CL or VH.CH1/VL.CI, fused ~nm~;n~ (see figs.6-9). This method comprises the following basic scheme:
(i) Cloning or synthesis of the antibody Fv and~or Fab genes from the antibodies which exhibit the desired binding properties. The genes should be inserted into plasmid or phage vectors suitable for proliferation and /or gene expression.
(ii) The addition of the VH or VL t30m;l; n genes by C-term;n~l gene fusion to the respective antibody partners such that on correct heterodimerization of these added ~o~;n~ the bispecific construct should be created.
(iii) Transfer of the complete individal gene constructs to an appropriate expression vector or vectors (if not performed previously) and subsequent introduction to the host organism.
(iv) Growth of the host organism and expression of the installed recombinant genes to achieve antibody production.
(v) Purification of the antibody/antibodies and any ensuing biochemical manipulation to obtain the bispecific construct.
Naturally, the details of the individual steps will 2 ~ S ~ 28 be dependant on the form of the bispecific antibody to be produced e.g (FV)2~V~/VL or F(ab) 2.VH/VL, (see figs 4 and 5) whether they are to be co- or individually expressed and the host organism (e.g prokaryote or eukaryote) which is to be used.
The invention is illustrated but not limited by the following examples.
Example 1 This example illustrates the production of a bispecific antibody from two different scFv fragments expressed in Escherichia coli.
(a) Starting with the mouse hybridoma cell lines producing the monoclonal antibodies of interest, a suitable method for determ; n; ng the corresponding heavy and light chain variable sequences from hybridoma in RNA
is described by Orlandi et al., Proc. Natl. Acad. Sci.
USA, 86, 3833-3837, 1989.
(b) Genes encoding reshaped human antibodies (comprising the necessary mouse CDR and framework residues) which correspond to the original mouse monoclonals can be produced by site directed mutagenesis (see Riechm~n et al.). Alternatively genes encoding the reshaped human antibodies can be asse-m~bled by gene synthesis (Jones et al.)(ref 20).
(c) These genes can be manipulated using various molecular biology techniques to form scFv fra~ments as described by Bird et al., Science 242, 423-426, 1988 (ref 21) or Huston et al., Proc. Natl. Acad. Sci. USA 85, ~CYO 94/09131 2 PC~r/G B93/02133 _ 29 ~ ~ 6 ~ S
5879-5883, 1988 (ref 22).
Complete human VH or VL domains (naturally paired but preferably showing no specific binding relative to the antibodies utilized) can be added by C-terminal gene fusion, one to either of the two constituent scFv constructs.
(d) The completed genes can then be cloned into an appropriate expression vector(s) depending on whether they are to be expressed as in a two cistron system on one vector under a single promotor, expressed singularly from each carried on its own vector yet together in a co-transformed host or each individual grown in different cultures. Examples of such vectors may be obtained from the literature of Skerra et al., Science, 240 1038-1041, 1988 (ref 3), Ward et al., Nature, 341, 544-546, 1989 (ref 13), Bird et al., Science 242, 423-426, 1988 (ref 21~, Hoogenboom et al., Nuc. Acid Res. 19, 4133-4137, 1991 (ref 23)., etc. Further changes to obtain vectors suited to the needs of bispecific production may be necessary.
(e) Depending on whether the bispecific formation is to be performed in vitro or in vivo and the state of the products formed it may be pertinent to incorporate an additional reduction and reoxidation of the purified antibody fragments in order to obtain the desired bispecific (Bird et al., Science 242, 423-426, 1988 (ref 21), Skerra et al., Science, 240 1038-1041, 1988 (ref3), Kostelny et al., J. Tmml~nQl. 148, 1547-1553, 1992 (ref WO94/09131 ~ ~ ~ PCT/GB93/0213 10 ) . ) Similarly F(ab) 2.VH.VL fragments can be produced in E.coli by similar means and the VH/VL association technique may utilized for for other antibody fragments and other dimerization purposes (see fig 5) Example 2 This example illustrates the production of a bispecific antibody from two different Fab fragments expressed mAmm~ 1; an cell lines. The initial procedures are quintessencally the same as in example 1 (steps a and b) The construction of the bispecific shown in fig.5, could be made by the same technique as was used for the F(ab) 2 Jun.Fos. construction as described by Kostelny et al., J. Tmmllnol~ 148, 1547-1553, 1992 (ref 10). in which the one the additional dimerization ~m~ inS were fused to the first few residues of the CH2 intron such that it would be spliced to the CH1 of the VH gene thus replacing the nonmal CH2 and CH3. The VL/CL genes are expressed from a separate vector which is co-transfected with the VH carrying plasmid.
Again various strategies for expression and bispecific production may be attempted as have ~een detailed previously for E. coli expression.
094/09l3l 1~6 ~ PCT/GB93/02133 REF~RENCES
The following references are all here~y incorporated by reference.
1. Kohler G., & Milstein C., (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 265, 495-497.
2. Verhoeyen M., Milstein C., & Winter G., (1988).
Reshaping human antibodies: grafting an anti-lysozyme activity. Science, 239, 1098-1104.
3. Skera A., and Pluckthun A., (1988). Assembly of a functional immllnoglobulin Fv fragment in E.coli Science, 240, 1038-1041.
4. Better M., Chang C.P., Robinson R.R., and Horwitz A.H., (1988). E.coli secretion of an active chimaeric antibody fragment. Science, 240, 1041-1043.
5. Winter G., & Milstein C., (1991). Man-made antibodies. Nature, 349, 293-299.
6. Nolan O., and O'~enn~y R., (1992). Bifunctional antibodies and their potential clinical applications Int. J Clin Lab Res, 22, 21-27.
WO94/U9131 ¦ ~ 6 ~ ~ 4 PCT/GB93/~213 7. Yokota T ., Milenic D. E., Whitlow M., and Schlom J., (1992) Rapid tumor penetration of a single-chain Fv and comparison with other ;mmllnoglobulin forms.
Cancer Res, 52, 3402-3408.
WO94/U9131 ¦ ~ 6 ~ ~ 4 PCT/GB93/~213 7. Yokota T ., Milenic D. E., Whitlow M., and Schlom J., (1992) Rapid tumor penetration of a single-chain Fv and comparison with other ;mmllnoglobulin forms.
Cancer Res, 52, 3402-3408.
8. Skerra A., and Pluckthun A., (1991). Secretion and in vivo folding of the Fab fragment of the antibody MPC603 in E.coli influence of disulphides and cis prolines. Protein Engineering, 4, 971-979.
9. Bird R.E., and Walker B.W., (1991) Single chain antibody variable regions. TIBTECH, 9, 132-137.
10. Kostelny S.A., Cole M.S., and Tso J.Y., (1992).
Formation of a bispecific antibody by the use of leucine zippers J. Tmmllnol, 148, 1~47-lS53.
Formation of a bispecific antibody by the use of leucine zippers J. Tmmllnol, 148, 1~47-lS53.
11. Pack P., and Pluckthun A., (1992). Miniantibodies use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in E.coli Biochemistry, 31, 1579-1584.
12. Carter P., Kelley R.F., Rodrigues M.L., Snedecor B., Covarrubias M., Velligan M.D., Wong W.L.T., Rowland A.M., Knotts C.E., Carver M.E., Yang M., Bourell J.H., Shepard H.M., and Henner D., (1992) High level Escherichia coli expression and production of a bivalent humanised antibody fragment.
094/U9131 ~683'~ PCT/GB93/02133 Biotechnology, 10, 163-167.
094/U9131 ~683'~ PCT/GB93/02133 Biotechnology, 10, 163-167.
13. Ward E.S., Gussow D.H., Griffiths A.D., Jones P.T., and Winter G., (1989) Binding activities of a repoertoire of single ;mm~noglobulin variable ~om~;n~ secreted from E.coli Nature, 341, 544-546.
14. Horne C., Klein M., Polidoulis I., and Dorrington K.D., (1982). Noncovalent association between heavy and light chains of ;mmllnoglobins~ III Specific interactions between VH and VL J Tmmllnol., 129, 660-664.
15. Chaudary V.K., Batra J.K., Glallow M.G., Wilingham M.C., FitzGerald D.J., and Pastan I., (1990) A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single chain ;mml~notoxins Proc. natl. Acad. Sci. U.S.A., 87, 1066-1070.
16. Hiquchi R., (1990) Recombinant PCR. In PCR
protocols: A guide to methods and applications, (Edited by Innes M.A., Gelfand D.H., Sninsky J.J., and White T.J.,) pp 177-183 Academic Press, London.
protocols: A guide to methods and applications, (Edited by Innes M.A., Gelfand D.H., Sninsky J.J., and White T.J.,) pp 177-183 Academic Press, London.
17. Ornstein L., (1964) Disc electrophesis - I
Background and theory. Ann N.Y. Acad Sci, 121, 321-349.
WO94/09131 j ~ PCT/GB93/021 ~
2~4~54 34 18. Hamel P.A., Klein M.H., Smith-Gill S.J., and Dorrington K.D., (1987) Relative noncovalent association constant between immlln~globulins H andL
ch~;n~ iS unrelated to their expression of antigen-bin~;nq activity J Tmmllnol~ 139, 3012-3020.
Background and theory. Ann N.Y. Acad Sci, 121, 321-349.
WO94/09131 j ~ PCT/GB93/021 ~
2~4~54 34 18. Hamel P.A., Klein M.H., Smith-Gill S.J., and Dorrington K.D., (1987) Relative noncovalent association constant between immlln~globulins H andL
ch~;n~ iS unrelated to their expression of antigen-bin~;nq activity J Tmmllnol~ 139, 3012-3020.
19. Glockshuber R., Malia M., Pfitzinger I., and Pluckthun A., (1990). A comparison of strategies to stabilize Immunoglobulin Fv-Fragments.
Biochemistry, 29, 1362-1367.
Biochemistry, 29, 1362-1367.
20. Jones P.T., Dear P.H., Foote J Neuberger M.S., and Winter G., (1986) Replacing the complementary det~rmi n; ng regions in a hllmAn antibody for those from a mouse. Nature, 321, 522-525.
21. Bird R.E., Hardman K.D., Jacobson J.W., Johnson S., Kallfm~nn B.M., Lee S Lee T., Pope S H., Riordan G.S., and Whitlow M., (1988) Single chain antigen binding proteins. Science, 242, 423-426.
22. Huston J.S., Levinson D., Mudgett-Hunter M., Tai M., Novotny J., Margolies M.N., Ridge R.J., Bruccoleri R.E., Haber E., Cera R., and Oppenman H., (1988) Protein engineering of antibody binding sites:
Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in E.coli. Proc.
NatR. Acad. Sci U.S.A., 85, 5879-5883.
094/09131 1~68S~ PcT/GB93/o2l33 23. Hoogenboom H.R., Griffiths A.D., Johnson K.S., Chiswell D.J., Hudson P., and Winter G., Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light ch~;n~ N.A.R. 4133-4137.
Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in E.coli. Proc.
NatR. Acad. Sci U.S.A., 85, 5879-5883.
094/09131 1~68S~ PcT/GB93/o2l33 23. Hoogenboom H.R., Griffiths A.D., Johnson K.S., Chiswell D.J., Hudson P., and Winter G., Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light ch~;n~ N.A.R. 4133-4137.
Claims (22)
1. A specific binding protein having first and second binding regions which specifically recognise and bind to target entities, said binding regions being contained at least in part on respectively first and second polypeptide chains, said chains additionally incorporating respectively first and second associating domains which are capable of binding to each other, causing the first and second polypeptide chains to combine, thereby providing a single protein incorporating the binding specificities of said first and second binding regions.
2. A specific binding protein according to claim 1 wherein said first and second binding regions recognise different target entities.
3. A specific binding protein according to claim 1 or claim 2, wherein said associating domains are derived from an antibody.
4. A specific binding protein according to any one of the preceding claims wherein the associating domains are derived from a human protein (ie one which has been exposed to the human immune system).
5. A specific binding protein according to any one of the preceding claims wherein said associating domains are two identical domains which are capable of association, even if they do not normally associate in nature.
6. A specific binding protein according to any one of claims 1 to 4, wherein said associating domains are selected from:
antibody VH and VL regions, antibody CH1 and CL regions, antibody VH.CH1 and VL.CL regions.
antibody VH and VL regions, antibody CH1 and CL regions, antibody VH.CH1 and VL.CL regions.
7. A specific binding protein according to any one of the preceding claims, wherein the first and second binding regions are antibody antigen-binding domains.
8. A specific binding protein according to claim 7, wherein the antigen binding domains comprise VH and VL
regions.
regions.
9. A specific binding protein according to claim 8, wherein the VL and VH regions are contained in a Fab fragment.
10. A specific binding protein according to claim 8, wherein the VL and VH regions are contained in a single-chain Fv fragment.
11. A specific binding protein according to claim 7, wherein either or both of the binding domains are derived from just one of the VH or VL regions of an antibody.
12. A specific binding protein according to claim 11, wherein either or both of the binding domains are derived from the VH region of an antibody.
13 . DNA encoding the polypeptides of the specific binding protein of any one of the preceding claims.
14. DNA according to claim 13 contained in one or more expression vectors.
15. A host cell containing recombinant DNA according to claim 13 and capable of expression of the DNA to produce the polypeptides of the specific binding protein.
16. A host cell according to claim 13 wherein the polypeptides on expression associate to form the specific binding protein.
17. A process which comprises expressing the recombinant DNA in a host cell of claim 15 to produce the polypeptides encoded thereby, and where necessary performing post-translation manipulation or processing of the polypeptides, to produce the specific binding protein.
18. A process for producing a specific binding protein of any one of claims 1 to 12, which comprises:
(i) cloning or synthesising DNA encoding said first and second binding regions, (ii) joining in reading frame to the DNA of said first and second binding regions DNA encoding respectively said first and second associating domains, (iii) providing one or more expression vectors containing said fused DNA constructs of (i) and (ii), (iv) inserting the expression vectors into a host organism, (v) culturing the host organism to express the encoded polypeptides, and (vi) causing or allowing said first and second associating domains to combine to form the specific binding protein.
(i) cloning or synthesising DNA encoding said first and second binding regions, (ii) joining in reading frame to the DNA of said first and second binding regions DNA encoding respectively said first and second associating domains, (iii) providing one or more expression vectors containing said fused DNA constructs of (i) and (ii), (iv) inserting the expression vectors into a host organism, (v) culturing the host organism to express the encoded polypeptides, and (vi) causing or allowing said first and second associating domains to combine to form the specific binding protein.
19. A pharmaceutical preparation comprising a specific binding protein according to any one of the preceeding claims.
20. A method comprising using the preparation of claims 19 to treat a human or animal patient.
21. A diagnostic reagent comprising a specific binding protein according to any one of claims 1 to 18.
22. A method comprising using the reagent of claim 21 in a diagnostic technique.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9221657.1 | 1992-10-15 | ||
| GB929221657A GB9221657D0 (en) | 1992-10-15 | 1992-10-15 | Recombinant bispecific antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2146854A1 true CA2146854A1 (en) | 1994-04-28 |
Family
ID=10723495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2146854 Abandoned CA2146854A1 (en) | 1992-10-15 | 1993-10-15 | Recombinant specific binding protein |
Country Status (5)
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| JP (1) | JPH08505761A (en) |
| AU (1) | AU5283793A (en) |
| CA (1) | CA2146854A1 (en) |
| GB (1) | GB9221657D0 (en) |
| WO (1) | WO1994009131A1 (en) |
Families Citing this family (132)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0739984A1 (en) * | 1995-04-26 | 1996-10-30 | San Tumorforschungs-Gmbh | Bivalent polypeptides containing at least two domains |
| US7973137B1 (en) * | 1996-03-28 | 2011-07-05 | Johns Hopkins University | Cell compositions comprising molecular complexes that modify immune responses |
| JP2000508892A (en) | 1996-04-04 | 2000-07-18 | ユニリーバー・ナームローゼ・ベンノートシャープ | Multivalent and multispecific antigen binding proteins |
| EP1049787B1 (en) * | 1998-01-23 | 2004-11-24 | Vlaams Interuniversitair Instituut voor Biotechnologie | Multipurpose antibody derivatives |
| DE10021678A1 (en) * | 2000-05-05 | 2002-04-18 | Stefan Duebel | Recombinant polyspecific antibody constructs, useful for diagnosis and treatment of cancer, comprises three antibody fragments,where at least one comprises a disulfide bridge |
| AU2001283496A1 (en) * | 2000-07-25 | 2002-02-05 | Immunomedics, Inc. | Multivalent target binding protein |
| WO2004041865A2 (en) | 2002-11-08 | 2004-05-21 | Ablynx N.V. | Stabilized single domain antibodies |
| EP1867661A1 (en) * | 2006-06-12 | 2007-12-19 | Diatos | Compositions and methods for delivering anti-activated ras antibodies into cells |
| WO2009018386A1 (en) * | 2007-07-31 | 2009-02-05 | Medimmune, Llc | Multispecific epitope binding proteins and uses thereof |
| EP2535349A1 (en) * | 2007-09-26 | 2012-12-19 | UCB Pharma S.A. | Dual specificity antibody fusions |
| CL2008003561A1 (en) * | 2007-11-30 | 2010-02-05 | Glaxo Group Ltd | Il-13 binding construct comprising at least one antibody single domain (dab) grafted onto a monoclonal antibody (mabdab); polynucleotide and host cell; procedure to prepare the construction; pharmaceutical composition comprising it and used to treat cancer or inflammatory diseases. |
| US20090162359A1 (en) | 2007-12-21 | 2009-06-25 | Christian Klein | Bivalent, bispecific antibodies |
| US9266967B2 (en) | 2007-12-21 | 2016-02-23 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
| CN102037007A (en) | 2008-01-25 | 2011-04-27 | 奥尔胡斯大学 | Selective exosite inhibition of PAPP-A activity against IGFBP-4 |
| DK2732823T3 (en) | 2008-06-25 | 2019-09-16 | H Lundbeck As | Modulation of TrpV: the Vps10p domain receptor system for the treatment of pain |
| US10407513B2 (en) | 2008-09-26 | 2019-09-10 | Ucb Biopharma Sprl | Biological products |
| US8703125B2 (en) | 2008-12-19 | 2014-04-22 | H. Lundbeck A/S | Modulation of the Vps10p-domain receptor family for the treatment of mental and behavioural disorders |
| BRPI1014089A2 (en) | 2009-04-02 | 2016-04-19 | Roche Glycart Ag | multispecific antibodies comprising full length antibodies and single chain fab fragments |
| WO2010112034A2 (en) | 2009-04-02 | 2010-10-07 | Aarhus Universitet | Compositions and methods for treatment and diagnosis of synucleinopathies |
| JP5616428B2 (en) | 2009-04-07 | 2014-10-29 | ロシュ グリクアート アクチェンゲゼルシャフト | Trivalent bispecific antibody |
| SG176219A1 (en) * | 2009-05-27 | 2011-12-29 | Hoffmann La Roche | Tri- or tetraspecific antibodies |
| US9676845B2 (en) | 2009-06-16 | 2017-06-13 | Hoffmann-La Roche, Inc. | Bispecific antigen binding proteins |
| NZ598962A (en) | 2009-09-16 | 2014-12-24 | Genentech Inc | Coiled coil and/or tether containing protein complexes and uses thereof |
| GB201005063D0 (en) * | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
| US9588121B2 (en) | 2009-11-06 | 2017-03-07 | Go Therapeutics, Inc. | Method for early detection of cancer |
| AR080793A1 (en) | 2010-03-26 | 2012-05-09 | Roche Glycart Ag | BISPECIFIC ANTIBODIES |
| JP5964300B2 (en) * | 2010-08-02 | 2016-08-03 | マクロジェニクス，インコーポレーテッド | Covalently bonded diabody and its use |
| CA2807278A1 (en) | 2010-08-24 | 2012-03-01 | F. Hoffmann - La Roche Ag | Bispecific antibodies comprising a disulfide stabilized - fv fragment |
| WO2012085111A1 (en) | 2010-12-23 | 2012-06-28 | F. Hoffmann-La Roche Ag | Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery |
| CA2824824A1 (en) | 2011-02-28 | 2012-09-07 | F. Hoffmann-La Roche Ag | Monovalent antigen binding proteins |
| MX341921B (en) | 2011-02-28 | 2016-09-07 | Hoffmann La Roche | Antigen binding proteins. |
| CN107936121B (en) * | 2011-05-16 | 2022-01-14 | 埃泰美德(香港)有限公司 | Multispecific FAB fusion proteins and methods of use thereof |
| KR101723273B1 (en) * | 2011-08-23 | 2017-04-04 | 로슈 글리카트 아게 | Fc-free antibodies comprising two fab fragments and methods of use |
| RU2605390C2 (en) | 2011-08-23 | 2016-12-20 | Рош Гликарт Аг | Bispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use |
| RS56773B1 (en) * | 2012-01-13 | 2018-04-30 | Univ Wuerzburg J Maximilians | Dual antigen-induced bipartite functional complementation |
| WO2013119966A2 (en) | 2012-02-10 | 2013-08-15 | Genentech, Inc. | Single-chain antibodies and other heteromultimers |
| BR112014028368A2 (en) | 2012-06-27 | 2017-11-14 | Hoffmann La Roche | method of producing antibody fc region conjugate, antibody fc region conjugate and pharmaceutical formulation |
| EP2867253B1 (en) | 2012-06-27 | 2016-09-14 | F. Hoffmann-La Roche AG | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
| WO2015028027A1 (en) | 2013-08-29 | 2015-03-05 | University Of Copenhagen | Anti-ADAM12 antibodies for the treatment of cancer |
| BR112016006929A2 (en) | 2013-10-11 | 2017-09-19 | Hoffmann La Roche | ANTIBODY, NUCLEIC ACID, EXPRESSION VECTOR, HOST CELL, METHODS OF PREPARING ANTIBODY, TREATMENT OF PATIENTS AND GENERATION OF AN ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE OF THE ANTIBODY |
| JOP20200094A1 (en) | 2014-01-24 | 2017-06-16 | Dana Farber Cancer Inst Inc | Antibody molecules to pd-1 and uses thereof |
| JOP20200096A1 (en) | 2014-01-31 | 2017-06-16 | Children’S Medical Center Corp | Antibody molecules to tim-3 and uses thereof |
| EP3116909B1 (en) | 2014-03-14 | 2019-11-13 | Novartis Ag | Antibody molecules to lag-3 and uses thereof |
| EP3593812A3 (en) | 2014-03-15 | 2020-05-27 | Novartis AG | Treatment of cancer using chimeric antigen receptor |
| GB201411320D0 (en) | 2014-06-25 | 2014-08-06 | Ucb Biopharma Sprl | Antibody construct |
| JP2017528433A (en) | 2014-07-21 | 2017-09-28 | ノバルティス アーゲー | Low immunoenhancing dose of mTOR inhibitor and CAR combination |
| CN112481283A (en) | 2014-07-21 | 2021-03-12 | 诺华股份有限公司 | Treatment of cancer using CD33 chimeric antigen receptor |
| US11542488B2 (en) | 2014-07-21 | 2023-01-03 | Novartis Ag | Sortase synthesized chimeric antigen receptors |
| TWI750110B (en) | 2014-07-21 | 2021-12-21 | 瑞士商諾華公司 | Treatment of cancer using humanized anti- bcma chimeric antigen receptor |
| EP3174546B1 (en) | 2014-07-31 | 2019-10-30 | Novartis AG | Subset-optimized chimeric antigen receptor-containing t-cells |
| JP6919118B2 (en) | 2014-08-14 | 2021-08-18 | ノバルティス アーゲー | Treatment of cancer with GFRα-4 chimeric antigen receptor |
| EP2985294A1 (en) * | 2014-08-14 | 2016-02-17 | Deutsches Krebsforschungszentrum | Recombinant antibody molecule and its use for target cell restricted T cell activation |
| MY189028A (en) | 2014-08-19 | 2022-01-20 | Novartis Ag | Anti-cd123 chimeric antigen receptor (car) for use in cancer treatment |
| AU2015317608B2 (en) | 2014-09-17 | 2021-03-11 | Novartis Ag | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
| EP4245376A3 (en) | 2014-10-14 | 2023-12-13 | Novartis AG | Antibody molecules to pd-l1 and uses thereof |
| WO2016087416A1 (en) | 2014-12-03 | 2016-06-09 | F. Hoffmann-La Roche Ag | Multispecific antibodies |
| WO2016090034A2 (en) | 2014-12-03 | 2016-06-09 | Novartis Ag | Methods for b cell preconditioning in car therapy |
| EP3271402B1 (en) | 2015-03-16 | 2021-04-28 | Aarhus Universitet | Antibodies towards an extracellular region of nbcn1 |
| EP3973958A3 (en) | 2015-03-20 | 2022-06-22 | Aarhus Universitet | Inhibitors of pcsk9 for treatment of lipoprotein metabolism disorders |
| SI3280729T1 (en) | 2015-04-08 | 2022-09-30 | Novartis Ag | Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell |
| WO2016172583A1 (en) | 2015-04-23 | 2016-10-27 | Novartis Ag | Treatment of cancer using chimeric antigen receptor and protein kinase a blocker |
| US20180207273A1 (en) | 2015-07-29 | 2018-07-26 | Novartis Ag | Combination therapies comprising antibody molecules to tim-3 |
| EP3328418A1 (en) | 2015-07-29 | 2018-06-06 | Novartis AG | Combination therapies comprising antibody molecules to pd-1 |
| LT3317301T (en) | 2015-07-29 | 2021-07-26 | Novartis Ag | Combination therapies comprising antibody molecules to lag-3 |
| US20200261573A1 (en) | 2015-12-17 | 2020-08-20 | Novartis Ag | Combination of c-met inhibitor with antibody molecule to pd-1 and uses thereof |
| MX2018007423A (en) | 2015-12-17 | 2018-11-09 | Novartis Ag | Antibody molecules to pd-1 and uses thereof. |
| EP3851457A1 (en) | 2016-01-21 | 2021-07-21 | Novartis AG | Multispecific molecules targeting cll-1 |
| WO2017149515A1 (en) | 2016-03-04 | 2017-09-08 | Novartis Ag | Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore |
| WO2017157305A1 (en) | 2016-03-15 | 2017-09-21 | Generon (Shanghai) Corporation Ltd. | Multispecific fab fusion proteins and use thereof |
| EP3432924A1 (en) | 2016-03-23 | 2019-01-30 | Novartis AG | Cell secreted minibodies and uses thereof |
| BR112018071096A2 (en) | 2016-04-15 | 2019-02-26 | Novartis Ag | compositions and methods for selective protein expression |
| US20210177896A1 (en) | 2016-06-02 | 2021-06-17 | Novartis Ag | Therapeutic regimens for chimeric antigen receptor (car)- expressing cells |
| US20190336504A1 (en) | 2016-07-15 | 2019-11-07 | Novartis Ag | Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor |
| KR20230100748A (en) | 2016-07-28 | 2023-07-05 | 노파르티스 아게 | Combination therapies of chimeric antigen receptors adn pd-1 inhibitors |
| MX2019001469A (en) | 2016-08-01 | 2019-10-02 | Novartis Ag | Treatment of cancer using a chimeric antigen receptor in combination with an inhibitor of a pro-m2 macrophage molecule. |
| CA3035875A1 (en) | 2016-09-20 | 2018-03-29 | Aarhus Universitet | Compounds for treatment of lipoprotein metabolism disorders |
| TW202340473A (en) | 2016-10-07 | 2023-10-16 | 瑞士商諾華公司 | Treatment of cancer using chimeric antigen receptors |
| WO2018140725A1 (en) | 2017-01-26 | 2018-08-02 | Novartis Ag | Cd28 compositions and methods for chimeric antigen receptor therapy |
| US20200048359A1 (en) | 2017-02-28 | 2020-02-13 | Novartis Ag | Shp inhibitor compositions and uses for chimeric antigen receptor therapy |
| US11965021B2 (en) | 2017-04-26 | 2024-04-23 | Eureka Therapeutics, Inc. | Cells expressing chimeric activating receptors and chimeric stimulating receptors and uses thereof |
| WO2018201051A1 (en) | 2017-04-28 | 2018-11-01 | Novartis Ag | Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor |
| WO2018201056A1 (en) | 2017-04-28 | 2018-11-01 | Novartis Ag | Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor |
| PE20200717A1 (en) | 2017-06-22 | 2020-07-21 | Novartis Ag | ANTIBODY MOLECULES THAT BIND AND USES CD73 |
| EP3645037A1 (en) | 2017-06-27 | 2020-05-06 | Novartis AG | Dosage regimens for anti-tim-3 antibodies and uses thereof |
| KR20200035966A (en) | 2017-07-11 | 2020-04-06 | 콤파스 테라퓨틱스 엘엘씨 | Effector antibodies that bind to human CD137 and uses thereof |
| US20200172617A1 (en) | 2017-07-20 | 2020-06-04 | Novartis Ag | Dosage regimens of anti-lag-3 antibodies and uses thereof |
| WO2019089753A2 (en) | 2017-10-31 | 2019-05-09 | Compass Therapeutics Llc | Cd137 antibodies and pd-1 antagonists and uses thereof |
| WO2019089798A1 (en) | 2017-10-31 | 2019-05-09 | Novartis Ag | Anti-car compositions and methods |
| MX2020004756A (en) | 2017-11-16 | 2020-08-20 | Novartis Ag | Combination therapies. |
| EP3713961A2 (en) | 2017-11-20 | 2020-09-30 | Compass Therapeutics LLC | Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof |
| AU2019215031A1 (en) | 2018-01-31 | 2020-08-20 | Novartis Ag | Combination therapy using a chimeric antigen receptor |
| US20210147547A1 (en) | 2018-04-13 | 2021-05-20 | Novartis Ag | Dosage Regimens For Anti-Pd-L1 Antibodies And Uses Thereof |
| EP3784351A1 (en) | 2018-04-27 | 2021-03-03 | Novartis AG | Car t cell therapies with enhanced efficacy |
| CA3099308A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Compositions and methods for enhancing the killing of target cells by nk cells |
| WO2019226658A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Multispecific antigen-binding compositions and methods of use |
| US20210213063A1 (en) | 2018-05-25 | 2021-07-15 | Novartis Ag | Combination therapy with chimeric antigen receptor (car) therapies |
| US20210214459A1 (en) | 2018-05-31 | 2021-07-15 | Novartis Ag | Antibody molecules to cd73 and uses thereof |
| KR20210020932A (en) | 2018-06-13 | 2021-02-24 | 노파르티스 아게 | BCMA chimeric antigen receptor and uses thereof |
| PE20210418A1 (en) | 2018-06-19 | 2021-03-08 | Atarga Llc | COMPLEMENT COMPONENT 5 ANTIBODY MOLECULES AND THEIR USES |
| AR116109A1 (en) | 2018-07-10 | 2021-03-31 | Novartis Ag | DERIVATIVES OF 3- (5-AMINO-1-OXOISOINDOLIN-2-IL) PIPERIDINE-2,6-DIONA AND USES OF THE SAME |
| WO2020021465A1 (en) | 2018-07-25 | 2020-01-30 | Advanced Accelerator Applications (Italy) S.R.L. | Method of treatment of neuroendocrine tumors |
| AU2019379576A1 (en) | 2018-11-13 | 2021-06-03 | Compass Therapeutics Llc | Multispecific binding constructs against checkpoint molecules and uses thereof |
| WO2020128892A1 (en) | 2018-12-20 | 2020-06-25 | Novartis Ag | Extended low dose regimens for mdm2 inhibitors |
| KR20210106437A (en) | 2018-12-20 | 2021-08-30 | 노파르티스 아게 | Dosage regimens and pharmaceutical combinations comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives |
| EP3924055B1 (en) | 2019-02-15 | 2024-04-03 | Novartis AG | Substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| US10871640B2 (en) | 2019-02-15 | 2020-12-22 | Perkinelmer Cellular Technologies Germany Gmbh | Methods and systems for automated imaging of three-dimensional objects |
| US20220144807A1 (en) | 2019-02-15 | 2022-05-12 | Novartis Ag | 3-(1-oxo-5-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| WO2020172553A1 (en) | 2019-02-22 | 2020-08-27 | Novartis Ag | Combination therapies of egfrviii chimeric antigen receptors and pd-1 inhibitors |
| AU2020253833A1 (en) | 2019-03-29 | 2021-10-28 | Atarga, Llc | Anti FGF23 antibody |
| BR112022007376A2 (en) | 2019-10-21 | 2022-07-05 | Novartis Ag | COMBINATION THERAPIES WITH VENETOCLAX AND TIM-3 INHIBITORS |
| WO2021079195A1 (en) | 2019-10-21 | 2021-04-29 | Novartis Ag | Tim-3 inhibitors and uses thereof |
| EP4065158A2 (en) | 2019-11-26 | 2022-10-05 | Novartis AG | Chimeric antigen receptors binding bcma and cd19 and uses thereof |
| WO2021123996A1 (en) | 2019-12-20 | 2021-06-24 | Novartis Ag | Uses of anti-tgf-beta antibodies and checkpoint inhibitors for the treatment of proliferative diseases |
| EP4090762A1 (en) | 2020-01-17 | 2022-11-23 | Becton, Dickinson and Company | Methods and compositions for single cell secretomics |
| JP2023510393A (en) | 2020-01-17 | 2023-03-13 | ノバルティス アーゲー | A combination comprising a TIM-3 inhibitor and a hypomethylating agent for use in treating myelodysplastic syndrome or chronic myelomonocytic leukemia |
| EP4110377A2 (en) | 2020-02-27 | 2023-01-04 | Novartis AG | Methods of making chimeric antigen receptor-expressing cells |
| CN115916199A (en) | 2020-06-23 | 2023-04-04 | 诺华股份有限公司 | Dosing regimens comprising 3- (1-oxoisoindolin-2-yl) piperidine-2, 6-dione derivatives |
| IL299586A (en) | 2020-07-16 | 2023-03-01 | Novartis Ag | Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules |
| WO2022026592A2 (en) | 2020-07-28 | 2022-02-03 | Celltas Bio, Inc. | Antibody molecules to coronavirus and uses thereof |
| US20230271940A1 (en) | 2020-08-03 | 2023-08-31 | Novartis Ag | Heteroaryl substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| US20230338587A1 (en) | 2020-08-31 | 2023-10-26 | Advanced Accelerator Applications International Sa | Method of treating psma-expressing cancers |
| WO2022043557A1 (en) | 2020-08-31 | 2022-03-03 | Advanced Accelerator Applications International Sa | Method of treating psma-expressing cancers |
| JP2023547499A (en) | 2020-11-06 | 2023-11-10 | ノバルティス アーゲー | Antibody Fc variant |
| CA3198447A1 (en) | 2020-11-13 | 2022-05-19 | Novartis Ag | Combination therapies with chimeric antigen receptor (car)-expressing cells |
| JP2024505049A (en) | 2021-01-29 | 2024-02-02 | ノバルティス アーゲー | Administration modes for anti-CD73 and anti-ENTPD2 antibodies and their uses |
| TW202304979A (en) | 2021-04-07 | 2023-02-01 | 瑞士商諾華公司 | USES OF ANTI-TGFβ ANTIBODIES AND OTHER THERAPEUTIC AGENTS FOR THE TREATMENT OF PROLIFERATIVE DISEASES |
| AR125874A1 (en) | 2021-05-18 | 2023-08-23 | Novartis Ag | COMBINATION THERAPIES |
| WO2023044483A2 (en) | 2021-09-20 | 2023-03-23 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
| WO2023092004A1 (en) | 2021-11-17 | 2023-05-25 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of tau-related disorders |
| US20230383010A1 (en) | 2022-02-07 | 2023-11-30 | Visterra, Inc. | Anti-idiotype antibody molecules and uses thereof |
| WO2023220695A2 (en) | 2022-05-13 | 2023-11-16 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
| WO2024030976A2 (en) | 2022-08-03 | 2024-02-08 | Voyager Therapeutics, Inc. | Compositions and methods for crossing the blood brain barrier |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4444878A (en) * | 1981-12-21 | 1984-04-24 | Boston Biomedical Research Institute, Inc. | Bispecific antibody determinants |
| IL97459A0 (en) * | 1990-03-09 | 1992-06-21 | Hybritech Inc | Trifunctional antibody-like compounds as a combined diagnostic and therapeutic agent |
| GB9012995D0 (en) * | 1990-06-11 | 1990-08-01 | Celltech Ltd | Multivalent antigen-binding proteins |
-
1992
- 1992-10-15 GB GB929221657A patent/GB9221657D0/en active Pending
-
1993
- 1993-10-15 CA CA 2146854 patent/CA2146854A1/en not_active Abandoned
- 1993-10-15 AU AU52837/93A patent/AU5283793A/en not_active Abandoned
- 1993-10-15 JP JP6509777A patent/JPH08505761A/en active Pending
- 1993-10-15 WO PCT/GB1993/002133 patent/WO1994009131A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994009131A1 (en) | 1994-04-28 |
| AU5283793A (en) | 1994-05-09 |
| GB9221657D0 (en) | 1992-11-25 |
| JPH08505761A (en) | 1996-06-25 |
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